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https://openalex.org/W4390957082	https://storage.googleapis.com/jnl-up-j-ijic-files/journals/1/articles/8418/65713f84a1856.pdf	English	null	Measuring goal progress using the goal-based outcome (GBO) measure in Youth Wellness Hubs Ontario – an integrated youth mental health service	International journal of integrated care	2,023	cc-by	599	Measuring goal progress using the goal-based outcome (GBO) measure in Youth Wellness Hubs Ontario – an integrated youth mental health service 23rd International Conference on Integrated Care, Antwerp, Flanders, 22-24 May 2023 Debbie Chiodo1, Karleigh Darnay, Jo Henderson 1: Centre for Addiction and Mental Health, Toronto, Canada Problem and Context. There is a growing body of evidence indicating that the prevalence of mental health difficulties, in particular anxiety and depression, is increasing among youth, and many young people in this age group (12-25 years) do not get adequate mental health support. Recently, a global movement to transform youth mental health services is underway with the establishment of integrated youth services (IYS) that offer youth-specific care and emphasize early intervention and prevention, developmentally and culturally- informed services, community engagement, evidence-based and evidence-informed interventions, and youth and family engagement. While outcome data has an important role in enhancing the effectiveness of care for youth mental health, having young people define for themselves the area they feel should be the focus of in an intervention are also common measures used in IYS settings. Goal-setting has been used in therapy for several decades, and agreement on goals between young person and their clinician is thought to be central to successfully building a good therapeutic relationship and improving outcomes. Who is it for? Service providers, researchers, clinicians, youth and families. Who did you involve and engage with? Participants were young people (>5000) who engaged with a Youth Wellness Hub service between April 2020 and October 2022. The data for this study comprised over 15,000 goals. Youth can set up to one, two or three goals at each visit using the Goal-Based Outcome (GBO) tool. Inductive thematic analysis was employed on the types of goals set by young people. What did you do? The aim of this study was to explore the type of service goals set by young people who are engaging with integrated mental health services. The study also looked at whether services facilitated progress in goals as set by young people. What results did you get? What impact did you have? The analysis identified a number of goal- related themes related to improving mental health, reducing substance use and other addictions, improving physical health, service navigation, and self-improvement. Data from presentation to last assessment will be reviewed to examine change in progress on goals over time. What is the learning for the international audience? Chiodo D et al, 2023 Measuring goal progress using the goal-based outcome (GBO) measure in Youth Wellness Hubs Ontario – an integrated youth mental health service. International Journal of Integrated Care 23(S1):582 DOI: doi.org/10.5335/ijic.ICIC23582 Chiodo D et al, 2023 Measuring goal progress using the goal-based outcome (GBO) measure in Youth Wellness Hubs Ontario – an integrated youth mental health service. International Journal of Integrated Care 23(S1):582 DOI: doi.org/10.5335/ijic.ICIC23582 Measuring goal progress using the goal-based outcome (GBO) measure in Youth Wellness Hubs Ontario – an integrated youth mental health service 23rd International Conference on Integrated Care, Antwerp, Flanders, 22-24 May 2023 Understanding the clinical utility of an idiographic measure like the GBO with young people accessing community-based services is critical because the tool allows for capturing aspects of young people’s lives that are of importance to them that may not be as easily captured on standardized outcome tools. Chiodo: Measuring goal progress using the goal-based outcome (GBO) measure in Youth Wellness Hubs Ontario – an integrated youth mental health service What are the next steps? Future directions include continuous learning and evaluation of the GBO in integrated care settings and examining the association between goal progress and standardize outcomes.
https://openalex.org/W3014859581	https://iiste.org/Journals/index.php/RHSS/article/download/52183/53920	English	null	Relevance of In-service-Teacher Training in Tanzania: Lessons from Licensed and Normal Trained Teachers	Research on Humanities and Social Sciences	2,020	cc-by	5,149	Relevance of In-service-Teacher Training in Tanzania: Lessons from Licensed and Normal Trained Teachers Benjamin Mbeba Meli Dares Salaam University College of Education Faculty of Humanities and Social sciences, Department of History, political Science and Development Studies P.o Box 2329 Dares Salaam Tanzania, East Africa Email: benjaminmbeba@gmail.com Benjamin Mbeba Meli Dares Salaam University College of Education Faculty of Humanities and Social sciences, Department of History, political Science and Development Studies P.o Box 2329 Dares Salaam Tanzania, East Africa Email: benjaminmbeba@gmail.com Abstract The focus of this paper is to examine the efficacy of various in-service teacher training programs offered to both licensed and normal trained teachers in Tanzania. The in-service programs includes effective ways of improving teaching performance for in-service training students, types of in-service teacher training offered to both licensed and normal trained teachers, self-initiatives taken by both types of teachers to undergo in-service training, challenges and problems facing licensed and normal trained teachers, conclusion and recommendations. The paper advances some recommendations that should be taken by the government and other stake-holders to minimize the challenges and problems facing these two types of teachers as far as the education sector in Tanzania are concerned. Key Words: Licensed and Normal Trained Teachers, Pedagogical Skills, In-service Teacher, Teacher training DOI: 10.7176/RHSS/10-6-11 Publication date:March 31st 2020 www.iiste.org www.iiste.org Research on Humanities and Social Sciences Research on Humanities and Social Sciences ISSN 2224-5766 (Paper) ISSN 2225-0484 (Online) Vol.10, No.6, 2020 1.0 Introduction In-service training (INSET) is a situation in which teachers have a chance to attain new knowledge and skills in a more dynamic way (Galabawa et al., 2000). The Ministry of Education, Science and Technology (MoEST) in Tanzania has made sure that both licensed and normal trained teachers are getting in-service training courses in order to upgrade their skills and knowledge. Special emphasis was put on licensed trained teachers that by 2010 all licensed trained teachers should have upgraded their levels of education otherwise they will be terminated from their employment. Therefore, the Open University of Tanzania was requested to enrol all licensed trained teachers. It is during INSET where teachers’ professional knowledge, professional skills and professional attitudes develop and mature. It is also through INSET that teachers’ academic standard is raised, participants acquire skills, attitudes and values intended for particular professionals. The importance of INSET is expressed by Swazin and Monk (2000) who revealed that, INSET which is conducted from areas of work and using facilities which do not really exist in the teachers working places yield very little results. In most cases, teachers fail to transfer knowledge and skills to their students. On the other hand, Linde (1998) suggested that, INSET programs that take place within working places, using the coaching approach, to a large extent yield positive results. Teachers are able to attain and deploy in their classrooms large amount of knowledge and skills acquired. The literature indicates that the good fruits which are expected from INSET depend on the way it is planned and conducted. 2.0 Effective ways of improving Teaching Performance for In-service Training Students Teachers’ performance is the basic foundation in enhancing quality of education in any society. Teachers whose performance is good always possess knowledge of pedagogy so as to address issues related to classrooms practices (Cochran, 1993; Kanu, 1996). Dunn (1981), Dye (1978) and Meli (2014) support the statement that poor training of the teachers results into teachers’ poor performance and the solution for solving that problem is the provision of in-service trainings for teachers. Osaki (1996) pointed out that good teachers’ preparation during in-service training, where skills to create a conducive learning environment and to motivate and improve students self esteem are acquired lead to student achievements in learning. 1.0 Introduction Thus , in order to attain effective training, the in-service training programs must be of high quality of three inter-dependent components namely, facilitators, learners and materials as illustrated in Figure 1 below. 2.0 Effective ways of improving Teaching Performance for In-service Training Students Teachers’ performance is the basic foundation in enhancing quality of education in any society. Teachers whose performance is good always possess knowledge of pedagogy so as to address issues related to classrooms practices (Cochran, 1993; Kanu, 1996). Dunn (1981), Dye (1978) and Meli (2014) support the statement that poor training of the teachers results into teachers’ poor performance and the solution for solving that problem is the provision of in-service trainings for teachers. Osaki (1996) pointed out that good teachers’ preparation during in-service training, where skills to create a conducive learning environment and to motivate and improve students self esteem are acquired lead to student achievements in learning. Thus , in order to attain effective training, the in-service training programs must be of high quality of three inter-dependent components namely, facilitators, learners and materials as illustrated in Figure 1 below. 84 Research on Humanities and Social Sciences Research on Humanities and Social Sciences ISSN 2224-5766 (Paper) ISSN 2225-0484 (Online) Vol.10, No.6, 2020 ISSN 2224-5766 (Paper) ISSN 2225-0484 (Online) Vol.10, No.6, 2020 Figure 1: Components for Effective Teacher Education and Training. Source: Osaki (1996). Materials Facilitators Learners Learners Materials Facilitators Figure 1: Components for Effective Teacher Education and Training. Source: Osaki (1996). Kiwia (1999) revealed that among the problems facing training programs is the facilitator. This problem ranges from experience to mastery of subject matter. The trainers who are mandated to train effective teachers to teach in secondary schools should be competent enough in their subject mastery (Kiwia, 1999) and teachers should acquire the requisite , pedagogical skills during the in-service training. On the other hand, Heyneman and Loxley (1983) pointed out that, in-service training must meet two crucial criteria: first, it must produce learning changes with efficiency and efficacy and secondly, the programs for imparting knowledge for in-service students must be well prepared, and if participants find them entertaining and enjoyable, the chances of learning are greater to occur. Therefore, the training for teachers during in-service training must result in some benefits to teachers, students, schools and the education sector in general. It must be carried out effectively, efficiently and it must create excellence in learning. 1.0 Introduction It further must be employee/teacher centred and more committed to reducing the turnover and grievances of teachers and help them to do their jobs more accurately and efficiently as indicated in figure 2 below below. Figure 2: The Logic of Training. Source: Heyneman and Loxley (1983) Likewise, Joyce and Shower (1988) proposes the main training outcomes if a person attending in-service training s: awareness of education theories and practices, new curriculum; change in attitudes; self role perception changes; cademic content; development of skills and the ability to perform discrete behaviours such as designing and delivering questions of various cognitive levels, as well as transfer of training and executive control with consistent nd appropriate use of new skills and strategies for classroom instruction. Participants talking skills, knowledge & required attitudes. Training Program Participants learn new skills, knowledge, & attitudes. Participants with new skills knowledge & attitudes. Work place Participants use new knowledge, skills & attitudes. Payoff to organization. elow. Figure 2: The Logic of Training. Source: Heyneman and Loxley (1983) Participants talking skills, knowledge & required attitudes. Training Program Participants learn new skills, knowledge, & attitudes. Participants with new skills knowledge & attitudes. Work place Participants use new knowledge, skills & attitudes. Payoff to organization. Figure 2: The Logic of Training. Source: Heyneman and Loxley (1983) Likewise, Joyce and Shower (1988) proposes the main training outcomes if a person attending in-service training as: awareness of education theories and practices, new curriculum; change in attitudes; self role perception changes; academic content; development of skills and the ability to perform discrete behaviours such as designing and delivering questions of various cognitive levels, as well as transfer of training and executive control with consistent and appropriate use of new skills and strategies for classroom instruction. Likewise, Joyce and Shower (1988) proposes the main training outcomes if a person attending in-service training as: awareness of education theories and practices, new curriculum; change in attitudes; self role perception changes; academic content; development of skills and the ability to perform discrete behaviours such as designing and delivering questions of various cognitive levels, as well as transfer of training and executive control with consistent and appropriate use of new skills and strategies for classroom instruction. 3.0 Types of In-service Teacher Training Offered to both Licensed and normal Trained Teachers Furthermore, Swazin and Monk (2000: pg?) commenting on the importance of attending in-service teacher training courses argues that: ‘‘....teachers who attend in-service teacher training courses, their academic performance is more than those who do not attend, because it keeps teachers up to date in their knowledge, skills, methods and classrooms management...” Table 2 and Table 3 below summarize the types of in-service teacher training attended to both types of teachers. Table 2 and Table 3 below summarize the types of in service teacher training attended to both types of teachers. Table 2: Municipal Secondary Education Officers’ Responses towards In-Service Teacher Training Courses for Licensed Teachers (2008-2010). Municipal/Districts Types of Courses Offered Diploma in Education Bachelor Degree in Education Post Graduate Studies Total Morogoro 30 46 2 78 Iringa 28 50 3 81 Songea 23 45 1 69 Bunda 38 31 1 70 Total 119 172 07 298 Source: Computed from field data (2014). Table 3: Municipal Secondary Education Officers’ Responses towards In-Service Teacher Training Courses for Normal Teachers (2008-2010) Municipal/Districts Types of Courses Offered Diploma in Education Bachelor Degree in Education Post Graduate Studies Total Morogoro - 34 14 48 Iringa - 38 18 56 Songea - 37 15 52 Bunda - 29 11 40 Total - 138 58 196 Source: Computed from field data (2014). Municipal Secondary Education Officers’ Responses towards In-Service Teacher Training Courses for Licensed Teachers (2008-2010). Table 2: Municipal Secondary Education Officers’ Responses towards In-Service Teacher Training Courses for Licensed Teachers (2008-2010). for Licensed Teachers (2008 2010). Municipal/Districts Types of Courses Offered Diploma in Education Bachelor Degree in Education Post Graduate Studies Total Morogoro 30 46 2 78 Iringa 28 50 3 81 Songea 23 45 1 69 Bunda 38 31 1 70 Total 119 172 07 298 Source: Computed from field data (2014) Table 3: Municipal Secondary Education Officers’ Responses towards In-Service Teacher Training Courses for Normal Teachers (2008-2010) Municipal/Districts Types of Courses Offered Diploma in Education Bachelor Degree in Education Post Graduate Studies Total Morogoro - 34 14 48 Iringa - 38 18 56 Songea - 37 15 52 Bunda - 29 11 40 Total - 138 58 196 Source: Computed from field data (2014). 3.0 Types of In-service Teacher Training Offered to both Licensed and normal Trained Teachers 3.0 Types of In-service Teacher Training Offered to both Licensed and normal Trained Teachers Findings from respondents in the four municipals through interview and focus group discussion showed that both types of teachers had undergone in-service teacher training program depending on the level of their education. However, the costs of the training were not met by the government but by the teachers themselves. What the government did was to aloow them to attend the training. The Municipal Secondary Education Officers interviewed, indicated that their teachers had attended further studies in various programs such as Diploma in Education course, Bachelor degree and Post graduate studies. The main aim of sending them for in-service courses was to make them more competent in teaching skills. On the other hand, one of the Ministry of Education, Science and Technology (MoEST) respondents made the following statement on the importance of sending teachers for in-service training that: 85 Research on Humanities and Social Sciences www.iiste.org ISSN 2224-5766 (Paper) ISSN 2225-0484 (Online) Vol.10, No.6, 2020 ”...an effective teacher learns as he/she teaches, there is not any type of teacher education that can prepare him/her for the varied experiences he/she faces year to year. Only in-service training can keep a teacher up to date in his/her knowledge, skills, methods and classrooms management. He/she needs in-service training in order to remain effective and successful as a teacher...” www.iiste.org ISSN 2224-5766 (Paper) ISSN 2225-0484 (Online) Vol.10, No.6, 2020 ”...an effective teacher learns as he/she teaches, there is not any type of teacher education that can prepare him/her for the varied experiences he/she faces year to year. Only in-service training can keep a teacher up to date in his/her knowledge, skills, methods and classrooms management. He/she needs in-service training in order to remain effective and successful as a teacher...” This is in line with Clandinin’s (1995) argument that the process of becoming a professional teacher is a “life long process” which usually involves years of acquiring knowledge on teaching, learning process, trying out new teaching style that engage learners, observing others, effective classroom management, good preparation, effective use of teaching and learning materials, receiving and giving feedback. 3.0 Types of In-service Teacher Training Offered to both Licensed and normal Trained Teachers Table 3: Licensed and Normal Trained Teachers’ Responses on why they enrolled for In-Service Teacher Training. Reasons for Joining In-Service Teacher Training Courses Normal Trained Teachers=30 Licensed Trained Teachers=30 * It enables them to design tasks that will address learners’ abilities, interests and needs. 06 08 * It leads them to develop intellectual Skills eg: inquiry, problem solving, analysis and synthesis. 17 15 * It enables them to know how to organize learning groups of different abilities. 09 11 * It enables them to use and emphasize interactive methods of teaching and learning. 06 08 * It enables them to increase salary, promotion and categorization. 28 27 * It makes teachers use a variety of teaching aid which are available in their environment. 12 14 Source: Compiled from field data (2014). Research on Humanities and Social Sciences ISSN 2224-5766 (Paper) ISSN 2225-0484 (Online) Vol.10, No.6, 2020 ...”the government through the Ministry of Education, and Vocational and Training issued directives to licensed trained teachers instructing them that within five years (2004-2009) of starting the program, they are supposed to enrol for further studies (Diploma or Degree courses) and government will sponsor them...” This was supported by the Coordinator of Induction course for licensed secondary school teachers from the MoEST who commented that: who commented that: “within five years (2004-2009) licensed teachers should enrol themselves with the Open University of Tanzania (OUT) for a degree or Diploma program in education and MoEST will sponsor the respective programs”. In addition, the respondents were asked why they decided to join in-service programs. Both types of teachers had the following reasons as indicated in Table 3 below. Table 3: Licensed and Normal Trained Teachers’ Responses on why they enrolled for In-Service Teacher Training. Reasons for Joining In-Service Teacher Training Courses Normal Trained Teachers=30 Licensed Trained Teachers=30 * It enables them to design tasks that will address learners’ abilities, interests and needs. 06 08 * It leads them to develop intellectual Skills eg: inquiry, problem solving, analysis and synthesis. 17 15 * It enables them to know how to organize learning groups of different abilities. 09 11 * It enables them to use and emphasize interactive methods of teaching and learning. 06 08 * It enables them to increase salary, promotion and categorization. 28 27 * It makes teachers use a variety of teaching aid which are available in their environment. 3.0 Types of In-service Teacher Training Offered to both Licensed and normal Trained Teachers 12 14 Source: Compiled from field data (2014). nsed and Normal Trained Teachers’ Responses on why they enrolled for In-Service Teacher T i i ble 3: Licensed and Normal Trained Teachers’ Responses on why they enrolled for In-Service Teacher Training. Source: Compiled from field data (2014). Table 3 indicates that both categories of teachers had similar ranking on the reasons for joining in-service teacher training as follows: In terms of prioritization, first, it enables them to increase salary, promotion and categorization; second, it leads them to develop intellectual skills such inquiry, problem-solving, analysis and synthesis; third, it makes teachers use a variety of teaching aids which are available in their environment; Fourth, it enables them to know how to organize learning groups of different abilities; Fifth, it enables them to design tasks that will address learners’ abilities, interests and needs. In this respect, Mbunda (1992) point out that in-service teacher training is always provided in order to upgrade academic as well as professional competencies among teachers. The duration and type of in-service courses, involve residential courses leading to certificates of attendance while others are long-term residential courses that lead to diploma and degree certificates to promote salary increase and knowledge skills. 3.0 Types of In-service Teacher Training Offered to both Licensed and normal Trained Teachers nicipal Secondary Education Officers’ Responses towards In-Service Teacher Training Courses for Normal Teachers (2008-2010) Table 2 and Table 3 above show that the number of the normal trained teachers who joined in-service teacher training courses for both Bachelor and postgraduate degrees were more than the licensed trained teachers. There were larger numbers of licensed trained teachers who enrolled for degrees in Education than normal teachers. For example, Morogoro municipal had 30 licensed trained teachers who joined Diploma course and 46 licensed trained teachers who enrolled for Bachelor degree against 34 of normal trained teachers who enrolled for the bachelor degree. Iringa municipal had 28 licensed trained teachers who joined for Diploma in Education and another 50 for Bachelor degree. This was different from the normal trained teachers where there was no one enrolled for Diploma in education while there were 38 teachers who were enrolled for Bachelor degree. Songea had 23 licensed trained teachers for Diploma in education and 45 for Bachelor degree, while for the normal trained teachers in the same municipal there were only 37 for Bachelor degree and none enrolled for Diploma course. Bunda municipal indicated that there were 38 licensed trained teachers for Diploma in education and another 31 for Bachelor degree while for normal trained teachers there were only 29 who enrolled for Bachelor degree. However, one of the respondents from headmasters/mistresses commenting on why the licensed trained teachers had joined more in the in-service training than normal trained teachers had this to say: 86 Research on Humanities and Social Sciences www.iiste.org ISSN 2224-5766 (Paper) ISSN 2225-0484 (Online) Vol.10, No.6, 2020 ...”the government through the Ministry of Education, and Vocational and Training issued directives to licensed trained teachers instructing them that within five years (2004-2009) of starting the program, they are supposed to enrol for further studies (Diploma or Degree courses) and government will sponsor them...” This was supported by the Coordinator of Induction course for licensed secondary school teachers from the MoEST who commented that: “within five years (2004-2009) licensed teachers should enrol themselves with the Open University of Tanzania (OUT) for a degree or Diploma program in education and MoEST will sponsor the respective programs”. In addition, the respondents were asked why they decided to join in-service programs. Both types of teachers had the following reasons as indicated in Table 3 below. 0 Challenges and Problems Facing Licensed and Normal Trained Teachers 5.0 Challenges and Problems Facing Licensed and Normal Trained Teachers Findings revealed that there were several challenges and problems that face licensed and normal trained teachers in teaching students in their classes. Both licensed and normal trained teachers maintained that their classes were too overcrowded and this led to poor teaching. They argued that increasing number of enrolment for students in form one had brought a negative impact to the teaching profession. All of the schools visited had higher teacher- student ratio in classroom than that of the national education standard which is 1:40. This made both types of teachers fail to impart knowledge effectively to the students. Frequent changes in secondary schools curricula had brought a negative impact on the teaching profession. Education curricula had been changing frequently without having any teaching orientation to the teachers. As a result, teachers were appointed to teach subjects without any knowledge of the subject concerned. For example, one of the respondents from headmasters/mistress had the following arguments: “teachers have been forced to teach certain topics from curricula that were unfamiliar to them and without being given any orientation. This situation is an impediment to respective teachers as regards teaching/learning materials preparations and use”. On other side of the coin, there was a shortage of teaching and learning facilities in secondary schools which hindered their teaching evaluation. The absence of learning and teaching materials had made teachers to teach students without using teaching aids and sometimes had to look for assistance from neighbouring school in order to improve their teaching profession. For instance, some of the respondents from licensed and normal trained teachers went further to point out that: “we had to get some academic assistance from our fellow colleagues within the schoolsor from colleagues in the neighbouring schools to help us on how to teach the new curricula or topicsand sometimes, we relied heavily on school inspectors or secondary academic officersfor help.” Their arguments were supported by almost all Municipal Secondary Education Officers who concurred that there was a shortage of teaching and learning facilities in secondary schools which hindered their teaching evaluation. They argued that the absence of learning and teaching materials have forced teachers to teach students without using teaching aids. 0 Self-initiatives Taken by both Two Types of Teachers to Undergo In-service Training Course h li d t i d t h i t d t th t th t th t f d th t d f th 4.0 Self-initiatives Taken by both Two Types of Teachers to Undergo In-service Training Course The licensed trained teachers pointed out that there were two reasons that forced them to undergo further studies to up-date their knowledge and skills. First, there was a promise made by MoEST to sponsor those studies for all licensed teachers from the beginning of the program in 2004 to 2009 on conditions that anybody who did not undergo further studies his/her employment would be terminated by the government. Second, the society perceived them as unqualified teachers because of the short training which made them be nicknamed as voda fasta in relation to the Vodacom mobile phone company’s advertisement on how fast they can provide their services to their customers when compared to other mobile phone service providers. However, municipal secondary education officers and headmasters/mistresses reported that licensed trained teachers had more self-initiative in attaining in-service training course than normal trained teachers because the former were given a probation period of five years for up-grading their levels of education, otherwise, they would be terminated from employment. Therefore, it could be concluded that licensed trained teachers were likely to have 87 Research on Humanities and Social Sciences ISSN 2224-5766 (Paper) ISSN 2225-0484 (Online) Vol.10, No.6, 2020 more self-initiative for in-service training than normal trained teachers because normal were not employed under government conditions. 0 Challenges and Problems Facing Licensed and Normal Trained Teachers For example, one of the respondents had the following arguments: to teach subjects without any knowledge of the subject matter concerned. For example, one of the respondents had the following arguments: to teach subjects without any knowledge of the subject matter concerned. For example, one of the respondents had the following arguments: “teachers have been forced to teach certain topics from curricula that were unfamiliar to them and without being given any orientation. This situation is an impediment to respective teachers as regards teaching/learning materials preparations and use”. In addition, The heads of schools had it that poor working environment was a big challenge that faces both types of teachers in Tanzania. This situation had contributed to the decline of teachers’ morale for work as noted out by one of the respondents from students’ parents that: In addition, The heads of schools had it that poor working environment was a big challenge that faces both types of teachers in Tanzania. This situation had contributed to the decline of teachers’ morale for work as noted out by one of the respondents from students’ parents that: “ the quality of education in Tanzania will decline every year if the government does not improve the living and working conditions for teachers. Teachers are working and living in poor environment, they do not have proper and decent quarters or houses, good salaries and other fringe benefits ” The zonal secondary inspectorate officers reported that there was a lot of bureaucracy in releasing teachers from work. They argued that headmasters/ mistresses and municipal secondary school officers had a lot of bureaucracy for releasing their teachers to attend further studies. 6.0 General Conclusions The study tried to identify clearly the types of in-service teacher training programs offered to both two types of teachers to update and upgrade their knowledge and skills, self-initiatives taken by both two types of teachers to undergo in-service training course, challenges and problems facing them in their daily activities. It can be concluded that apart from student-teachers having the same academic levels, but they differ in how they acquired the knowledge and skills. For example, the licensed trained teachers showed more enthusiasm to upgrading themselves compared to the normal trained teachers. Furthermore, the duration of training program was not equal because licensed trained teachers were taught for three months only while normal trained teachers were taught for two years. This variation of training contributed much to the ineffectiveness of teaching. 7.0 Recommendations Based on the study findings and ensuing conclusion, the following recommendations are made: first and foremost, The MoEST should provide and support general and specific in-service training programs, especially for the crash trained teachers, so that they become more knowledgeable and skilled in the teaching and learning processes. Second, both moral and material incentives should be given to both types of teachers in order to motivate them including improvement of working environment, salaries, teaching allowances and other fringe benefits. 0 Challenges and Problems Facing Licensed and Normal Trained Teachers Both licensed and normal trained teachers pointed out that: g g p “we had to get some academic assistance from our fellow colleagues within the schools or in the neighbouring schools to help us on how to teach the new curricula or topics and sometime, we relied heavily on school inspectors or secondary academic officers for help”. The above testimonies were concurred by Tanzania Institute of Education (TIE) and MoEST officials who noted out that: “shortage of teaching and learning materials was a big problem in the schools because teachers had to teach students using their own experience. This made students acquire knowledge and skills in a shallow manner” In addition, poor working environment was a big challenge that faced both types of teachers in Tanzania. They had no houses for settling in with their families and salaries were not enough to sustain their families. This situation had contributed to the decline of teachers’ morale for work as pointed out by one of the respondents from students’ parents that: “the quality of education in Tanzania will decline every year if the government does not improve the living and working conditions for teachers. Teachers are working and living in poor environment, they do not have proper and decent quarters or houses, good salaries and other fringe benefits” Furthermore, zonal secondary inspectorate officers reported that there was a lot of bureaucracy in releasing teachers from work. They argued that headmasters/ mistresses and municipal secondary school officers had a lot of bureaucracy for releasing their teachers to attend further studies. They argued that teachers applied to join further studies and got admitted but their headmasters/mistresses did not allow unless they had attained five years of employment. Furthermore, headmasters/mistresses pointed out that those frequent changes in secondary schools curricula had brought a negative impact on the teaching profession. They contended that secondary education curricula had been changing frequently without having any teaching orientation to the teachers. As a result, teachers were appointed 88 Research on Humanities and Social Sciences www.iiste.org ISSN 2224-5766 (Paper) ISSN 2225-0484 (Online) Vol.10, No.6, 2020 to teach subjects without any knowledge of the subject matter concerned. For example, one of the respondents had Research on Humanities and Social Sciences ISSN 2224-5766 (Paper) ISSN 2225-0484 (Online) Vol.10, No.6, 2020 ( p ) ( ) Vol.10, No.6, 2020 to teach subjects without any knowledge of the subject matter concerned. 8.0 Reference Clandinin, J., (1995). Teachers’ Professional Land Scales. New York: Lincoln Inc. Cochran, K., (1993). Pedagogical Context Knowing: An Integrative Model for Teachers Preparation. Journal of Teachers Education. Vol. 44. No. 2. pp 19-32. Cochran, K., (1993). Pedagogical Context Knowing: An Integrative Model for Teachers Preparation. Journal of Teachers Education. Vol. 44. No. 2. pp 19-32. Dunn, W., (1981). Public Policy Analysis: An Introduction. Eaglewood Cliff: Prentice Hall. Dye, I., (1978). Understanding Public Policy. Eaglewood Cliff: Prentice Hall Dye, I., (1978). Understanding Public Policy. Eaglewood Cliff: Prentice Hall Galabawa, J. Senkoro, F and Lwaitama, F (2000). The Quality of Education in Tanzania. 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https://openalex.org/W4231085290	https://europepmc.org/articles/pmc6260120?pdf=render	English	null	Chemokines and Chemokine Receptors as Therapeutic Targets in Inflammatory Bowel Disease; Pitfalls and Promise	Journal of Crohn's and colitis	2,018	cc-by	364	Palak J. Trivedia,b,c,d, David H. Adamsa,b aNational Institute for Health Research (NIHR) Birmingham Biomedical Research Centre (BRC), University of Birmingham and University Hospitals Birmingham NHS Foundation Trust, UK bInstitute of Immunology and Immunotherapy, University of Birmingham, Birmingham, UK bInstitute of Immunology and Immunotherapy, University of Birmingham, Birmingham, UK cLiver Unit, University Hospitals Birmingham NHS Foundation Trust, UK cLiver Unit, University Hospitals Birmingham NHS Foundation Trust, UK dDepartment of Gastroenterology, University Hospitals Birmingham NHS Foundation Trust, UK dDepartment of Gastroenterology, University Hospitals Birmingham NHS Foundation Trust, UK Corresponding author: Prof. David H. Adams, MD, FRCP, FMedSci, National Institute for Health Research (NIHR), Birmingham Biomedical Research Centre (BRC), University of Birmingham, Birmingham, UK. Email: d.h.adams@bham.ac.uk In the original article, the affiliations were incorrect and should have read as per the above. The funding statement was also incorrect and should have read as follows. The funding statement was also incorrect and should have read as follows. Journal of Crohn’s and Colitis, 2018, 1508 doi:10.1093/ecco-jcc/jjy130 Advance Access publication September 29, 2018 Corrigendum Journal of Crohn’s and Colitis, 2018, 1508 doi:10.1093/ecco-jcc/jjy130 Advance Access publication September 29, 2018 Corrigendum © The Author(s) 2018. Published by Oxford University Press on behalf of European Crohn’s and Colitis Organisation. 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 reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Funding PJT and DHA received institutional funding from the NIHR BRC. This article is independent research supported by the NIHR Birmingham Liver Biomedical Research Centre. The views expressed in this publication are of the authors and not necessarily those of the National Health Service, the NIHR, or the Department of Health. The authors apologize for the errors. 1508 1508 © The Author(s) 2018. Published by Oxford University Press on behalf of European Crohn’s and Colitis Organisation. 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 reuse, distribution, and reproduction in any medium, provided the original work is properly cited. p ( p which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
https://openalex.org/W3205775502	https://www.researchsquare.com/article/rs-826873/latest.pdf	English	null	Performance Analysis of Large Intelligent Reflecting Surface Aided Moderate MIMO for 5G Communication	Research Square (Research Square)	2,021	cc-by	5,650	Research Article Noname manuscript No. (will be inserted by the editor) Noname manuscript No. (will be inserted by the editor) Performance Analysis of Large Intelligent Reﬂecting Surface Aided Moderate MIMO for 5G Communication Fitsum Dessalegn Mekonnen Muluneh Mekonnen Tulu and Sultan Feisso Received: date / Accepted: date Received: date / Accepted: date Abstract The recently completed 5G framework is the outcome of a few advanced technologies. Massive Multiple Input Multiple Output (MIMO), millimeter wave communication, and network densiﬁcation are examples of these technologies. How- ever, there are two disadvantages to this technology.1) the lack of control over the wireless channel, and 2) the wireless interface’s excessive power consumption. The concept of re-conﬁgurable Intelligent Reﬂecting Surfaces has emerged to answer the need for green and cost-effective future cellular networks. In this study, we’ll look at how using an Intelligent Reﬂecting Surface (IRS) improve the performance of mod- erate MIMO communication in terms of the rate, SINR, energy efﬁciency and trans- mit power metrics. Despite the fact that the underlying issue is non-convexity, we use Lagrangian dual transform and Quadratic Transform to change and rearrange the original issue. After that, active and passive beam forming improved alternatively us- ing an alternating Direction method of multiplier algorithm (ADMM.The IRS-aided Fitsum Dessalegn Mekonnen Department of Electrical & Computer Engineering, Addis Ababa Science and Technology University, Addis Ababa, Ethiopia. Tel.: +251-923242006 E-mail: dﬁtsum06@gmail.com Muluneh Mekonnen Tulu Tel.: +251-913123353 E-mail: mulumoke@yahoo.com Department of Electrical & Computer Engineering, Addis Ababa Science and Technology University, Addis Ababa, Ethiopia. Artiﬁcial & Robotics Intelligence, COE, Addis Ababa Science and Technology University, Addis Ababa, Ethiopia. · Sultan Feisso Tel.: +251-912635020 E-mail: sultan.feisso@aastu.edu.et Department of Electrical & Computer Engineering, Addis Ababa Science and Technology University, Addis Ababa, Ethiopia. Artiﬁcial & Robotics Intelligence, COE, Addis Ababa Science and Technology University, Addis Ababa, Ethiopia. Department of Electrical & Computer Engineering, Addis Ababa Science and Technology University, Addis Ababa, Ethiopia. A iﬁi l & b i lli CO Addi Ab b S i d h l i i Addi Ab b 2 Fitsum Dessalegn Mekonnen Muluneh Mekonnen Tulu and Sultan Feisso system with a reasonable number of antennas at the access point (AP) outperforms the massive MIMO without IRS in terms of sum rate, SINR, energy efﬁciency and transmit power metrics. Keywords 5G · IRS · ADMM · MIMO · SINR Keywords 5G · IRS · ADMM · MIMO · SINR 1 Introduction Through many creative developments, such as massive (MIMO), millimeter wave (mm Wave) communication, and network densiﬁcation, the Fifth Generation (5G) network standard promises to deliver enhanced bandwidth, accessibility, and incredi- bly low delay. Regardless of this advancement, providing services of consistent qual- ity to clients in difﬁcult propagation environments consumes a signiﬁcant amount of power. The total network energy consumption, for instance, is related to the number of base stations (BS) and active antenna elements at each BS. Communication in the mm Wave bands suffers from signiﬁcant path/penetration losses. As a result of these issues, future cellular networks must be green and sustainable, with control over the propagation environment. The smart radio environment (SRE) is a new concept that fulﬁlls this demand by changing the wireless propagation environment into an intel- ligent tunable space that engages in radio frequency transmission from sender to re- ceiver [1]. This concept can be realized by placing low-cost antenna arrays [2], smart reﬂect-arrays [3], and re-conﬁgurable meta-surfaces in the environment to passively shape intruding electromagnetic (EM) waves in the desired direction without produc- ing additional radio signals. In this paper, we will see the importance of Intelligent Reﬂecting Surfaces to improve the performance of moderate MIMO system. 1.1 Related Works In [4], the comparative analysis of intelligent Reﬂecting surfaces (IRS’s) as well as amplify and forward (AF) relaying wireless networks Considered, and it is demon- strated that IRS aided wireless systems has a higher spectral efﬁciency (SE) than the corresponding AF relaying wireless systems. In [5], a survey conducted on the IRS is presented this encompasses IRS-aided communications data rate and capacity as- sessments, deep learning-based design, power/spectral optimization’s, and reliability analysis they also look into IRS deployments and how IRS’s can be used for secure communication, terminal location, and other novel applications. The author in [6], investigate the multi user down-link MISO system, which is helped by the IRS. To optimize the WSR under the BS transmit power constraint, a hybrid active and pas- sive beam forming issue is deﬁned. An iterative solution based on the recently an- nounced fractional programming methodology has been designed to solve this non convex issue. Three low-complexity algorithms are also offered to solve the problem. In [7], massive MIMO designs for milli meter wave (mm Wave) communication is proposed It is shown that properly locating the active receiving antennas for the intel- ligent surface signiﬁcantly improves the spectral efﬁciency of RIS supported MIMO 3 Title Suppressed Due to Excessive Length structures. Furthermore, they advocate for a power usage framework for RIS sup- ported radio network that accounts for RIS effect imperfections, such as taper loss, spillover loss, phase shifter loss and aperture loss. In [8], the use of re-conﬁgurable intelligent surface (RIS) to enhance uplink beam formation gain in a massive MIMO system is investigated. It has also been demonstrated that IRS can improve network throughput. In [9], Performance analysis of Re-conﬁgurable intelligent surface (RIS) in multi-user MIMO systems was outlined. The capacity of both of the up link and down link channels has been discussed. In [10], after the concentrated weighted sum rate augmentation problem has been transformed to a manageable sub problem, the author presents an incremental alternate direction of multiplier (ADMM) calculation to up-date the beam former locally. The author in [11], investigated the problem of ”capacity-maximization for MIMO IRS-assisted point-to-point communication, us- ing joint IRS reﬂection coefﬁcients”. An alternative optimization strategy was pro- posed for frequency-ﬂat channels, which provides a locally optimized solution by optimizing a single optimization variable (”the transmit co-variance matrix”). 1.1 Related Works In [12], an IRS-assisted (MISO) remote framework was presented to consider where an IRS is sent to help a single radio wire client to receive the correspondence from the mul- tiple receiving wire. So, the customer receives the channel directly from the AP at the same time, just as the IRS does. The author in [13], propose a twofold IRS- assisted multiple input multiple output (MIMO) communication framework for LOS channels. Advance the transmit co-variance network and the inactive beam forming grids of the two agreeable IRSs to investigate the limit ampliﬁcation issue. The au- thor in [14], offered three analytic approaches to play out the ﬁnding in a variety of settings with varying IRS’s CSI requirements and computing complexities. In [15], the use of an IRS at the cell limit improved the cell-edge client execution of multi cell communication frameworks. They addressed the WSR augmentation problem by simplifying the passive shifts at the IRSs and Active Transmit Precoding (TPC) lat- tices at the AP, while ensuring that each BS’s power requirement and unit-modulus limitation at the IRS were met. In [16], in order to deal with inaccurate CSI estimates, the author devised as well as solved ”a robust probabilistic constrained optimization problem for IRS-aided MISO communication systems. The best beam forming vector there at BS as well as reﬂecting elements at the IRS are determined iteratively with the converging alternative optimization approach. 2.1 Down-link Moderate MIMO System with IRS Support 2.1 Down-link Moderate MIMO System with IRS Support We consider an Intelligent Reﬂecting Surface (IRS) supported Moderate MIMO com- munication framework as displayed in ﬁgure 1 in which there is a BS furnished with ’N’ Transmitting Antennas, M’ Reﬂecting passive components Serving ’K’ numer- ous antenna users each of has ’Q’ Antenna components Serving ’K’ numerous an- tenna users each of has ’Q’ Antenna components where ((N > 1,Q >= 1) indicate by S = [S1,. ....Sk]T ∈Ck×Q and ’the Gaussian information images in which every component is a free arbitrary variable with zero mean and unit variance, likewise Sk Fitsum Dessalegn Mekonnen Muluneh Mekonnen Tulu and Sultan Feisso 4 component image sent to the Kth client. An IRS element is thought to be with Max elements on a level plane and May elements in an upward direction.( M = Max by May). The smart controller also controlled the IRS unit, which arranges the reﬂecting Fig. 1 IRS aided Moderate MIMO Communication System [17] modes for The IRS unit. Where wk ∈CM×K is the matrix of Linear Transmit Precod- ing (TPC) used by BS to send its information vector Sk to the kth user. Also, Base band channels crossing through BS to IRS, and those from IRS to the Kth user, are denoted by G ∈CN×M and hr,k ∈CM×1 in the order given.We assume that the BS has “Perfect channel – state information (CSI)” [18] for all channels involved and all the channels are quasi-Static ﬂat-fading Rayleigh. In likewise, we Denote θn ∈F the RC of the nth reﬂection component, where F is the feasible set of RCs. The BS transmits a signal that is given by: Fig. 1 IRS aided Moderate MIMO Communication System [17] Fig. 1 IRS aided Moderate MIMO Communication System [17] modes for The IRS unit. Where wk ∈CM×K is the matrix of Linear Transmit Precod- ing (TPC) used by BS to send its information vector Sk to the kth user. Also, Base band channels crossing through BS to IRS, and those from IRS to the Kth user, are denoted by G ∈CN×M and hr,k ∈CM×1 in the order given.We assume that the BS has “Perfect channel – state information (CSI)” [18] for all channels involved and all the channels are quasi-Static ﬂat-fading Rayleigh. In likewise, we Denote θn ∈F the RC of the nth reﬂection component, where F is the feasible set of RCs. 2.2 Problem Formulation All signals from other users are treated as interference by the Kth user. As a result, yk = hr,kHφG wk 2 k ∑ i=1,i̸=k hr,kHφG wi 2 +σ2 (2.4) (2.4) Where wk = [w1,w2........wk] ∈CM×k and the major goal is to increase the WSR by creating the transmit beam forming matrix W at the BS and the phase shift matrix Phi at the IRS together. The Spectral Efﬁciency (SE) (bits/Hz) at user k is given by: SE = RK (W,φ) = log2(1+yk) (2.5) (2.5) And the Sum rate is given by: And the Sum rate is given by: SR = Ssum (W,φ) = K ∑ k=1 wkRk (w,φ) (2.6) (2.6) Therefore, the WSR maximization problem is become Therefore, the WSR maximization problem is become (P1)max w,φ f1 (w,φ) = K ∑ k=1 wklog2 (1+yk) (2.7) (P1)max w,φ f1 (w,φ) = K ∑ k=1 wklog2 (1+yk) (2.7) s.t K ∑ k=1 ∥wk∥2 ≤Pmax(t) θn ∈F,∀n = 1,......,M (2.7) s.t K ∑ k=1 ∥wk∥2 ≤Pmax(t) 2.1 Down-link Moderate MIMO System with IRS Support modes for The IRS unit. Where wk ∈CM×K is the matrix of Linear Transmit Precod- ing (TPC) used by BS to send its information vector Sk to the kth user. Also, Base band channels crossing through BS to IRS, and those from IRS to the Kth user, are denoted by G ∈CN×M and hr,k ∈CM×1 in the order given.We assume that the BS has “Perfect channel – state information (CSI)” [18] for all channels involved and all the channels are quasi-Static ﬂat-fading Rayleigh. In likewise, we Denote θn ∈F the RC of the nth reﬂection component, where F is the feasible set of RCs. The BS transmits a signal that is given by: The BS transmits a signal that is given by: x = k ∑ k=1 wksk x = k ∑ k=1 wksk (2.1) (2.1) We consider the following assumption for the feasible set of RCs: φ = diag(βθ1...βθn) ,θn ∈[0,2π] and β ∈[0,1] are the diagonal phase-shift lattice for the IRS, the com- bined incident signal’s phase shift and amplitude reﬂection coefﬁcient respectively. Now let’s deﬁned the total transmitted signal by: 5 5 Title Suppressed Due to Excessive Length Yk = hr,kHφGX +nk (2.2) Yk = hr,kHφGX +nk (2.2) Where nk =CN 0,σ2 represents additive white Gaussian noise (AWGN) at the Kth user receiver.Then by substituting (2.1) in to (2.2) we get Where nk =CN 0,σ2 represents additive white Gaussian noise (AWGN) at the Kth user receiver.Then by substituting (2.1) in to (2.2) we get Yk = hr,kHφG K ∑ k=1 wksk +nk (2.3) (2.3) 2.2 Problem Formulation 2.2 Problem Formulation 2.3 Beam forming Design with Active and Passive Components 2.3 Beam forming Design with Active and Passive Components First, we have to make decouple the optimization of TPC matrix W and the phase shift matrix φ in to several tractable sub problems. So by using Lagrangian dual transform we can address the logarithm problem in the objective function of (P1). Hence, we use the proposed Lagrangian dual transform in [19] then (P1) becomes in the form, (2.8) (P1∗) max w,φ f2 (w,φ,α) (P1∗) max w,φ f2 (w,φ,α) Fitsum Dessalegn Mekonnen Muluneh Mekonnen Tulu and Sultan Feisso 6 s.t K ∑ k=1 ∥wk∥2 ≤Pmax(t) θn ∈F,∀n = 1,......,M s.t K ∑ k=1 ∥wk∥2 ≤Pmax(t) θn ∈F,∀n = 1,......,M Where α refers to [α1,α2,........αk]Tis an extra vector introduced by the Lagrangian dual transform. The WSR of Problem (2.7) can be written as: f2 (W,φ,α) = K ∑ k=1 wklog2 (1+αk)− K ∑ k=1 wkαk + K ∑ k=1 wk (1+αk)yk 1+yk (2.9) (2.9) Thus solving (2.7) is the same as solving (2.9) so, we provide a solution (2.9) by using an alternating iterative method that yields three alternating steps i.e., the auxiliary variable α, Active Beam Forming and Passive Beam Forming. So, in (Pl*) when w and φ hold ﬁxed and setting d f2 dαk = 0 The optimal α is: αk0 = yk (2.10) (2.10) αk0 = yk Then for afﬁxed α optimizing W and φ is reduced to: Then for afﬁxed α optimizing W and φ is reduced to: Then for afﬁxed α optimizing W and φ is reduced to: Then for afﬁxed α optimizing W and φ is reduced to: (P1new)max w,φ K ∑ k=1 αkyk 1+yk (2.11) s.t K ∑ k=1 ∥wk∥2 ≤Pmax(t) θn ∈F,∀n = 1,......,M (P1new)max w,φ K ∑ k=1 αkyk 1+yk (2.11) s.t K ∑ k=1 ∥wk∥2 ≤Pmax(t) θn ∈F,∀n = 1,......,M s.t K ∑ k=1 ∥wk∥2 ≤Pmax(t) θn ∈F,∀n = 1,......,M Here, αk = wk(1 + αk) Since,W and θ are related only to the SINR term the other variables in (2.9) will be ignored.Then we are going solve ‘W’ by holding φ and solve φ by holding ‘W’ respectively. 2.4 Active Beam forming scheme Let us ﬁrst optimize The ABF by ﬁxing the PBF matrix. 2.3 Beam forming Design with Active and Passive Components For Problem 2.11 let denote the combined channel for user k by: hk = hr,kHφGX (2.12) hk = hr,kHφGX (2.12) hk = hr,kHφGX (2.12) Then the SINR in (2.4) becomes: Then the SINR in (2.4) becomes: yk = hkHwk 2 k ∑ i=1,i̸=k hkHwi 2 +σ2 (2.13) (2.13) So,by using 2.13 the primary function of problem 2.11 can be expressed as a function of ’W’. f3(w) = K ∑ k=1 αkyk 1+yk f3(w) = K ∑ k=1 αkyk 1+yk (2.14) (2.14) 7 7 Title Suppressed Due to Excessive Length Substituting (2.13) in to (2.14) we get: Substituting (2.13) in to (2.14) we get: K ∑ k=1 αk hkHwk 2 k ∑ i=1,i̸=k hkHwi 2 +σ2 K ∑ k=1 αk hkHwk 2 k ∑ i=1,i̸=k hkHwi 2 +σ2 (2.15) (2.15) Given this ′α’ and ′φ’ enhancing ’W’ becomes: Given this ′α’ and ′φ’ enhancing ’W’ becomes: (P2)max w f3(w) (2.16) (P2)max w f3(w) (2.16) (2.16) (P2)max w f3(w) (P2)max w f3(w) s.t K ∑ k=1 ∥wk∥2 ≤Pmax(t) θn ∈F,∀n = 1,......,M θn ∈F,∀n = 1,......,M We not that (2.14) is a fractional programming problem with multiple ratios. As a result, by employing the quadratic transform proposed technique in [19] we can write (2.15) as: f4(w,β) = k ∑ k=1 2 p αk Re{βkhkHwk}− K ∑ k=1 \|βk\|2 k ∑ i=1 hkHwi 2 +σ2 (2.17) (2.17) Where, β = [β1,....βk]Tis the additional variable introduced by using the quadratic transform. and (2.17) is a problem of biconvex enhancement.we can rewrite 2.17 as: f(w,θ,α,β) = K ∑ k=1 2 p αk Re{βkhkHwk}− K ∑ k=1 \|βk\|2( k ∑ i=1 hkHwi 2 +σ2) (2.18) (2.18) The ADMM method is then used to solve problem (P2) perfectly [20]. Problem (P2) augmented ’s Lagrangian is: f(w,θ,α,β,λ) = f(w,θ,α,β)+( K ∑ k=1 ∥wk∥2−Pt)+ N ∑ n=1 IF(θn)+ p 2 ∥θn +λ∥2 2 (2.19) Then the variables can be updated incrementally by: αi0+1 = argmaxL(θ i0,wi0,α,β i0,λ i0) (2.20a) β i0+1 = argmaxL(θ i0,wi0,αi0+1,β,λ i0) (2.20b) wi0+1 = argmaxL(θ i0,w,αi0+1,β i0+1,λ i0) (2.20c) θ i0+1 = argmaxL(θ,wi0+1,αi0+1,β i0+1,λ i0) (2.20e) λ i0+1 = λ i0 +P(θ i0+1) (2.20 f) The optimal solution of α for problem (2.20a) is α0 = yk (2.21) Fitsum Dessalegn Mekonnen Muluneh Mekonnen Tulu and Sultan Feisso 8 This is obtained when W and θ hold ﬁxed. The optimal solution of β for problem (2.20b) is: This is obtained when W and θ hold ﬁxed. Substituting (2.13) in to (2.14) we get: The optimal solution of β for problem (2.20b) is: β 0 = √αkhkHwk K ∑ i=1,i̸=k hkHwi 2 +σ2 β 0 = √αkhkHwk K ∑ i=1,i̸=k hkHwi 2 +σ2 (2.22) (2.22) This is obtained by setting d f4/dβk = 0 The optimal solution of W for problem (2.20c) is: This is obtained by setting d f4/dβk = 0 The optimal solution of W for problem (2.20c) is: wk0 = p αkβk(λ0IN + k ∑ i=1 \|βi\|2hihiH) −1 hkH (2.23) (2.23) This is obtained by setting d f4/dwk = 0 Where λ0 ≥0 is the best dual variable to use in the transmit power constraint λ0 = min{λ0 ≥0 : K ∑ k=1 ∥wk∥2 ≤Pmax(t)} (2.23b) (2.23b) 2.5 Passive Beam forming scheme 2.5 Passive Beam forming scheme To ﬁnd the optimal solution of θ we ﬁrst rearrange some equations with some math- ematical manipulation so,(2.15) can be re written as: hkHw = θ Hdiaghr,kHGW (2.24) Where (2.24) Where Where Where φ H = θ Hdiaghr,kH (2.24b) i lif l φ H = θ Hdiaghr,kH (2.24b) To simplify let; (2.24b) vk, j = diaghr,kHGW (2.24c) Then we rewrite (2 19) as: Then we rewrite (2.19) as: Then we rewrite (2.19) as: max f5(θ) = K ∑ k=1 αk θ Hvk,k 2 k ∑ i=1,i̸=k θ Hvk, j 2 +σ2 (2.25) (2.25) s.t 0 ≤θn ≤2π,∀n = 1,......,M (2.25b) s.t 0 ≤θn ≤2π,∀n = 1,......,M (2.25b) (2.25b) s.t 0 ≤θn ≤2π,∀n = 1,......,M Then using QT- we can write(2.25) as Then using QT- we can write(2.25) as max f6 (θ,r) = K ∑ k=1 2 p αk Re{rkθ Hvk,k}− rk2( k ∑ i=1 θ Hvk, j 2 +σ2) (2.26) 9 Title Suppressed Due to Excessive Length Where ‘r’ is the auxiliary variable introduced by the Lagrange multiplier-based Quadratic Transform, and setting d f6(θ,r)/drk the optimal ’r’ is given by: rk0 = √αkθ Hvk,k k ∑ j=1 θ Hvk, j 2 +σ2 rk0 = √αkθ Hvk,k k ∑ j=1 θ Hvk, j 2 +σ2 (2.27) (2.27) Given ‘r’ the optimization of θ can be written as: max f7(θ,r) = −θ HAθ +2Re{θ Hb}− K ∑ k=1 rk2σ2 (2.28) (2.28) Algorithm [1. Input: G,hr,vk [2. Output: w,θ,Sum rate [3. i0 ←0 [4. While stopping criterion not satisﬁed do [5. Update αi0+1 ,∀kusing(2.21) [6. Update β i0+1 ,∀kusing(2.22) [7. Update wi0+1 ,∀kusing(2.23) [8. Update θ i0+1 ,byusing(2.20e) [9. Update λ i0+1 ,byusing(2.20 f) [10. With given θ updateφ [11. i0 ←i0 +1 [12. Until the Stopping Criterion is Met. [13. end while [1. Input: G,hr,vk [2 O t t θ S t [1. Input: G,hr,vk [4. While stopping criterion not satisﬁed do [4. While stopping criterion not satisﬁed do [12. Until the Stopping Criterion is Met. Where Where [A= K ∑ k=1 rk02 K ∑ j=1 vk,jvk, j [A= K ∑ k=1 rk02 K ∑ j=1 vk,jvk, j [A= K ∑ k=1 rk02 K ∑ j=1 vk,jvk, j and and [b= K ∑ k=1 √αkrk0vk,k [b= K ∑ k=1 √αkrk0vk,k [b= K ∑ k=1 √αkrk0vk,k Then, by removing the constant terms that are unrelated to θ, we can rewrite: Then, by removing the constant terms that are unrelated to θ, we can rewrite: max f8(θ,r) = −θ HAθ +2Re{θ Hb} (2.29 (2.29) [s.t 0≤θn ≤2π,∀n = 1,....,M(2.29b) Problem (2.29) is an example of a convex optimization problem. The Lagrangian dual decomposition method can be used to solve (2.29). Lagrange’s dual of (2.29) can be expressed as [21]: minimize λ L(λ) = max θ G(θ,λ) (2.3 (2.30) s.t λn ≥0,∀n = 1,.......,M (2.30b) s.t λn ≥0,∀n = 1,.......,M s.t λn ≥0,∀n = 1,.......,M where λ = [λ1,.........λn] is the dual variable for the constraint θ Henenθ ≤1 andG(θ,λ) denotes the dual objective function given by: G(θ,λ) = f8(θ)− N ∑ n=1 λn(θ Henenθ −1) (2.31) (2.31) Where en ∈Rm×1primary level vector with a one in the kth position and zeros everywhere else. Then by setting dG/dθ = 0 The optimal θ can be obtained as: θ 0 = (A+ M ∑ k=1 λnenenH) −1 b = D(λ)b (2.32) θ 0 = (A+ M ∑ k=1 λnenenH) −1 b = D(λ)b (2.32) Fitsum Dessalegn Mekonnen Muluneh Mekonnen Tulu and Sultan Feisso 10 Table 1 Simulations and Algorithmic Parameters Parameters Values Algorithm and convergence parameter 0.01 Transmission bandwidth BW 20MHz Maximum transmit Power at the BS 30dBm AWGN -85dBm Table 1 Simulations and Algorithmic Parameters Utilizing Schur complement technique proposed in [22], the problem in (2.30) could be designed as a problem of Semi Deﬁnite Programming(SDP). Utilizing Schur complement technique proposed in [22], the problem in (2.30) could be designed as a problem of Semi Deﬁnite Programming(SDP). Utilizing Schur complement technique proposed in [22], the problem in (2.30) could be designed as a problem of Semi Deﬁnite Programming(SDP) maximizeε ελ −tr(diag(λ)) (2.33) s.t A+tr(diag(λ)) b bH −ε ≥0 (2.33B) maximizeε ελ −tr(diag(λ)) (2.33) s.t A+tr(diag(λ)) b bH −ε ≥0 (2.33B) (2.33) s.t A+tr(diag(λ)) b bH −ε ≥0 (2.33B) (2.33B) Where D(λ) = (A+diag(λ))−1 So, by solving (2.33) using CVX tool box we get the optimal value of λ 3.1 Introduction In this section, simulation results and corresponding analysis are presented following the Algorithm analysis discussed in section 2. Simulation results are carried out to compare the performance of massive MIMO and IRS aided Moderate MIMO systems 11 Title Suppressed Due to Excessive Length 10 20 30 40 50 60 70 Number of Antenna Elements:N 0 1 2 3 4 5 6 7 8 9 Sum Rate (bps/Hz) Sum-Rate Comparision IRS Aided Mimo with - ADMM mMIMO with-MMSE mMIMO with-ZF IRS Aided Mimo w/o ADMM Fig. 2 A comparison of the four schemes’ sum rates. 3.2 Performance Metrics Performance measures such as Sum Rate, SINR, Energy efﬁciency and Transmit Power are used to analyze and validate the proposed system. Sum-Rate Comparision Sum-Rate Comparision Fig. 2 A comparison of the four schemes’ sum rates. 3.2 Performance Metrics Performance measures such as Sum Rate, SINR, Energy efﬁciency and Transmit Power are used to analyze and validate the proposed system. Performance measures such as Sum Rate, SINR, Energy efﬁciency and Transmit Power are used to analyze and validate the proposed system. 3.5 SINR The other metrics used to compare massive MIMO and IRS-assisted moderate MIMO communication systems is SINR. So, we examine the network SINR achieved by the following Three schemes: N= 40 with M = 80,N = 40, and N=70, total amount of antenna elements at the BS and the total amount of reﬂecting elements at the IRS, respectively, are N and M. The number of Users K =20. So, as we can see in ﬁgure 4 Passive IRS elements helps to reduce the number of active antennas (see N=40 with M=80 vs N=40 and N=80 without IRS). 3.4 Transmit Power As shown in Figure 3, The overall transmit power is reduced by using large intelligent reﬂecting surfaces. So, there is a signiﬁcant power saving with IRS. For example, with 40 number of Reﬂecting elements the Transmitted power is 4.5 dBm and when we increase the number of IRS elements to 60 the transmitted power is dropped to 2 dBm and as we continually increase the number of reﬂecting elements to 160 the transmitted power will drop to around -11 dBm. 3.3 Sum Rate One of the measures used to compare the performance of massive MIMO and IRS- assisted moderate MIMO communication systems is the user sum rate. So, to com- pare the network throughput achieved by the four techniques below, we present a nu- merical example: IRS-assisted MIMO communication with and without the ADMM optimization algorithm and massive MIMO with MMSE and ZF beamforming algo- rithm without IRS. We assume the BS is installed with N = 70 antennas, K = 3 users, and the number of IRS elements varies between 8 and 80 with ﬁve number of points. Other important parameters are available in table 1.Fig.2 illustrates the sum-rate per- formance of the three users characterized by various IRS element counts under the four schemes. Initially, it is noticed that the sum-rate performance of the IRS-assisted MIMO system is poorer than that of the massive MIMO system without IRS. Next, it is observed that under the optimal ADMM beamforming method described in (2.23 and 2.28), The sum-rate obtained in the IRS- assisted MIMO system is signiﬁcantly higher than that obtained in the massive MIMO system without IRS. Fitsum Dessalegn Mekonnen Muluneh Mekonnen Tulu and Sultan Feisso 12 40 60 80 100 120 140 160 M -12 -10 -8 -6 -4 -2 0 2 4 6 Pt(dBm) Transmit Power vs M IRS Aided MIMO with-ADMM Fig. 3 Transmit power vs M 3.4 Transmit Power As shown in Figure 3, The overall transmit power is reduced by using large intelligent reﬂecting surfaces. So, there is a signiﬁcant power saving with IRS. For example, with 40 number of Reﬂecting elements the Transmitted power is 4.5 dBm and when we increase the number of IRS elements to 60 the transmitted power is dropped to 2 dBm and as we continually increase the number of reﬂecting elements to 160 the transmitted power will drop to around -11 dBm. 40 60 80 100 120 140 160 M -12 -10 -8 -6 -4 -2 0 2 4 6 Pt(dBm) Transmit Power vs M IRS Aided MIMO with-ADMM Transmit Power vs M IRS Aided MIMO with-ADMM Pt(dBm) Fig. 3 Transmit power vs M 3.6 Energy Efﬁciency Another criterion used to compare the performance of IRS-aided moderate MIMO versus massive MIMO without the IRS is energy efﬁciency. as we can see in ﬁgure 5 IRS aided communication is critical to create energy efﬁcient wireless system.(where K=20,N=70 and M=120). 13 Title Suppressed Due to Excessive Length -55 -50 -45 -40 -35 -30 Pt(dBm) 20 25 30 35 40 45 SINR (dB) SINR vs Pt mMIMO N-70 mMIMO with N=40 IRS Aided with N=40,M=80 Fig. 4 SINR vs Transmit power 0 2 4 6 8 10 SE [bit/s/Hz] 0 10 20 30 40 50 60 70 80 90 100 EE [Mbit/Joule] EE vs SE IRS Aided MIMO mMIMO w/o IRS Fig. 5 Energy Efﬁciency vs Spectral Efﬁciency 4 Conclusion In this paper we have suggested the IRSs to enhance the performance of massive MIMO system. Speciﬁcally, to enhance the user rate, SINR,Energy efﬁciency and lowering the overall transmit power at the access point are all goals. The IRS aided system does not have satisfactory result compared to massive MIMO system if it has not optimized by a proper beam forming algorithm. We have two different beam- -55 -50 -45 -40 -35 -30 Pt(dBm) 20 25 30 35 40 45 SINR (dB) SINR vs Pt mMIMO N-70 mMIMO with N=40 IRS Aided with N=40,M=80 Fi 4 SINR T i SINR vs Pt Fig. 4 SINR vs Transmit power Fig. 4 SINR vs Transmit power 0 2 4 6 8 10 SE [bit/s/Hz] 0 10 20 30 40 50 60 70 80 90 100 EE [Mbit/Joule] EE vs SE IRS Aided MIMO mMIMO w/o IRS EE [Mbit/Joule] Fig. 5 Energy Efﬁciency vs Spectral Efﬁciency 4 Conclusion In this paper we have suggested the IRSs to enhance the performance of massive MIMO system. Speciﬁcally, to enhance the user rate, SINR,Energy efﬁciency and lowering the overall transmit power at the access point are all goals. The IRS aided system does not have satisfactory result compared to massive MIMO system if it has not optimized by a proper beam forming algorithm. We have two different beam- In this paper we have suggested the IRSs to enhance the performance of massive MIMO system. Speciﬁcally, to enhance the user rate, SINR,Energy efﬁciency and lowering the overall transmit power at the access point are all goals. The IRS aided system does not have satisfactory result compared to massive MIMO system if it has not optimized by a proper beam forming algorithm. We have two different beam- Fitsum Dessalegn Mekonnen Muluneh Mekonnen Tulu and Sultan Feisso 14 forming schemes the ﬁrst is active beam forming at the AP, and the second is inactive reﬂecting beam forming at the IRS. So, we have proposed an ADMM algorithm to jointly optimize the two beam forming schemes. We demonstrated, using this solu- tion, that the IRS-aided moderate MIMO system achieve higher user rate, SINRs, energy efﬁciency and low transmit power compared to its counterpart without IRS. Because of this, IRS aided MIMO has proven to be very effective in improving the efﬁciency of massive MIMO system. Declaration Currently there is no any fund support this research work.Also,there is no conﬂicts of interest/Competing interests within the authors. In addition,the data sets generated during/or analyzed during the current study are available from the corresponding au- thor on reasonable request. References 1. Marco Di Renzo, Merouane Debbah,Dinh-Thuy Phan-Huy,Zappone,Mohamed-SlimAlouini,Chau Yuen, Vincenzo Sciancalepore, George C. 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Boulogeorgos and Angeliki Alexiou,”Performance Analysis of Reconﬁg- urable Intelligent Surface-Assisted Wireless Systems and Comparison With Relaying,” IEEE Access, vol. 8, no. Received May 7,pp. 2-7, June 2, 2020. 5. Jun Zhao and Yang Liu, ”A Survey of Intelligent Reﬂecting Surfaces (IRSs):Towards 6G Wireless Communication Networks,” http://arxiv.org/abs/1907.04789, vol. 3,pp. 5-7,12 December 2019. 6. Huayan Guo, Ying-Chang Liang,Jie Chen, and Erik G. Larsson, ”Weighted Sum-Rate Optimization for Intelligent Reﬂecting Surface Enhanced Wireless Networks,” http://arxiv.org/abs/1905.07920, vol. 2,pp. 1-12, 30 May,2019. 7. Vahid Jamali, Antonia M. Tulino, Georg Fischer, Ralf Muller, and Robert Schober, ”Intelligent Reﬂect- ing and Transmitting Surface Aided Millimeter Wave Massive MIMO,” arXiv:1902.07670 [cs.IT] , vol. 2,pp. 6, 23 Sep 2019. 8. Zhaorui Wang, Liang Liu and Shuguang Cui, ”Intelligent Reﬂecting Surface Assisted Massive MIMO Communications,” arXiv:2002.05899 [cs.IT] , vol. 3,pp. 5-9, 5 May 2020. 9. S. Liu, ”Performance Analysis of Intelligent Reﬂecting Surface in Multi-user MIMO Systems,” J. Phys.: Conf. Ser. 1575 012078,pp. 3-6 2020. 10. S. Huang, ”Rate, Reliability and Secrecy Performance Analysis and Optimization for Millimeter Wave Communications,” Doctoral Thesis, pp. 39-44, Stockholm, Sweden 2020. 11. Shuowen Zhang and Rui Zhang, ”Capacity Characterization for Intelligent Reﬂecting Surface Aided MIMO Communication,” arXiv:1910.01573 [cs.IT] , vol. 1,pp. 4-6, 3 Oct 2019. 12. Qingqing Wu and Rui Zhang, ”Intelligent Reﬂecting Surface Enhanced Wireless Network: Joint Ac- tive and Passive Beamforming Design,” http://arxiv.org/abs/1809.01423, vol. 1,pp. 2-4, 5 Sep 2018. 13. Yitao Han, Shuowen Zhang, Lingjie Duan and Rui Zhang, ”Double-IRS Aided MIMO Communica- tion under LoS Channels: Capacity Maximization and Scaling,” arXiv:2102.13537 [cs.IT] , vol. 1,pp. 2-7, 26 Feb 2021. Title Suppressed Due to Excessive Length 15 14. References Rui Sun, Weidong Wang, Li Chen, Guo Wei, and Wenyi Zhang, ”Diagnosis of Intelligent Reﬂecting Surface in Millimeter-wave Communication Systems,” arXiv:2101.03792 [eess.SY] , vol. 1,pp. 3-7, 11 Jan 2021. 15. Cunhua Pan, Hong Ren, Kezhi Wang, Wei Xu, Maged Elkashlan, Arumugam Nallanathan and Lajos Hanzo, ”Multicell MIMO Communications Relying on Intelligent Reﬂecting Surfaces,” arXiv:1907.10864 [eess.SP] , vol. 4,pp. 5-7, 24 Apr 2020. pp p 16. Tuan Anh Le, Trinh van Chien and Marco Di Renzo, ”Robust Probabilistic-Constrained Optimization for IRS-Aided MISO Communication Systems,” IEEE wireless communications letters, IEEE comsoc, In press, ff10.1109/LWC, vol. 1, 23 Nov 2020. 17. Chongwen Huang,Alessio Zappone,George C. 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https://openalex.org/W2583118017	https://www.nature.com/articles/srep41719.pdf	English	null	Dynamic evolution of hepatitis C virus resistance-associated substitutions in the absence of antiviral treatment	Scientific reports	2,017	cc-by	5,818	Dynamic evolution of hepatitis C virus resistance-associated substitutions in the absence of antiviral treatment received: 30 August 2016 accepted: 28 December 2016 Published: 31 January 2017 Auda A. Eltahla1, Preston Leung1, Mehdi R. Pirozyan1, Chaturaka Rodrigo1, Jason Grebely2 Tanya Applegate2, Lisa Maher2, Fabio Luciani1, Andrew R. Lloyd1 & Rowena A. Bull1 Auda A. Eltahla1, Preston Leung1, Mehdi R. Pirozyan1, Chaturaka Rodrigo1, Jason Grebely2, Tanya Applegate2, Lisa Maher2, Fabio Luciani1, Andrew R. Lloyd1 & Rowena A. Bull1 Resistance against new hepatitis C virus (HCV) antivirals is an area of increasing interest. Resistance- associated substitutions (RASs) have been identified in treatment-naïve individuals, but pressures driving treatment-independent RAS emergence are poorly understood. We analysed the longitudinal evolution of RASs in twelve participants with early acute HCV infections. Full-genome deep sequences were analysed for changes in RAS frequency within NS3, NS5A and NS5B-coding regions over the course of the infection. Emergence of RASs relevant only to the polymerase non-nucleoside inhibitors (NNI) was detected, and these lay within CD8+ T-cell epitopes. Conversely, the loss of NNI RASs over time appeared likely to be driven by viral fitness constraints. These results highlight the importance of monitoring CD8+ T cell epitope-associated RASs in populations with dominant HLA types. Antiviral therapy for hepatitis C virus (HCV) has undergone a recent revolution with the approval of several direct-acting antivirals (DAA) targeting the HCV protease (NS3), phosphoprotein (NS5A) and polymer- ase (NS5B). Interferon-free regimens, which contain multiple DAAs, have been approved in several coun- tries to treat infections with different HCV genotypes1. Each agent has been shown to promote emergence of resistance-associated substitutions (RASs) when studied in vitro, and depending on the DAA, the presence of these substitutions could reduce the efficacy of antiviral treatment in vivo2. Monitoring the emergence of HCV RASs in vivo is therefore of particular importance to both clinical practice and public health strategies. As a consequence of an error-prone viral polymerase, and a high replication rate, a swarm of mutations con- stantly get generated during HCV infection. Some of these natural mutations encode for RASs and have been reported in many studies in treatment-naïve patients3–6. Such naturally occurring RASs could negatively impact treatment success rates, particularly in patients with cirrhosis, those infected with genotype 3, and those who have failed interferon-based therapies. In the context of antiviral treatment, the emergence of RASs is initially limited by the genetic barrier to resistance, defined by the number and type of nucleotide changes required for amino acid substitutions7. 1School of Medical Sciences, Faculty of Medicine, UNSW Australia, Sydney, NSW 2052, Australia. 2The Kirby Institute, UNSW Australia, Sydney, NSW 2052, Australia. Correspondence and requests for materials should be addressed to R.A.B. (email: r.bull@unsw.edu.au) www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Scientific Reports \| 7:41719 \| DOI: 10.1038/srep41719 Methods S bj Subjects and samples. Viremic blood samples were obtained from prospective cohorts of high-risk, HCV- uninfected subjects recruited between 2005 and 2014 in NSW Australia. Sample were obtained from two cohorts; the Hepatitis C Incidence and Transmission Study in prisons (HITS-p) and in the community (HITS-c)11,12. Participants were tested every three to six months for HCV seroconversion, and then followed regularly post-in- fection until spontaneous clearance or persistence was determined when antiviral treatment was offered if they remained infected. For this study, twelve participants with early acute HCV infection were included. An early infection case was defined by the availability of at least one viremic sample prior to seroconversion. The estimated date of infection was calculated for each subject by subtracting the recognized mean pre-seroconversion window period of 51 days from the midpoint between the last HCV RNA-positive/HCV Ab-negative time point and the first seropositive time point11. Ethical approvals were obtained from the Human Research Ethics Committees of Justice Health (reference number G304/11), the New South Wales Department of Corrective Services (refer- ence number 05/0884), and the University of New South Wales (reference numbers 05094, 08081, 13237, 09075, 14170). Written informed consent was obtained from the participants. All methods were also performed in accordance with the relevant guidelines and regulations. Viral genome sequencing and analysis. Viral RNA was extracted from plasma samples and amplicons covering the full HCV genome were generated either as single near-full length products, or as three overlapping fragments, as described previously13,14. Next-generation sequencing of the amplicons was conducted using either the Roche 454 FLX, or the Illumina MiSeq sequencing platform, as previously reported11,13. Sequence alignments were generated using Bowtie 215 against the corresponding genotype reference sequence: GT1a (GenBank acces- sion numbers AF009606), GT1b (AJ238799), GT2b (AB030907) and GT3a (D17763). Single-nucleotide poly- morphism (SNP) analysis was performed using the Geneious software package version 816 with minimum variant frequency threshold of 0.001, maximum variant P-value of 10−6, and a minimum coverage of 1,000 (Illumina) or 400 (Roche 454). Sixty eight positions across the genome where RASs have previously been reported2 were analysed. These covered RASs impacting NS3, NS5A and NS5B inhibitors. Consensus sequences for each HCV genotype were obtained from the Los Alamos Hepatitis C Sequence Database (http://hcv.lanl.gov). Analysis of autologous T cell responses across known RAS sites. Methods S bj HCV residue positions where RASs were gained over the course of infection were examined to determine whether their site of occurrence fell within potential CD8+ T cell epitopes. Potential HLA-I restricted epitopes were identified from previous experimental validation, and via bioinformatic predictions using NetMHC (www.iedb.org net). Predictions were obtained from HLA-I typing of the subject as well as from the autologous viral consensus sequence, generated via next generation sequencing. As predicted epitopes could represent false positives, strict selection criteria were applied as previously described13. Selected autologous HLA-1 restricted peptides were synthesised and epitope-specific interferon gamma (IFN-γ) production assessed via enzyme-linked immunospot (ELISpot) assays as previously described11. A positive response towards an autologous peptide was defined as >20 spot-forming units (SFU)/million peripheral blood mononuclear cells (PBMC). Dynamic evolution of hepatitis C virus resistance-associated substitutions in the absence of antiviral treatment The potential for particular RASs to persist in the host ultimately depends on the trade-off between the loss of replicative fitness and the survival advantage under strong antiviral drug selection pressure. In the absence of treatment, RASs may be retained if they increase viral fitness, are unable to revert back to wild-type2, or potentially if the relevant sites are under other selective pressures including host immune responses. For instance, some RAS sites within NS3 and NS5B have been shown to fall within experimentally-confirmed or pre- dicted CD8+ T cell epitopes6,8,9. In addition, the prevalence of an NS5A RAS was recently shown to be associated with the host interferon λ4 (IFNL4) genotype10. These findings therefore suggest that both innate and adaptive immune responses play a role in the emergence of HCV DAA resistance in the absence of antiviral treatment. As primary HCV infection is typically asymptomatic, it has been challenging to characterise the evolution of HCV mutations, and hence RASs, in the early phase of infection. Furthermore, the lack of well-characterised samples collected over the course of infection has limited longitudinal analyses of the interplay between the host immune response and viral fitness in relation to RAS development. Characterisation of the emergence of RASs in the absence of antiviral pressures is critical to our understanding of their stability within the host, and their 1School of Medical Sciences, Faculty of Medicine, UNSW Australia, Sydney, NSW 2052, Australia. 2The Kirby Institute, UNSW Australia, Sydney, NSW 2052, Australia. Correspondence and requests for materials should be addressed to R.A.B. (email: r.bull@unsw.edu.au) Scientific Reports \| 7:41719 \| DOI: 10.1038/srep41719 1 www.nature.com/scientificreports/ potential influence on treatment options. The aim of this study was to examine the longitudinal emergence of RASs in the early phase of primary HCV infection. Specifically, the aim was to understand the interplay between viral replicative fitness and the host T cell responses in driving the emergence of RASs in the absence of antiviral treatment. potential influence on treatment options. The aim of this study was to examine the longitudinal emergence of RASs in the early phase of primary HCV infection. Specifically, the aim was to understand the interplay between viral replicative fitness and the host T cell responses in driving the emergence of RASs in the absence of antiviral treatment. Results and Discussionh Longitudinal analysis of HCV resistance-associated substitutions (RAS) in early HCV infection RASs are shown in longitudinally sampled infections across HCV NS3, NS5A and NS5B. Rows represent individual samples and columns indicate amino acid positions, grouped by different coding regions and numbered at the bottom. Sample identification numbers are shown together with the estimated days post infection (DPI) to the left of each row. Samples are grouped by HCV genotype (GT). The frequency of RASs is indicated by the color according to the scale bar. RASs that are fixed within an HCV genotype34 (i.e. consensus RASs) are presented with 100% frequency across time-points. Figure 1. Longitudinal analysis of HCV resistance-associated substitutions (RAS) in early HCV infections. RASs are shown in longitudinally sampled infections across HCV NS3, NS5A and NS5B. Rows represent individual samples and columns indicate amino acid positions, grouped by different coding regions and numbered at the bottom. Sample identification numbers are shown together with the estimated days post infection (DPI) to the left of each row. Samples are grouped by HCV genotype (GT). The frequency of RASs is indicated by the color according to the scale bar. RASs that are fixed within an HCV genotype34 (i.e. consensus RASs) are presented with 100% frequency across time-points. over time, autologous CD8+ T cell responses were tested via IFN-γ ELISpot assays. In parallel, selection by rep- lication fitness was defined as the emergence of a mutation that was identical to the consensus genome for that sample’s corresponding genotype, which was termed to be a reversion event.i p p g g yp In all three cases where fixation events resulted in the gain of RASs, M414T (Cl_277), A421V (Ch_86MX) and V499A (Ch_485FX), the mutations were not reversion events, suggesting that viral fitness was not driving selection of these residues (Table 1). In relation to T cell driven pressures, M414T was identified to lie within the published HLA-A2 restricted epitope 2838WLGNIIMYA2846 (RAS site underlined)17. When this peptide was examined by ELISpot, a moderate IFN-γ response was detected for participant Cl_277 at 74 DPI, with 20 SFU/ million PBMC. At 116 DPI, the time-point at which the RAS was detected, a slightly stronger T cell response was detected, with 45 SFU/million PBMC. Results and Discussionh The longitudinal prevalence of RASs was analysed in 12 participants, each with 2–6 time-points (Fig. 1). The median number of days post-infection (DPI) at the earliest time-points was estimated at 34 (range 2–50). Four of these participants demonstrated spontaneous clearance (Cl), and eight developed chronic (Ch) infection (Fig. 1). HCV genotype distribution was GT1a (n = 5), GT1b (n = 1), GT2b (n = 1) and GT3a (n = 5). Longitudinal analysis of HCV RASs. Sixty-eight previously known RAS sites within NS3, NS5A and NS5B were screened in a total of 40 samples sequenced by next generation sequencing. Across the entire data- set, RASs were detected in 30/68 genome positions at frequencies >1% spanning NS3, NS5A and NS5B-coding regions (Fig. 1). While low-frequency variants (<1%) were detected within 51/68 (75%) of the examined sites at the earliest time-points, longitudinal analysis revealed that RASs within the majority of these sites (49/51, 96%) did not reach frequencies >10% over the course of the infection (Fig. 1). q ( g ) Within NS3 and NS5A, all RASs detected at the consensus-level in the earliest time-points were conserved over time, and no new RASs emerged at high frequency or were lost over the course of the infection (Fig. 1). By contrast, within NS5B non-synonymous mutations resulting in emergence of consensus-level RASs M414T, A421V and V499A were observed in three participants infected with HCV GT3a, GT1a and GT1b, respectively (Fig. 1 and Table 1). Additionally, loss of consensus NS5B RASs S556G and P496S was observed in two partici- pants infected with HCV GT1b and GT3a, respectively (Fig. 1 and Table 1). Interestingly, two NS5B RASs, A553V and S556G, were transiently detected at 61 DPI with frequencies 17–18% in a participant infected with GT1a (Cl_686FX), but these RASs were both lost within two weeks (Fig. 1). In summary, RASs affecting HCV DAAs targeting NS3 and NS5A are stable over the course of primary infection in this study, but emergence and loss of RASs were detected within NS5B of multiple genotypes. Factors associated with the emergence of HCV RASs. In order to examine selective pressures that could be associated with the gain of NS5B RASs, we assessed whether these substitutions fell within known, or predicted, CD8+ T cell epitopes. For the three subjects with evidence of RAS emergence and reaching fixation Scientific Reports \| 7:41719 \| DOI: 10.1038/srep41719 2 www.nature.com/scientificreports/ Figure 1. Results and Discussionh Despite this, the 2841ARMVMMTHF2849 peptide was tested via ELISpot in Ch_684MX and found to be weakly positive (30 SFU/million PBMC). A number of factors could explain the lack of emergence of A421V in subject Ch_684MX, including differences in peptide affinity or T cell exhaustion20. Investigation of these possibilities was beyond the scope of this study. y p y The RAS V499A was detected in participant Ch_485FX, a site which falls within the known HLA-B57-restricted epitope, 2912LGVPPLRAWR2921 21. However, the autologous circulating virus carried a sub- stitution at position 2919, and the predicted epitope was the nonamer 2913GVPPLRVWR2921. This subject was not HLA-B57 positive, and the only HLA allele of Ch_485FX predicted to bind to this peptide was HLA-A01:01, which had an IC50 score of 22 mM. The ELISpot response against the predicted transmitted founder derived, HLA-A01:01-restricted autologous epitope, 2913GVPPLRVWR2921 was negative (Table 1). Therefore, the role of T cell responses in driving the emergence of this RAS could not be ascertained. p g g Overall, these results suggest that RASs M414T and A421V emerged in the early phase of HCV infection as a esult of T cell responses in participants carrying relevant HLA-I alleles. Factors associated with the loss of HCV RASs. Unlike emerging RASs, all mutations which resulted in the loss of HCV RASs in this study were reversion events (Table 1). The loss of RAS S556G was observed as a result of a fixation event around 93 DPI in the same GT1b infected participant in whom another RAS, V499A, emerged (Ch_485FX, Fig. 1). Peptides encompassing this RAS were screened for CD8 T cell responses in previ- ous studies, and found to be negative22,23. The loss of the second RAS, P496S, was observed in a GT3a infected participant (Ch_240) as a gradual decrease in frequency from 33% to 1.5% between 44 DPI and 538 DPI (Fig. 1). The site of this RAS, P496, was found to be conserved across all HCV GTs. This suggests that the loss of the RAS could be driven by fitness costs of the substitution. In the participant where two RASs emerged at low frequency, Cl_686FX, both RASs also reverted to the consensus sequence (Table 1). One of these RASs, S556G, overlapped with that detected in another participant, Ch_485FX (Fig. 1). Results and Discussionh aRAS; resistance-associated substitution. Gain or loss or RAS is determined longitudinally with reference to the earliest sample. bAmino acid position with reference to strain H77 (AF009606) for GT1a, Con1 (AJ238799) for GT1b, and NZL1 (D17763) for GT3a. cSpot-forming units per million peripheral blood mononuclear cells. dEstimated days post infection (DPI) at which analysis was performed. resistance associated substitution. Gain or loss or RAS is determined longitudinally with reference to the earliest sample. bAmino acid position with reference to strain H77 (AF009606) for GT1a, Con1 (AJ238799) for GT1b, and NZL1 (D17763) for GT3a. cSpot-forming units per million peripheral blood mononuclear cel dEstimated days post infection (DPI) at which analysis was performed. Despite the lack of longitudinal evidence of an ongoing T cell response against this epitope, this immunodom- inant epitope is reported to be targeted by almost all subjects with the HLA-B27 genotype that are infected with HCV, and the A421V substitution has been shown to be HLA-driven18,19. These data support the hypothesis that the A421V substitution in subject Ch_086MX could be driven by T cell responses. Despite the lack of longitudinal evidence of an ongoing T cell response against this epitope, this immunodom- inant epitope is reported to be targeted by almost all subjects with the HLA-B27 genotype that are infected with HCV, and the A421V substitution has been shown to be HLA-driven18,19. These data support the hypothesis that the A421V substitution in subject Ch_086MX could be driven by T cell responses. j y To further validate the hypothesis that the evolution of RASs within a single infection could be driven by the T cell response, two participants, infected with the same transmitted founder virus and with longitudinal infor- mation on viral mutations were examined. Participants Ch_086MX and Ch_684MX were siblings who con- currently became infected. The consensus viral sequences for these subjects shared 99.9% nucleotide similarity across the transmitted founder virus genome (Fig. 1). At the earliest time-points analysed (16 DPI and 2 DPI for Ch_086MX and Ch_684MX, respectively), both subjects carried the variant sequence 2841ARMVMMTHF2849 (Fig. 1), which is the known HLA-B27-restricted CD8+ T cell epitope. Notably, only the virus in the the subject carrying the HLA-B27:05 allele developed the A421V substitution, while the sibling carried the HLA-B27:10, for which the predicted binding score for the same epitope is significantly lower (157 μM and 2,619 μM, respectively). Results and Discussionh These findings support the hypothesis that CD8+ T cell responses exerted selective pressure on this RAS site (Table 1).hi The second RAS, A421V, reached fixation in participant Ch_086MX at 72 DPI, and is recognised to lie within an HLA-B27-restricted CD8+ T cell epitope 2841ARMILMTHF2849 18,19. In the subject analysed here (Ch_086MX), the autologous epitope present in the transmitted/founder virus that established the infection differed from this known epitope at two positions, 2844 and 2845 (2841ARMVMMTHF2849). Experimental validation of this epitope was positive at 72 DPI, with a low CD8+ T cell frequency response of 25 SFU/million PBMCs (Table 1). When the putative escape variant epitope, 2841VRMVMMTHF2849 was tested using ELISpot, no response could be detected. Scientific Reports \| 7:41719 \| DOI: 10.1038/srep41719 3 www.nature.com/scientificreports/ Participant HCV Genotype Substitution detected RAS outcomea Positionb Reversion CD8 T cell epitope analysis Epitope sequence HLA Result (SFU/106 PBMC)c Time-point (DPI)d Ch_086MX 1a A421V Gain 2841 No 2841ARMVMMTHF2849 HLA-B27:05 25 72 Ch_485FX 1b V499A Gain 2918 No 2913GVPPLRVWR2921 HLA-A01:01 Negative 79 S556G Loss 2975 Yes — — — — Cl_277 3a M414T Gain 2844 No 2838WLGNIIMYA2846 HLA-A02:01 45 116 Ch_240 3a P496S Loss 2926 Yes — — — — Cl_686FX 1a A553V Gain/Loss 2973 Yes 2967LSGWFTAGY2975 HLA-A01:01 Negative 117 S556G Gain/Loss 2976 Yes — — — — Table 1. Summary of the RAS analysis in this study with corresponding ELISpot responses. aRAS; resistance-associated substitution. Gain or loss or RAS is determined longitudinally with reference to the earliest sample. bAmino acid position with reference to strain H77 (AF009606) for GT1a, Con1 (AJ238799) for GT1b, and NZL1 (D17763) for GT3a. cSpot-forming units per million peripheral blood mononuclear cells. dEstimated days post infection (DPI) at which analysis was performed. Participant HCV Genotype Substitution detected RAS outcomea Positionb Reversion CD8 T cell epitope analysis Epitope sequence HLA Result (SFU/106 PBMC)c Time-point (DPI)d Ch_086MX 1a A421V Gain 2841 No 2841ARMVMMTHF2849 HLA-B27:05 25 72 Ch_485FX 1b V499A Gain 2918 No 2913GVPPLRVWR2921 HLA-A01:01 Negative 79 S556G Loss 2975 Yes — — — — Cl_277 3a M414T Gain 2844 No 2838WLGNIIMYA2846 HLA-A02:01 45 116 Ch_240 3a P496S Loss 2926 Yes — — — — Cl_686FX 1a A553V Gain/Loss 2973 Yes 2967LSGWFTAGY2975 HLA-A01:01 Negative 117 S556G Gain/Loss 2976 Yes — — — — Table 1. Summary of the RAS analysis in this study with corresponding ELISpot responses. aRAS; Table 1. Summary of the RAS analysis in this study with corresponding ELISpot responses. Results and Discussionh The second RAS, A553V, fell within a predicted HLA-A01:01 restricted epitope, however, no response could be detected against this epitope in the ELISpot assays (Table 1). Overall, these results indicate that T cell responses do not appear to be driving the loss of HCV RASs detected in early acute HCV infection. Instead, reversion of residues carried by the virus towards consensus sequences indicates that the observed loss of RASs could be driven by reduced viral fitness.h i The occurrence of RASs in the transmitted founder sequences within these subjects could be attributed to immune responses within the original host (i.e the transmission source) that were not investigated in this study, or to bottleneck selection in the new host (i.e the recipient) during viral transmission. They could also be due to transmission of drug-resistance variants from a treatment-experienced source. However, the participants in this study were recruited and sampled prior to the availability of HCV NNIs, and while the participants were not part of DAA clinical trials, these were being conducted concurrently in the Australian community, and there remains the possibility that the source was treatment-experienced. Scientific Reports \| 7:41719 \| DOI: 10.1038/srep41719 4 www.nature.com/scientificreports/ Relevance of detected evolving RASs. All six RASs with longitudinal evolution in this study are rele- vant to one class of DAAs; non-nucleoside inhibitors (NNI) of the HCV NS5B. The RAS V499A, causes a minor reduction to some thumb-binding HCV NNIs24 but both none of those in development. A421V is associated with resistance to thumb-binding NS5B inhibitors, such as beclabuvir25; A421V, was identified upon treatment of GT1 patients with beclabuvir25, and with the combination daclatasvir/asunaprevir/beclabuvir, but had no significant association with virologic outcome26. M414T confers resistance to palm-binders, including the recently approved NNI dasabuvir27. Viral breakthrough and relapse after treatment in an IFN-free combination including dasabuvir have been associated with substitutions with M414I/T28–30. It should be noted, however, that the M414T substi- tution is observed in a GT3a infected participant and dasabuvir is known to have significantly reduced activity against GT3a in general27. hi g g The fitness of these RASs in cell culture models is variable, and depends on the examined genotypes. A421V does not seem to impact the replication of GT1a31 or GT1b32 replicons, whilst M414T affects the fitness of GT1b but not GT1a in replicon studies27. V499A was shown to impact the replication of GT1b in vitro24. Results and Discussionh This RAS, however, is not uncommon in GT1b infections, and therefore its impact on viral fitness could not be ascertained. hi p g yp does not seem to impact the replication of GT1a31 or GT1b32 replicons, whilst M414T affects the fitness of GT1b but not GT1a in replicon studies27. V499A was shown to impact the replication of GT1b in vitro24. This RAS, however, is not uncommon in GT1b infections, and therefore its impact on viral fitness could not be ascertained. Of the RASs that were observed to be lost over the analysed course of infection, A553T (in GT1a), A553V (in GT1b) and S556G (in both GT1a and GT1b) are key substitutions associated with reduced response to dasabuvir in vitro and in vivo27. These substitutions have been associated with reduced fitness of GT1 HCV in vitro27, poten- tially explaining their loss over time. pi Of the RASs that were observed to be lost over the analysed course of infection, A553T (in GT1a), A553V (in GT1b) and S556G (in both GT1a and GT1b) are key substitutions associated with reduced response to dasabuvir in vitro and in vivo27. These substitutions have been associated with reduced fitness of GT1 HCV in vitro27, poten- tially explaining their loss over time. Summary. Selection of HCV RASs via immune factors has been proposed in a number of studies. Emergence and loss of an NS3 RAS, R155K, has been previously reported in a single patient co-infected with both HIV-1 and GT1a HCV when sampled over time33. This site was later confirmed to fall within a CD8+ T cell epitope and the RAS was proposed to be associated with loss of recognition by these cells9. Resistance to other HCV DAAs has been proposed to arise through immune-driven selection in some putative HLA-restricted epitopes8, but these responses were not verified experimentally. Our study describes the evolution of six RASs during early acute HCV infections with three different genotypes, and proposes that immune selection pressures contribute to the emergence of HCV RASs in early HCV infections, particularly within NS5B, even in the absence of treatment. This study also supports previous reports highlighting the incurred fitness cost of RASs, and suggests that this cost contributes to their loss. These results suggest that populations with particular HLA dominant types should be closely monitored for changes in prevalence of immune-related RASs. References 1. Dore, G. J. & Feld, J. J. Hepatitis C virus therapeutic development: in pursuit of “perfectovir”. Clin. Infect. Dis. 60, 1829–1836 (2 2 Pa lotsk J M & Hepatitis C Virus Resistance to Direct Acting Anti iral Drugs in Interferon Free Regimens Gastroenter 1. Dore, G. J. & Feld, J. J. Hepatitis C virus therapeutic development: in pursuit of “perfectovir”. Clin. Infect. Dis. 60, 1829–1836 (2015) 2. Pawlotsky, J.-M. & Hepatitis, C. Virus Resistance to Direct-Acting Antiviral Drugs in Interferon-Free Regimens. Gastroenterology (2016) 1. Dore, G. J. & Feld, J. J. Hepatitis C virus therapeutic development: in pursuit of perfectovir . Clin. Infect. Dis. 60, 1829 1836 (2 2. Pawlotsky, J.-M. & Hepatitis, C. Virus Resistance to Direct-Acting Antiviral Drugs in Interferon-Free Regimens. Gastroenter (2016). ( ) 3. Eltahla, A. et al. Analysis of resistance‐associated substitutions in acute hepatitis C virus infection by deep sequencing across six genotypes and three continents. J. Viral Hepat. (2016). g yp p 4. Applegate, T. L. et al. 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Acknowledgements g Research support for the HITS-p cohort includes National Health and Medical Research Council of Australia (NHMRC) - Project No. 222887, Partnership No. 1016351, Program Nos. 510488 and 1053206; The HITS-c cohort is supported by the UNSW Hepatitis C Vaccine Initiative and NHMRC Project Grant No. 630483. The Kirby Institute is funded by the Australian Government Department of Health and Ageing. AAE, JG, ARL and RAB are supported by NHMRC research fellowships. The views expressed in this publication do not necessarily represent the position of the Australian Government. g Research support for the HITS-p cohort includes National Health and Medical Research Council of Australia (NHMRC) - Project No. 222887, Partnership No. 1016351, Program Nos. 510488 and 1053206; The HITS-c cohort is supported by the UNSW Hepatitis C Vaccine Initiative and NHMRC Project Grant No. 630483. The Kirby Institute is funded by the Australian Government Department of Health and Ageing. 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Scientific Reports \| 7:41719 \| DOI: 10.1038/srep41719 5 © The Author(s) 2017 Additional Informationi Competing financial interests: Jason Grebely is a consultant/advisor and has received research grants from AbbVie, Bristol–Myers Squibb, Gilead Sciences and Merck/MSD. Andrew Lloyd has received grants for investigator-initiated research from Gilead and Merck/MSD. The remaining authors have no conflicts of interest to disclose. How to cite this article: Eltahla, A. A. et al. Dynamic evolution of hepatitis C virus resistance-associated substitutions in the absence of antiviral treatment. Sci. Rep. 7, 41719; doi: 10.1038/srep41719 (2017). Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This work is licensed under a Creative Commons Attribution 4.0 International License. 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https://openalex.org/W3138763871	https://arts.units.it/bitstream/11368/2993099/1/ianza%20a%202.pdf	English	null	Role of the IGF-1 Axis in Overcoming Resistance in Breast Cancer	Frontiers in cell and developmental biology	2,021	cc-by	9,141	Role of the IGF-1 Axis in Overcoming Resistance in Breast Cancer Anna Ianza1, Marianna Sirico2,3, Ottavia Bernocchi1 and Daniele Generali1,3,4 1 Department of Medical, Surgery and Health Sciences, Azienda Sanitaria Universitaria Giuliano Isontina, Trieste, Italy, 2 Department of Surgery and Cancer, Faculty of Medicine, Imperial College London, London, United Kingdom, 3 Breast Cancer Unit and Translational Research Unit, ASST Cremona, Cremona, Italy, 4 Department of Medicine, Surgery and Health Sciences, University of Trieste, Trieste, Italy Over the last two decades, many studies have demonstrated that the insulin-like growth factor-1 (IGF-1) is involved in a number of patho-physiological processes, as well as in the development of different types of solid tumors, including breast cancer (BC). Preclinical and clinical data showed that IGF-1 receptor (R) is overexpressed and hyper- phosphorylated in several subtypes of BCs. The central implications of this pathway in tumor cell proliferation and metastasis make it an important therapeutic target. Moreover, the IGF-1 axis has shown strong interconnection with estrogen regulation and endocrine therapy, suggesting a possible solution to anti-estrogen resistance. IGF- 1R might also interfere with other pivotal therapeutic strategies, such as anti HER2 treatments and mTOR inhibitors; several clinical trials are ongoing evaluating the role of IGF-1R inhibition in modulating resistance mechanisms to target therapies. Our aim is to offer an overview of the most recent and signiﬁcant ﬁeld of application of IGF-1 inhibitors and relevant therapeutic strategies, weighing their possible future impact on clinical practice. Edited by: Marzia Di Donato, University of Campania Luigi Vanvitelli, Italy Reviewed by: Leena Hilakivi-Clarke, Georgetown University, United States Chunchun Han, Sichuan Agricultural University, China Correspondence: Anna Ianza annaianzamiccoli@gmail.com Keywords: IGF1, IGF-1R, clinical trial, therapy resistance, breast cancer INTRODUCTION The insulin-like growth factor-1 (IGF-1) is an insulin-like protein with anabolic eﬀects, whose production is stimulated by growth hormone (GH), and is one of the main mediators of GH eﬀects. Its circulating levels vary during childhood and reach its highest levels during puberty (Grimberg et al., 2016). The insulin-like growth factors (IGF-1 and IGF-2), their receptors, and a system of six insulin-growth factor binding proteins (IGFBP-1 to IGFBP-6) form a network involved in the activation of many downstream pathways (Allard and Duan, 2018). Multiple factors might activate IGF-1 receptor (R) tyrosine kinase activity (Llak, 2008) leading to interaction with its substrate, as insulin receptor (IR) substrate and the Drc-homology-2 containing protein SH2 (Radhakrishnan et al., 2011). After phosphorylation, this protein, acting as docking molecules, activates cellular kinases and initiates diﬀerent downstream signaling pathways. Speciﬁcally, IGF-IR activates the PI3K/AKT/mTOR and ras/raf/MEK signaling pathways that promote cell proliferation and, at the same time, inhibits programmed cell death, through the activation of the B-cell lymphoma 2 (Bcl2)/Bcl2 antagonist of cell death (BAD) pathway, leading to carcinogenesis (Hakuno and Takahashi, 2018). The transcription of IGF-1 enables the activation of the STAT3 pathway, which enhances the invasive ability of tumor cells in prostate cancer (Ma et al., 2020); Wang et al. (2020) Specialty section: This article was submitted to Molecular and Cellular Oncology, a section of the journal Frontiers in Cell and Developmental Biology Received: 14 December 2020 Accepted: 04 March 2021 Published: 22 March 2021 Citation: Ianza A, Sirico M, Bernocchi O and Generali D (2021) Role of the IGF-1 Axis in Overcoming Resistance in Breast Cancer. Front. Cell Dev. Biol. 9:641449. doi: 10.3389/fcell.2021.641449 Specialty section: This article was submitted to Molecular and Cellular Oncology, a section of the journal Frontiers in Cell and Developmental Biology Received: 14 December 2020 Accepted: 04 March 2021 Published: 22 March 2021 MINI REVIEW MINI REVIEW published: 22 March 2021 doi: 10.3389/fcell.2021.641449 Citation: Ianza A, Sirico M, Bernocchi O and Generali D (2021) Role of the IGF-1 Axis in Overcoming Resistance in Breast Cancer. Front. Cell Dev. Biol. 9:641449. doi: 10.3389/fcell.2021.641449 Ianza A, Sirico M, Bernocchi O and Generali D (2021) Role of the IGF-1 Axis in Overcoming Resistance in Breast Cancer. Front. Cell Dev. Biol. 9:641449. doi: 10.3389/fcell.2021.641449 Ianza A, Sirico M, Bernocchi O and Generali D (2021) Role of the IGF-1 Axis in Overcoming Resistance in Breast Cancer. Front. Cell Dev. Biol. 9:641449. doi: 10.3389/fcell.2021.641449 March 2021 \| Volume 9 \| Article 641449 Frontiers in Cell and Developmental Biology \| www.frontiersin.org 1 Ianza et al. IGF-1 in Breast Cancer demonstrated that IGF-1 activates NFkB signaling inﬂammation via cytosolic ROS in various cell cultures. An overview of the signaling pathways is described in Figure 1. demonstrated that IGF-1 activates NFkB signaling inﬂammation via cytosolic ROS in various cell cultures. An overview of the signaling pathways is described in Figure 1. Many diﬀerent factors aﬀect IGF-1 plasma concentrations: GH activity, nutritional status, sex, estrogen levels, and age (Clemmons and Van Wyk, 1984). Circulating IGF-1 is one of the major risk factors associated with increased BC risk (Lann and LeRoith, 2008). Previous in vitro studies demonstrated that IGF-1 stimulates the growth of human BC cell lines (Sasi et al., 2014; de Groot et al., 2020) and the in vitro blocking of IGF-1 system inhibits the response of human BC cell lines (Zha and Lackner, 2010). In the 1980s, the initial report by Furlanetto and DiCarlo (1984) highlighted the possible role of IGF-1 in the development of BC. g g p y g Insulin-like growth factor-1 and its system of binding proteins and receptors are physiologically involved in the development of many human tissues (Slepicka et al., 2020). It has been suggested that IGF-1 plays a signiﬁcant role in the ductal and mammary gland formation, function, and maintenance (Christopoulos et al., 2015). Preclinical and clinical data have shown that IGF-IR is overexpressed and hyper-phosphorylated in several subtypes of breast cancers (BCs) (Law et al., 2008), from which its role in BC development has stemmed. High plasma levels of IGF-1 and IGFBP-3 represent a risk factor for the development and recurrence of BC in the general population (Key et al., 2010). This is particularly veriﬁed for the incurrence of estrogen receptor positive (ER+) tumors, independent from menopausal status (Key et al., 2010). Citation: (2019) demonstrated a positive association between circulating IGFBP2 and mammographic density particularly among women with lower BMI, but no strong correlation with IGF-1. Citation: Whether it constitutes an additional risk for women with a family history of disease is not yet clariﬁed (Monson et al., 2020). However, an Italian study associated an increased risk of BC in patients with BRCA mutation (hereditary BC) with high serum IGF-1 levels (Pasanisi et al., 2011). Moreover, its role and level regulation naturally reveal a strong connection with dysmetabolism and body mass index (BMI), especially being a risk factor in HER2 positive (HER2+) overweight patients (Tong et al., 2020). Furthermore, it has been recently suggested that this factor could hold a negative prognostic signiﬁcance in BC (Hartog et al., 2013), overall and in patients undergoing endocrine therapy (Duggan et al., 2013; Hartog et al., 2013). Our aim is to oﬀer a focused review of the possible clinical role of IGF-1 as a therapeutic target and/or as part of combination therapy in BC. Later, many epidemiological and prospective studies have reported a positive correlation between circulating IGF-1 levels and BC development. A case-controlled study reported higher IGF-1 plasma concentrations in women with BC than patients without (Bruchim et al., 2009). Additionally, Werner and Laron (2020) reported a positive association between circulation concentrations of IGF-1 and BC risk for premenopausal women, but not for postmenopausal women. In the meta-analysis conducted by Renehan et al. (2004), high concentrations of IGF- 1 and IGFBP3 were associated with an increased risk of incident premenopausal BC but not with postmenopausal BC. A pooled data analysis of 4790 cases from 17 prospective studies from 12 countries clearly showed that women with relatively high circulating IGF-1 had a 30% higher risk of BC than women with relatively low circulating IGF-1. This positive association was found in ER+ but not estrogen-receptor negative (ER−) tumors. In addition, this correlation was independent of IGFBP3 and menopausal status (Key et al., 2010). Murphy et al. (2020) in their observational and Mendelian randomization analyses with 430,000 women found evidence that supports a probable causal relationship between circulating IGF-1 concentrations and BC. g Mammographic density is another BC risk factor. With regard to the association between mammographic density and serum IGF-1, there are controversial ﬁndings: Diorio et al. (2005) found a positive association in premenopausal women, but other studies did not support this result (Rice et al., 2012; Rinaldi et al., 2014). Recently, Hada et al. PLASMA LEVELS OF IGF-1 AND BREAST CANCER The concentrations of IG1 in plasma are approximately 150– 400 ng/mL, where it is present mostly as protein-bound form (Clemmons, 2007b). The free ligand concentration is less than 1% (Clemmons, 2007b). A family of high aﬃnity IGF binding proteins (IGFBPs) has the role of protecting IGFs from degradation through the formation of the complex IGFBP-IGF (Firth and Baxter, 2002). Even if IGFBPs were originally described as passive circulating transport proteins for IGF-I and IGF- II, now they are recognized as playing an important role in BC and IGF-1 action (Firth and Baxter, 2002). The major IGF transport function might be attributed to IGFBP-3, which is the most abounding IGF binding protein in the blood stream, followed by IGFBP-2 (Firth and Baxter, 2002). Once removed from the circulation, the binary complexes of IGFBP-IGF cross the endothelium to reach the target tissue and to interact with cell surface receptors. As the IGFBPs have a higher aﬃnity for the IGFs than the receptors, they could sequestrate IGFs away from the type I IGF receptor, blocking their interaction. On the other hand, IGFBPs may increase IGF cellular functions in the local microenvironment by acting as a reservoir that could slowly unbind the ligands (Brahmkhatri et al., 2015). Studies investigating the association between the IGF system and BC prognosis are limited and controversial. Some ﬁndings suggest a positive correlation (Duggan et al., 2013), others an inverse (Kalledsøe et al., 2019), or no clear association of the biomarkers of the IGF system with all causes of mortality or BC-speciﬁc mortality and recurrence (Al-Delaimy et al., 2011; Hartog et al., 2013). Zhu et al. (2020) in their large prospective study showed an inverse and independent association between circulating IGF-1 and all-cause mortality in invasive BC patients, with association being consistent across all clinical risk factors. Frontiers in Cell and Developmental Biology \| www.frontiersin.org IGF AS A TARGET OF THERAPY In hormone-responsive BC cells, IGF-1R function is crucially linked with ER action. In particular, both the IGF-1R and the March 2021 \| Volume 9 \| Article 641449 Frontiers in Cell and Developmental Biology \| www.frontiersin.org 2 IGF-1 in Breast Cancer Ianza et al. FIGURE 1 \| A schematic diagram of insulin growth factor-1 receptor (IGF-IR) activation and regulation. The IGF axis consists of ligands as insulin, insulin-like growth factor 1 and 2 (IGF-1, IGF-2), receptor, IGF binding proteins (IGFBPs) 1–7, and IGFBP proteases. The IGF ligands bind their receptors and binding proteins with high afﬁnity. IGFBPs bind tightly to IGF ligands, inﬂuencing binding to their receptors; IGFBP proteases cleave the IGFBPs into fragments with lower afﬁnity for the IGF ligands, thereby increasing free IGF-1 and IGF-2 bioavailability. Activation of IGF-1R promotes cellular growth, proliferation, survival, and metastasis via activation of molecular pathways downstream; among them the phosphatidylinositol 3-kinase (PI3K)-AKT and RAS-extracellular signal-regulated kinase (ERK) pathways. FIGURE 1 \| A schematic diagram of insulin growth factor-1 receptor (IGF-IR) activation and regulation. The IGF axis consists of ligands as insulin, insulin-like growth factor 1 and 2 (IGF-1, IGF-2), receptor, IGF binding proteins (IGFBPs) 1–7, and IGFBP proteases. The IGF ligands bind their receptors and binding proteins with high afﬁnity. IGFBPs bind tightly to IGF ligands, inﬂuencing binding to their receptors; IGFBP proteases cleave the IGFBPs into fragments with lower afﬁnity for the IGF ligands, thereby increasing free IGF-1 and IGF-2 bioavailability. Activation of IGF-1R promotes cellular growth, proliferation, survival, and metastasis via activation of molecular pathways downstream; among them the phosphatidylinositol 3-kinase (PI3K)-AKT and RAS-extracellular signal-regulated kinase (ERK) pathways. The role of the IGF-1R pathway in promoting tumor growth and survival is well established. Targeting the IGF signaling pathway represents a promising approach in the development of novel anti-cancer therapy. The rationale for targeting the IGF-1R is derived widely from cell culture experiments that demonstrate the importance of IGF-IR signaling in promoting proliferation, inhibiting apoptosis, and its involvement and impact on BC cells that are resistant to radiation and chemotherapy (Jones et al., 2009). In BC, speciﬁcally the expression of IGF-1R is at least 50% (Ekyalongo and Yee, 2017), much more compared to HER2+ positive BC, which represents 20–25% BC (Wang and Xu, 2019); besides, there is a broader potential group of patients that could be candidates for targeted therapy. IGF AS A TARGET OF THERAPY In the last few years, diﬀerent therapeutic strategies have been evaluated to inhibit the IGF-1R signaling pathway. These can be divided into three categories: monoclonal anti-IGF1R antibodies, small molecule tyrosine kinase inhibitors (TKIs), and IGF ligand antibodies. Based on preclinical data, these classes of drug have diﬀerent proﬁles of selectivity, eﬃcacy, and toxicity which might have some implications in clinical ER are expressed and act in synergy with estrogen steroid hormone to increase cell proliferation (Radhakrishnan et al., 2011). Otherwise in ER-BC cells, a more aggressive subtype of BC, the levels of the IGF-1R and IRS-1 are often low and IGF is not mitogenic, although IGF-1R is still required for metastatic spread (Radhakrishnan et al., 2011). Additionally, in ER+ cells, estrogens stimulate the expression of the IGF-IR and its major signaling substrate, IR substrate-1 (IRS-1), that promotes estrogen-independence for growth and transformation (Skandalis et al., 2014). Furthermore, IGF-1R and its substrate IRS-1 might induce drug and radio resistance of BC, cells leading to relapse (Pollak, 2012). Besides, high IGF-1R levels in primary tumor samples have been reported to be predictors of shorter disease-free survival, but data on the prognostic value of the IGF-1R for overall survival are contradictory (Pollak, 2012; Yerushalmi et al., 2012). Regarding these evidences, several strategies used to target the IGF axis have been clinically developed for cancer prevention and treatment. ER are expressed and act in synergy with estrogen steroid hormone to increase cell proliferation (Radhakrishnan et al., 2011). Otherwise in ER-BC cells, a more aggressive subtype of BC, the levels of the IGF-1R and IRS-1 are often low and IGF is not mitogenic, although IGF-1R is still required for metastatic spread (Radhakrishnan et al., 2011). Additionally, in ER+ cells, estrogens stimulate the expression of the IGF-IR and its major signaling substrate, IR substrate-1 (IRS-1), that promotes estrogen-independence for growth and transformation (Skandalis et al., 2014). Furthermore, IGF-1R and its substrate IRS-1 might induce drug and radio resistance of BC, cells leading to relapse (Pollak, 2012). Besides, high IGF-1R levels in primary tumor samples have been reported to be predictors of shorter disease-free survival, but data on the prognostic value of the IGF-1R for overall survival are contradictory (Pollak, 2012; Yerushalmi et al., 2012). Regarding these evidences, several strategies used to target the IGF axis have been clinically developed for cancer prevention and treatment. IGF-1R Antibodies Another strategy, employed with several agents, is tyrosine kinase inhibition (TKI). This kind of therapy is able to inhibit the kinase domains of the β-subunits of both immunoglobulin and IRs, as their primary sequences share 84% identity in the kinase domains maintaining a relatively intact ATP binding pocket (Munshi et al., 2003). There are only two exceptions to this, NVP-AEW541 and NVD-ADW742. NVP-AEW541 is a small molecular weight pyrrolo-[2,3]-pyrimidine derivative kinase inhibitor of IGF-IR (García-Echeverría et al., 2004), while NVP-ADW742 is an ATP-competitive inhibitor that prevents IGF-IR phosphorylation (Warshamana-Greene et al., 2005). These two inhibitors have 15–30 fold increased potency for IGF-1R kinase inhibition compared to IR kinase inhibition in cellular assay, but they are able to distinguish between the IGF-IR and the closely related InsR (Mitsiades et al., 2004; Serra et al., 2008). TKIs’ lack of selectivity might have some beneﬁt—upregulated serum levels of insulin after IGF-1R monoclonal antibody treatment may not have as much eﬀect on the tumor if both IGF-I1 and IR are blocked. Several studies demonstrated that these TKIs inhibit IGF-IR/IR phosphorylation and AKT activation, Monoclonal antibodies that target IGF-1R have shown beneﬁt in early-stage clinical trials (Gualberto and Pollak, 2009). IGF-1 antibodies block ligand binding, inducing receptor internalization and degradation. A few IGF-1R-speciﬁc antibodies can also partially aﬀect the IR-A signaling pathway by targeting IGF-1R/IRA hybrid receptors (Wang et al., 2005; Gualberto, 2010). However, they do not inhibit IGF-II activation of IR-A homodimers. One example is MEDI-573 (AstraZeneca), a fully humanized antibody able to neutralize both IGF-I and IR- A pathways in vitro and in mice. However, compared to the other human monoclonal antibodies, MEDI-573 selectively inhibits the activation of both the IGF-1R and the IR-A signaling, without cross-reactivity with insulin, sparing the insulin/IR pathway; besides glucose metabolism remains stable (Gao et al., 2011). However, after the completion of phase 2 study in metastatic BC AstraZeneca discontinued the investigation. More recently, another novel IGF ligand neutralizing antibody, Xentuzumab (BI836845) (Boehringer Ingelheim Pharmaceuticals) showed preclinical antitumor eﬃcacy of rapamycin by suppressing TABLE 1 \| Key clinical trial targeting IGF-1 axis in solid tumors. IGF AS A TARGET OF THERAPY IGF activities are mediated through substrate binding and subsequent activation of IGF-1R (Weroha and Haluska, 2012). March 2021 \| Volume 9 \| Article 641449 Frontiers in Cell and Developmental Biology \| www.frontiersin.org 3 Ianza et al. IGF-1 in Breast Cancer IGFs’ bioactivity and inhibiting rapamycin-induced PI3K AKT activation (Adam et al., 2012). IGFs’ bioactivity and inhibiting rapamycin-induced PI3K AKT activation (Adam et al., 2012). practice (Burtrum et al., 2003; Maloney et al., 2003). The main clinical trials targeting IGF-1 axis in solid tumors are detailed in Table 1. IGF-1R Antibodies Title ID number Drug regimen Phase and design Primary outcome Status A dose escalating clinical trial of the IGF-1 receptor inhibitor AXL1717 in patients with advanced cancer NCT01062620 AXL1717 Ia/b Single arm, open label RPTD, MTD Completed, results published A phase I study of the oral mTOR inhibitor ridaforolimus (RIDA) in combination with the IGF-1R antibody dalotozumab (DALO) in patients (pts) with advanced solid tumors. NCT00730379 Ridaforolimus plus dalotozumab I Single arm, open label Optimal dose, MTD Completed, results published A phase 2 study of ridaforolimus (RIDA) and dalotuzumab (DALO) in estrogen receptor positive (ER+) breast cancer NCT01605396 Ridaforolimus + dalotozumab VS examestane II Randomized, parallel assignment, open label PFS Completed, results published A phase I trial of the IGF-1R antibody ganitumab (AMG 479) in combination with everolimus (RAD001) and panitumumab in patients with advanced cancer NCT01061788 AMG 479 + RAD001 VS AMG 479 + RAD001 + panitumumab I Single center, dose escalation trial MTD, RPTD Completed Phase I study of everolimus (E, RAD001) and ganitumab (GANG 479) in patients (pts) with advanced solid tumors NCT01122199 Everolimus + ganitumab I Single arm, open label MTD, RPTD Completed A phase Ib/II study of the combination of BYL719 plus AMG 479 in adult patients with selected solid tumors NCT01708161 BYL719 (alpelisib) and AMG 479 (ganitumab) I/II Multicenter, open label, single arm DLT, ORR Terminated The XENERATM 1 study tests xentuzumab in combination with everolimus and exemestane in women with hormone receptor positive and HER2-negative breast cancer that has spread NCT03659136 Everolimus + exemestane VS everolimus + exemestane + xentuzumab II Two arm, open label PFS Recruiting Capecitabine and lapatinib ditosylate with or without cixutumumab in treating patients with previously treated HER2-positive stage IIIB-IV breast cancer NCT00684983 Capecitabine plus lapatinib ± cixutumumab II Randomized, parallel assignment, open label PFS Completed MTD, maximum tolerated dose; RPTD, recommended phase II dose; PFS, progression free survival; DLT, dose limiting toxicities. TABLE 1 \| Key clinical trial targeting IGF-1 axis in solid tumors. Capecitabine and lapatinib ditosylate with or without cixutumumab in treating patients with previously treated HER2-positive stage IIIB-IV breast cancer March 2021 \| Volume 9 \| Article 641449 Frontiers in Cell and Developmental Biology \| www.frontiersin.org Ianza et al. IGF-1 in Breast Cancer clinical problem (Nahta and Esteva, 2006; Abderrahman and Jordan, 2018). Drug resistance might be partially related to the crosstalk between the ER and the IGF pathways (Fagan et al., 2002). IGF-1R Antibodies For instance, HBL100 cells, under tamoxifen therapy, are not able to proliferate, but if they are treated concomitantly with IGF, they could survive (Christopoulos et al., 2018). However, the majority of clinical trials evaluating the combination of anti-IG-1R and anti-ER therapies in endocrine- resistance BC have yielded disappointing results, as they did not lead to any improvement in clinical outcome (Kaufman et al., 2010). Most of the women enrolled in these trials had already developed resistance and anti-IG-1R strategies were tested as the second and third line of therapy. The lack of clinical success of these trials implies that targeting just IGF-1R is not enough to overcome tumor growth. It has been reported that the continuous exposure of MCF-7 cells to tamoxifen resulted in the eventual emergence of resistant cells, called MCF-7 Tam- R, which lose IGF-IR expression but maintain IR expression for their growth (Fagan et al., 2002). Considering these results, targeting the IR pathway could be an alternative option to treat TamR BC. and consequently lead to increased apoptosis, decreased in vitro cell proliferation, and tumor suppression in xenografts models (Serra et al., 2008; Carboni et al., 2009). However, the potential beneﬁt of TKIs over antibody therapies targeting IGF-I1 might be their capacity to block also IR, which comes at the expense of metabolic alterations such as hyperglycemia and evidence of insulin resistance (Haluska et al., 2006). In 2015 Simon Ekman, in his phase 1a/b study, showed that the oral small molecule IGF-1-receptor pathway modulator had an acceptable safety proﬁle and demonstrated promising eﬃcacy in this heavily pretreated patient cohort, especially in patients with NSCLC (Ekman et al., 2016). IGF-1R and PI3K/Akt/mTOR Axis IGF 1R and PI3K/Akt/mTOR Axis Insulin-like growth factor-1 signaling is involved in complex cross-talk with other receptor tyrosine kinases (RTLs) and their downstream eﬀector which could likely confer resistance to inhibitors of a single class of receptor (Wilson et al., 2012). As known, the PI3K/AKT and RAS-MAPK axes are two well- established downstream pathways of IGF/insulin signaling. It is understood that the AKT pathway could be reactivated despite IGF-1R downregulation, mediated by anti-IGF-1R antibody or TKIs, leading to tumor progression (Cao et al., 2008). Based on this evidence, PI3K inhibitor such as LY294002 (Clark et al., 2002), S6K1 inhibitor H89 (Becker et al., 2011), MAPK inhibitor U0126 (Becker et al., 2011; Casa et al., 2012), and dual PI3K/mTOR inhibitor NVP-BEZ235 (Brachmann et al., 2009) have been studied in pre-clinical and clinical studies supported by the hypothesis that combinations of AKT and IGF-IR/InsR inhibitors would be an eﬀective treatment against hormone- independent ER + BC (Fox et al., 2013). Several studies have also shown that the dual inhibition of IGF-IR and m-TOR increased antitumor activity both in vitro and in BC. Di Cosimo et al. (2010), in a phase 1 clinical trial, have demonstrated clinical beneﬁt in 21.7% of BC patients combining ridaforolimus (a small molecule inhibitor of mTOR) and IGF-1R antibody dalotuzumab. The combination was feasible and well tolerated and a phase 2 was initiated, but accrual was prematurely interrupted due to a higher than expected incidence of stomatitis in the treated patients (Rugo et al., 2017). Chemotherapy py Cancers have the capacity to develop resistance to traditional therapies, and the increasing prevalence of these drug resistant cancers necessitates wider research and treatment development (Housman et al., 2014). Chemo-resistance is a common problem in the treatment of cancer patients, as cancer cells become resistant to chemical substances used in treatment, limiting the eﬃciency of chemotherapeutic agents (Phi et al., 2018). When tumor cells are treated with cytotoxic chemotherapy, susceptible cells die, while a subset of resistant cells will continue to proliferate (Weroha and Haluska, 2008). The IGF-pathway is implicated in the chemotherapy resistance process. For instance, IGF-I attenuated the response of theMCF-7 BC cell line to doxorubicin and paclitaxel by at least two mechanisms: induction of proliferation and inhibition of apoptosis (Clemmons, 2007a). Therefore, inhibition of IGF-I action could be useful to cytotoxic chemotherapy in BC. Moreover, it has been evaluated that also the timing of IGF-1R inhibition inﬂuences responses to chemotherapy. Zeng et al. showed that the administration of IGF1R inhibitors prior to doxorubicin therapy resulted in the best therapeutic responses registered in BC cell lines. The optimal dosage sequence was doxorubicin followed by an anti-IGF-1R antibody, while the opposite sequence decreased doxorubicin eﬀects (Zeng et al., 2009). Therefore, the timing of IGF-IR inhibition should be considered in the design of future clinical trials, combining IGF-IR blockade and chemotherapy. However, unlike other solid tumors (Goto et al., 2012), in BC, there are no results from clinical trials supporting the hypothesis of whether IGF-1R inhibition will enhance the activity of cytotoxic chemotherapy. March 2021 \| Volume 9 \| Article 641449 FUTURE DIRECTIONS Novel approaches to target IGF/insulin systems are related to small interfering RNA (siRNA) and microRNA (miRNA) in order to reduce IGF-IR expression and function (Jung and Suh, 2012). Durfort et al. showed that silencing IGF-IR using synthetic siRNA bearing 29-O-methyl nucleotides could induce cell-cycle arrest and decrease cell proliferation. Moreover, this study suggested that the crosstalk between the IGF-I axis and antitumor immune responses can mobilize pro-inﬂammatory cytokines, oﬀering a new clinical approach for treatment of mammary tumors expressing IGF-IR (Durfort et al., 2012). Nevertheless, this approach has two main problems: the ﬁrst one is that siRNA formulations for systemic application face a series of hurdles in vivo before reaching the cytoplasm of the target cell (Whitehead et al., 2009) and the second is the transient inhibition of the IGF pathway. However, preclinical in vivo studies showed that it might be possible to overcome at least the second obstacle, with the development of stable in vivo and inducible long-term expression of target short hairpin RNA using dimerizing drugs such as doxycycline or tetracycline (Jones et al., 2009). Furthermore, other miRNAs were investigated in the past few years (Guo et al., 2013). For instance, decreased levels of miR-139, which targets IGF-IR in colorectal cancer (CRC), were associated with disease progression and metastasis. This re- expression of miR-139 might suppress CRC cell invasion and metastasis by targeting IGF-IR (Shen et al., 2012). In esophageal squamous cell carcinoma (ESCC), it has been shown that miR- 375 inhibits tumor growth and metastasis through repressing IGF-1 receptor (Kong et al., 2012). Maybe in the future, siRNAs targeting IGF-IR will be modiﬁed in order to improve the eﬀect of IGF-IR downregulation and consequently modulate antitumor immune responses with the aim to oﬀer a new clinical approach for treatment of mammary tumors expressing IGF-IR. pp Recently, a new IGF-1 monoclonal antibody, Xentuzumab (Xen) has been investigated in the phase II XENERA-1 trial in combination with Ev and exemestane (Ex) in post-menopausal women with ER+ and HER2−metastatic BC (Crown et al., 2019). Crown et al., at San Antonio Breast Cancer Symposium in 2018, showed that in the overall (randomized) population, progression-free survival (PFS) was not signiﬁcantly improved in patients treated with Xen + Ev + Ex compared with Ev + Ex (Schmid et al., 2019). IGF-1R and Hormonal Therapies As discussed above, the crosstalk between IGF/IS pathway and estrogen receptors has been widely evaluated for potential new target drugs in ER + BC (Yee and Lee, 2000). ER + BC is the most common subtype, constituting almost 70% of all diagnosed BCs. Therefore, many trials have been performed to verify the eﬃcacy of the combination between anti-IGF-1R and anti- estrogen directed therapies. The overall eﬀect of hormonal agents on the IGF/insulin system is to regulate positively signaling. However, resistance to anti-estrogen therapies is still a pivotal Vlahovic et al. have evaluated the clinical beneﬁts of combining ganitumab, a monoclonal antibody directed versus IGF-1R, with everolimus (Ev) and panitumumab in patients with advanced cancers. However, the triplet regimen of ganitumab, Ev, and panitumumab was associated with unacceptable toxicity, and clinical activity has been demonstrated only in NSCLC and sarcoma (Vlahovic et al., 2018). Moreover, another phase I study of Ev and ganitumab in patients with advanced solid tumors has shown that this combination is safe; nevertheless, prolonged Frontiers in Cell and Developmental Biology \| www.frontiersin.org March 2021 \| Volume 9 \| Article 641449 5 Ianza et al. IGF-1 in Breast Cancer candidates for anti-HER2 and anti-IGF-1R combined therapies (Sanabria-Figueroa et al., 2015). clinical beneﬁt [stable disease (SD) ≥20 weeks] was noted only in refractory ﬁbrolamellar HCC, neuroendocrine, GIST, and urachal cancers (Jalal et al., 2013). A phase Ib/II study (NCT01708161) investigated the maximum tolerated dose (MTD) and response rate of the combination of ganitumab with alpelisib, a small molecule inhibiting the subalpha of PI3-kinase, in patients with ovarian and hormone receptor positive cancer carrying the somatic PIK3CA mutation. However, the recruitment has been stopped due to inconclusive results. candidates for anti-HER2 and anti-IGF-1R combined therapies (Sanabria-Figueroa et al., 2015). IGF-IR and HER2/erbB Receptor Therapy IGF-IR and HER2/erbB Receptor Therapy Most of the patients who obtain an initial response to trastuzumab-based therapy develop resistance within 1 year after commencing treatment (Albanell and Baselga, 2001). The possible existence of bi-directional crosstalk between the erbB family of receptors and IGF-1R may be implicated in resistance to targeted therapies including these receptors pathways (Weroha and Haluska, 2008). In BC cell models that overexpress HER2, an increased level of IGF-IR signaling might interfere with the action of trastuzumab (Lu et al., 2001). Moreover, BC cell lines, cultured in combination with an anti IGF-1 antibody, showed an increased cytotoxic eﬀect when treated with trastuzumab (Albanell and Baselga, 2001). Thus, strategies that co-target HER-2 and IGF-1R may prevent or postpone development of resistance to trastuzumab (Browne et al., 2012). In BC, an IgG1 monoclonal antibody that binds IGF-1R, cixutumumab (IMC-A12) is being investigated in combination with lapatinib in a phase II trial (Haluska et al., 2014). The mechanisms related to IGF-1R-driven HER-2 therapy are not well known; nevertheless, some studies showed that HER-2 therapy resistance may be associated with the downregulation of the PTEN/PI3K/AKT signaling pathway (Gallardo et al., 2012). Despite this, it has been shown that HER-2 overexpressing cancers treated with PI3K inhibitors developed AKT-mediated activation of other tyrosine kinase growth factors such as IGF1-R, Ins-R, and HER3 treatment. Besides, PI3K inhibitors should be combined with HER-2 targeted therapies including trastuzumab or lapatinib, in order to avoid AKT signaling activation (Chakrabarty et al., 2012). Moreover, in trastuzumab- resistant tumors, IGF-1R cell motility is related to the stimulation of FAK signaling and Forkhead box protein M1 (FoxM1). Furthermore, trastuzumab-resistant cancer cells might be the best FUTURE DIRECTIONS However, a pre-speciﬁed subgroup analysis showed that in the non-visceral metastases subgroup, the Xen + Ev + Ex arm demonstrated favorable PFS compared with the Ev + Ex arm. Speciﬁcally, an ongoing Phase II study (NCT03659136) is investigating the use of Xen + Ev + Ex in post-menopausal women with HR+/HER2−LA/mBC and non-visceral disease (Nahta et al., 2005). REFERENCES A phase I study of the oral mTOR inhibitor ridaforolimus (RIDA) in combination with the IGF-1R antibody dalotozumab (DALO) in patients (pts) with advanced solid tumors. J. Clin. Oncol. 28:15. Brachmann, S. M., Hofmann, I., Schnell, C., Fritsch, C., Wee, S., Lane, H., et al. (2009). 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CONCLUSION The IGF system has been involved in the oncogenesis of the majority of solid tumors. The central implications of this pathway in tumor cell proliferation and metastasis makes it an important therapeutic target. In BC, the IGF pathway has been implicated in resistance to the three cornerstones of BC therapy: hormonal agents, HER receptor targeting agents, and cytotoxic chemotherapy. Therefore, several clinical trials are currently evaluating the eﬃcacy of IGF-1R inhibition to overcome these resistance mechanisms. The competitive landscape for anticancer therapies in BC and the diﬃculty to recruit a suﬃcient number of patients limited de facto the continuation and validation of research with IGF-1R and GF inhibitors. That is why, even considering the encouraging initial results that we have illustrated, combined with the enormous potential clinical impact of the IGF axis, there is not yet an optimal combination therapy paradigm. 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Oncologist 23, 782–790. doi: 10.1634/theoncologist.2016- 0377 Copyright © 2021 Ianza, Sirico, Bernocchi and Generali. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Copyright © 2021 Ianza, Sirico, Bernocchi and Generali. 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, C., An, Y., Wang, Y., Wang, X., Luan, W., Ma, F., et al. (2020). Insulin- like growth factor-I activates NFκB and NLRP3 inﬂammatory signalling via March 2021 \| Volume 9 \| Article 641449 Frontiers in Cell and Developmental Biology \| www.frontiersin.org 9
W2004551913.txt	http://link.aps.org/pdf/10.1103/PhysRevSTAB.6.083201	en		Pipe cooling perspectives for superconducting accelerating cavities	Physical review special topics. Accelerators and beams	2,003	cc-by	4,849	PHYSICAL REVIEW SPECIAL TOPICS - ACCELERATORS AND BEAMS, VOLUME 6, 083201 (2003) Pipe cooling perspectives for superconducting accelerating cavities R. Ballantini, A. Chincarini,* G. Gemme, and R. Parodi Istituto Nazionale di Fisica Nucleare, via Dodecaneso 33, 16146 Genova, Italy (Received 12 June 2003; published 29 August 2003) We explore the rf characteristics of pipe cooled superconducting cavities versus bath cooled ones, using different pipe configurations and different liquid helium temperatures. Pipe cooled cavities can perform nearly as well as bath cooled ones, provided a suitable pipe configuration and cavity wall thickness is chosen. Pure thermal estimates and fits with experimental data show that pipe cooling is a viable solution for future cavities. DOI: 10.1103/PhysRevSTAB.6.083201 I. INTRODUCTION Superconducting rf cavities have been used in particle accelerators for several decades, these cavities being traditionally operated immersed in a liquid helium (lHe) bath. Nevertheless, several attempts have been made in the past to make use of the numerous operational and cost advantages of pipe cooling configuration: reduction in liquid helium inventory, minimized cooldown/warm-up times, and elimination of the lHe vessel, which reduces the sensitivity to microphonics and provides easier access to all cavity components [1]. Furthermore, the fact that cavities are operated in pulse mode makes the pipe cooling scheme more attractive since it provides extra stiffening to the cell. In this paper we compare the expected performances of bath cooled and pipe cooled single cells by means of a 2D computer code and we address the following questions: (1) What kind of reduction in Q0 values can we expect to have in a pipe cooled cavity? (2) What is the peak Bsurf field attainable if the losses were predominantly due to thermal effects? (3) If the losses in a bath cooled cavity were due to effects other than thermal (e.g., electron emission, multipacting, etc.), how would that cavity’s performance be influenced by pipe cooling? PACS numbers: 07.20.Mc, 41.75.Lx hoc parameters and functions added to the theoretical surface resistance. This problem is mainly due to the fact that most of the Q0 versus Eacc measurements are affected by dissipation mechanisms that are not related to Ohmic losses alone, not to mention the field dependence effect of sputtered niobium films on copper [5]. Our approach is then twofold: first we consider a purely thermal problem, with no other dissipation mechanisms, and compare the efficiency of different pipe configurations and lHe temperatures; then we take real measurements of the averaged surface resistance hRsurf i versus Eacc in bath cooled cavities and use this curve to extrapolate the hRsurf i in the case of pipe cooling. We carry out the calculation with the help of a 2D computer code that simulates the thermal behavior of axially symmetric structures, whose position dependent surface fields are given. The fields inside the structure come from OSCAR2D [6], a fast finite element code that computes all relevant rf quantities and field resonance distributions for axisymmetric cavities. A. Surface resistance When considering purely Ohmic dissipations, our calculations are based on an analytical model for the BCS resistance (Wilson formula), which has been proven to II. MODEL adequately match both the experimental data and the Thermal behavior of cavities has been computed in theoretical calculations [7]. The use of an analytical various ways [2 – 4], mainly using one dimensional modfunction has the advantage to considerably speed up the els. Despite several attempts, a coherent formulation that computation while preserving the correct parameters defits the measured data is still lacking, unless one uses ad pendence. The function is 2 f Tint f0 Tcr =Tint p cos=2Tint =Tcr 2 RBCS ; (1) e ln f0 Tcr Tint f where , , and are calculated to fit experimental data. Tint is the cavity’s inner surface temperature in K, f is the frequency in Hz, and f0 is the reference frequency at which experimental data are available. Tcr is a function of *Electronic address: andrea.chincarini@ge.infn.it 083201-1 1098-4402=03=6(8)=083201(8)$20.00 the surface magnetic field Bsurf and, together with Tint , it is also a function of the position. A small phenomenological residual resistance Rres , typically of the order of 10–50 n, is added and the overall position dependent surface resistance reads as follows:  2003 The American Physical Society 083201-1 PRST-AB 6 PIPE COOLING PERSPECTIVES FOR . . . Rs ~r; Tint ; f; Bsurf Rres RBCS T; (2) where r~ indicates the position vector on the cavity inner surface, and Bsurf is the surface magnetic field in tesla. Equation (2) though, once used to calculate the averaged hRs i, does not always correctly fit the experimental curves hRs i versus Eacc (or equivalently, versus peak Bsurf ). We took expression (2) only to look at the cavity from a purely Ohmic point of view, that is to study the cavity behavior in the hypothesis where the only limiting factors are the BCS surface resistance, the thermal conductivity of the material, and the way the cavity is cooled on the external surface. Considering experimental data though, one sees that the cavity performances are often limited by other processes (like field emission, multipacting, or surface defects) much earlier than the predicted limit given by Eq. (2) [8]. Since our aim is to look for applicability of pipe cooling and not to justify experimental hRs i curves, we can take the measured hRs i curves for a bath cooled cavity as given effective residual resistance, that is Rs ~r; Tint ; f; Bsurf R~res Eacc RBCS T; (3) ~ where Rres Eacc is a suitable function that fits the experimental data. We noted though that, for the cases we have considered, the main contribution to the hRs i came from field emission phenomena. It was therefore possible to use reasonable analytical functions to fit the measurements: R~ res Rres 1 Eacc 2 Eacc 3 Eacc 5=2 e4 =3 Eacc ; (4) where Rres limEacc !0 hRs i and i are free dimensional parameters. The term multiplying 1 takes into account the linear trend observed in sputtered Nb on Cu cavities at low fields [5] while the term multiplying 2 is the Fowler-Nordheim term [8]. Once the i are found through a fit, Eqs. (3) and (4) are used in the thermal calculation for a bath cooled cavity and the results are checked to verify that they match the experimental data again. It is then easy to modify the external condition to account for pipe cooling. Q0 values are then calculated by G (5) Q0 k ; hRs i 083201 (2003) properly scaled for the residual resistivity ratio (RRR) values when needed. The different values of the thermal conductivity used in the simulations have been computed scaling the measured curve (RRR 60) according to the Wiedmann-Franz [10] law: new RRR RRR 60 new RRR : 60 For Nb on Cu sputtered cavities, only Cu has been considered to account for heat transport, thus neglecting the sputtered Nb layer. Figure 1 shows both the Nb and Cu curves used in the model. C. Metal-lHe interface The power dissipated on the inner superconductor surface is transferred to the outer surface and then to the lHe medium. Across the interface between a solid and lHe there exists a temperature jump T Ts TB , where Ts is the solid outer surface temperature and TB is the bath temperature (both in K). T is related to the heat flux qe in Wm2 across the interface so that Ts can be described by the following relation [3]: 4 TB 4qe Rk TB3 1=4 TB 2:18 K; Ts (7) 20:18 < TB 4:2 K: TB qhe 1=a This expression shows that T is also dependent on the lHe regime: for superfluid lHe (TB 2:18 K), Ts is driven by the Kapitza resistance Rk that, for chemically polished and annealed Nb, has been measured to be [11]: 4 4:650:28 R1 k 0:020 0:00310 T (8) in W m2 K1 . Recent measures [12] report generally higher values for the Kapitza conductance, especially where G, in Ohm, is the geometry factor for the electromagnetic (e.m.) mode k, defined as R !k 0 V jHj2 dv R : (6) Gk 2 S jHj ds B. Thermal conductivity The thermal conductivities used in calculation come from experimental data [9], linearly interpolated and 083201-2 FIG. 1. (Color) Experimental thermal conductivity for Nb (solid line) and Cu (dashed line). Nb data refer to a RRR 60 sample. 083201-2 PRST-AB 6 R. BALLANTINI et al. for chemically treated surfaces. The values in Eq. (8) are therefore quite conservative. For the lHe-I regime, Ts is proportional to a power of qe , whose dimensional parameters have been experimentally determined to be h 1:23 104 and a 1:45 [13]. Equation (7) holds below a critical heat flux ( 104 W m2 ) at which film boiling sets on and the heat transfer rate sharply decreases by approximately 1 order of magnitude. Experimental data for the critical heat flux show a rather large scattering and are influenced by several factors such as the specimen geometry, submersion depth, and lHe bath temperature. The value of 104 W m2 is often assumed for He-II constrained in long tubes and we have taken this conservative approach in spite of the fact that critical fluxes up to 6 104 W=m2 have been observed [14]. D. Thermal modeling Our simulator computes the temperature profile as well as other relevant quantities of an axially symmetric structure whose e.m. fields distribution and properties are given. The structure symmetry allows a 2D simulation and integrates seamlessly with the e.m. computation from OSCAR2D (see Fig. 2). Figure 3 sketches the elementary surface elements on which the calculation is carried. si , so , sr , and st are surface elements at the curvilinear coordinate l, referring, respectively, to the inner surface (source of the rf heating), the outer surface (in contact with the bath), and the right and left surfaces, where lateral heat conduction takes place. FIG. 2. Electric field lines for the TE011 mode in a single cell. Graphical output by OSCAR2D. 083201-3 083201 (2003) FIG. 3. (Color) Sketch of the volume element used for the temperature modeling. The simulation starts with both the surfaces fsi g and fso g at the bath temperature and then, for each field value, calculates iteratively until convergence is reached. In the case of pipe cooling, the outer surface element so l which is not in contact with a pipe, is bound to reach the same temperature of the corresponding si l and the heat is therefore dissipated only through sr l and st l. Cavity thickness, material properties, and e.m. configuration as well as all relevant parameters can be varied at will. III. RESULTS A. Thermal effects This simulation set compares the expected results for a bath cooled versus a pipe cooled cavity when only BCS surface resistance and the material thermal conductivity are taken into account. Figures 4 and 5 show, respectively, the results for a TRASCO 0:85 Nb-Cu cavity and for a TRASCO 700 MHz Nb bulk cavity, both operated on the TM010 [15–17]. As expected, the maximum Bsurf predicted for bath cooling is much higher than the experimental values, thanks to the lack of other dissipation mechanisms. Pipe cooling performances are significantly different only in the high field region and exhibit almost no Q0 degradation in the range 0 –80 mT (TlHe 1:8 K) and in the range 0 –25 mT (TlHe 4:2 K). The pipe scheme used for one of these simulations is sketched in Fig. 6, where the pipes position is plotted versus the curvilinear abscissa running on the cavity’s profile. The normalized B profile pictured in Fig. 6 refers to the TRASCO 0:85 cavity. For this test, only three pipes of approximately 6 cm in diameter are used to cool the cavity, and they have been positioned in the highest B 083201-3 PRST-AB 6 10 PIPE COOLING PERSPECTIVES FOR . . . 11 083201 (2003) 1.2 Pipes 1 A 10 0.8 10 Q 0 B [Normalized units] B 10 9 D 0.6 0.4 C 0.2 0 10 8 0 20 40 60 80 100 peak B [ mT] 120 140 -0.2 160 0 0.2 0.4 surf FIG. 4. (Color) Performance comparison computed for a TRASCO 0:85 cavity. Simulation parameters are f 343 MHz, G 250 , Rres 10 n, Cu thickness 6 mm, and RRR 275. (A) and (B) are, respectively, bath and pipe cooled at TlHe 1:8 K, (C) and (D) are bath and pipe cooled at TlHe 4:2 K. field region yielding a cavity surface coverage of approximately 17%. We can also study the effect of different pipe cooling schemes, so that the best configuration can be chosen. The 10 11 10 10 1.4 1.6 configurations share approximately the same coverage on the cavity surface ( 20%), their difference consisting only in the number of pipes and their distribution. Figure 7 shows the results of this test for a 707 MHz cavity at 4.2 K. The choice in the cooling schemes does not yield an appreciable difference in the Q0 curves unless one wants to run the cavity near the (theoretical) transition point. 9 A Q 0 Q 0 1.2 FIG. 6. (Color) Pipes position relative to the magnitude of the magnetic field on the surface. Each pipe has a diameter of approximately 6 cm. 10 B 0.6 0.8 1 Curvilinear abscissa [m] 9 γ 10 α D C 8 β 10 0 50 100 peak B surf 150 [mT] 10 8 0 FIG. 5. (Color) Performance comparison computed for a TRASCO 700 MHz cavity. Simulation parameters are f 707 MHz, G 250 , Rres 10 n, Cu thickness 4 mm, and RRR 275. (A) and (B) are, respectively, bath and pipe cooled at TlHe 1:8 K, (C) and (D) are bath and pipe cooled at TlHe 4:2 K. 083201-4 10 20 30 40 peak B surf 50 60 70 [mT] FIG. 7. (Color) Simulation of a TRASCO 700 MHz cavity at 4.2 K with pipe cooling. The cavity surface coverage is 20% for all curves. Curve (): 3 pipes ; 1:8 cm; curve ( ): 5 pipes ; 1:0 cm; curve (%): 9 pipes ; 0:7 cm. 083201-4 PRST-AB 6 R. BALLANTINI et al. 083201 (2003) 11 10 9 10 Q Q 0 0 A B 10 10 C γ D α β 9 10 8 10 0 10 20 30 peak B surf 40 [mT] 50 60 70 FIG. 8. (Color) Simulation of a TRASCO 700 MHz cavity at 4.2 K with pipe cooling. The cavity is covered with nine pipes for a total of 20% of its surface. The curves relate to different Cu thickness. Curve (): thickness 2 mm; curve ( ): thickness 6 mm; curve (%): thickness 10 mm. A slight improvement, which favors a greater number of pipes, is nevertheless observable. Similarly, a thicker cavity wall improves the stability as shown in Fig. 8. These results support the idea that pipe cooling is a viable solution at least in the case where no dissipation mechanisms other than thermal breakdown are allowed. They also show that there is a non-negligible freedom in choosing the pipe distribution, provided enough surface coverage exists in the high field region. The simulations discussed above however, do not include pointlike surface defects. B. Frequency and RRR dependence Pipe cooling efficiency can also be tested as a function of several cavity parameters. We have run some simulations to study the thermal behavior dependence on the cavity’s frequency and the Nb RRR. Figure9 shows the Q0 performances with several RRR values and refers to a TRASCO 700 MHz cavity cooled at TlHe 1:8 K with the help of three pipes of diameter ; 2:2 cm, for a surface coverage of 23%. It may be of interest to look at the relative performance of a pipe cooled cavity versus a bath cooled one as function of the RRR. We have arbitrarily chosen to define this relative performance with the help of two parameters: &f 083201-5 maxBjpipes ; maxBjbath &q Q0 jbath : Q0 jpipes 0 20 40 60 peak B 80 surf 100 120 140 [mT] FIG. 9. (Color) Performances of a three-pipes cooled, 700 MHz single cell, with different Nb purity. Curve (A) is for a RRR 50, (B) for a RRR 100, (C) RRR 250, and (D) RRR 500. &f is a measure of how well the pipe cooled cavity is able to sustain high intensity fields. &q is the relative Q0 slope ratio, averaged over the lowest field range, that is over the range 0; maxBjpipes ]. Ideally, &q should approximate 1, meaning that the Q0 slope (versus B) for the pipe cooled cavity is comparable to the bath cooled one. A low value for both parameters indicates a low relative performance. Since the pipe cooled cavity performances are always lower than the bath cooled counterparts, we have 0 < &f ; &q < 1. Figure 10 shows these parameters as a function of the RRR, whose values (from 10 to 1000), although somewhat extreme, have been used to show the extent of the differences. Figure 10 confirms the need for a high thermal conductivity (RRR > 200) and shows that extreme values do not appreciably increase the stability. Frequency dependence has been tested by scaling the linear dimensions of a 700 MHz cavity in order to have its resonant frequency going from 500 MHz up to 3 GHz, all cavities cooled at TlHe 1:8 K. It is important to note that we have scaled the cavity linear dimensions and tube diameters (in order to keep the surface coverage a constant), whereas the wall thickness has been kept unchanged (2 mm). All other parameters such as residual resistance, geometry factor, and lHe temperature have been kept constant throughout all computations. We have simulated the cooling using three pipes, whose diameters ranged from 3.1 cm (for the 500 MHz cavity) to 5 mm (3 GHz cavity), keeping the surface coverage at 24%. Figure 11 shows the Q0 performances for the cavity scaled to operate at 500 MHz and 3 GHz. 083201-5 PRST-AB 6 PIPE COOLING PERSPECTIVES FOR . . . 1 1 0.8 0.8 0.6 0.6 0.4 0.4 0.2 0.2 083201 (2003) θq θq θf θf 0 0 10 100 1000 0.4 0.8 1.2 1.6 2 2.4 2.8 3.2 Frequency [GHz] RRR FIG. 10. (Color) Relative performance of a 700 MHz cavity as a function of the Nb RRR. Figure 12 shows the relative performance parameters versus the cavity frequency. Interestingly, the relative performances do not change appreciably in a quite wide frequency range. This means that, when pipe cooled, one could expect to achieve a maximum field of about 80% of the field attainable in a bath cooled cavity, almost regardless of the operating frequency. FIG. 12. (Color) Relative performance parameters as a function of the cavity frequency. Bulk Nb cavity, 2 mm thick, RRR 300, three pipes cooling. These results obviously are valid for a very ideal situation, especially regarding the cavity surface loss mechanism. Furthermore, keeping the thickness of the cavity as a constant over the whole frequency range, for example, would pose serious mechanical problems at low frequency. Similarly, scaling the pipe diameters to 5 mm may be unpractical. 11 10 C. Surface defects 10 10 A Q 0 B D 10 9 10 8 0 20 40 60 80 peak B surf C 100 120 140 160 [mT] FIG. 11. (Color) Performance comparison for a 2 mm thick, bulk Nb cavity whose dimensions were scaled to operate at 500 MHz and 3 GHz. RRR 300 for all curves. Curve (A): bath cooled, 500 MHz; (B): pipe cooled, 500 MHz; (C): bath cooled, 3 GHz; (D): pipe cooled, 3 GHz. 083201-6 One of the few published results of pipe cooled cavities versus bath cooled ones was given by Susta et al. in 1993 [1]. In their paper, they showed a very nice curve of Q0 versus peak Esurf in a bath cooled, bulk Nb cavity, accompanied by a pipe cooled measure, which suffered a reduction for peak Esurf of approximately 47% (peak Esurf 57 MV=m for bath cooling, peak Esurf 30 MV=m for pipe cooling, TlHe 1:8 K). In Kneisel’s paper, the cavity was cooled through a single, large pipe (71 mm wide) welded on the cell together with two other pipes welded on either side of the drift tubes. To check for a qualitative agreement with their experimental results, we have performed a simulation using their same pipe scheme and we have also added the simulation equivalent of a surface defect. In our code the surface defect can only be simulated by changing the properties of an annular surface, whose typical dimensions are 40 mm in radius and 1 mm in height. The results are shown in Fig. 13. The qualitative agreement with the measure (not reported here) reveals that the presence of a surface defect can be a valid explanation for 083201-6 PRST-AB 6 R. BALLANTINI et al. 083201 (2003) 11 11 10 10 a e c 10 Q 0 Q 0 10 10 10 a f d b c b 9 10 9 0 5 10 15 E acc 20 25 30 [MV/m] 10 0 5 10 15 E acc FIG. 13. (Color) Surface defect simulation for a 1500 MHz cavity. Curves are labeled as follows: (a) bath cooled, no surface defect; (b) bath cooled with surface defect; (c) five pipes ; 2 cm, surface coverage 38%, no defect; (d) same as (c) but with surface defect; (e) single pipe ; 7:1 cm, surface coverage 38%, no defect; (f) single pipe with surface defect. the severe loss in performance ( 44% reduction on the maximum attainable Eacc field). The simulation also shows that in the bath cooled case the presence of the defect does not deteriorate the achievable peak surface fields [curves (a) and (b) in Fig. 13]. This means that the cavity may perform very well in the bath and yet may turn out to be strongly affected by the defect when pipe cooled [curves (b) and (f ). The surface defect though, whose presence was taken into account in the original paper as well, is not the only cause responsible for the cavity behavior. A wiser choice for the pipes distribution could have helped; that is, a greater number of pipes suitably distributed perform better than a single big pipe, even if the single pipe surface coverage is a little greater than the multipipe configuration. A configuration involving several pipes is therefore more forgiving in case of a surface bad spot [compare curves (c), (d), and (f) in Fig. 13]. D. Measurement fits Real cavities performance curves often exhibit signatures of effects like electron field emission and multipacting. Because of their variability in strength and occurrence, it is difficult to account for them in a model. In this paper we have considered a set of measurements on a TRASCO 700 MHz cavity performed at Saclay, France [18]. 083201-7 20 25 [MV/m] FIG. 14. (Color) Performance simulation and experimental data for a 700 MHz cavity suffering from electron field emission. (a) (4) pipe cooled simulation; (b) (o) bath cooled simulation; (c) (+) measured data. These measurements clearly show a Q0 value degradation due to field emission, therefore we have used Eqs. (3) and (4) to fit the effective residual resistance, where the Fowler-Nordheim term accounts for the sharp increase of hRs i at relatively low accelerating fields. Figure 14 shows the measured data, the simulation for the bath cooled cavity, and the expected performance of the pipe cooling configuration on the same plot. In this test, we have simulated three pipes of approximately 1.5 cm in diameter equally spaced around the maximum value of the surface magnetic field. If we restrict our analysis to relatively low Eacc values ( < 20 MV=m), pipe cooling does not have any appreciable effect. The difference lies in the abrupt transition that takes place at a far lower field than the expected bath cooled counterpart ( 20 MV=m for pipe cooling, 28 MV=m for bath cooling). The simulations we performed, compared to the available real data on cavities limited by field emission, show that pipe cooling should not alter the overall cavity performance. Electron emission normally occurs at field’s values far lower than the one needed for thermal breakdown. It is therefore reasonable that, for those field’s values, pipe cooling should perform nearly as well as bath cooling. IV. APPLICABILITY Pipe cooling can also have interesting applications outside the world of particle accelerators. As an 083201-7 PRST-AB 6 PIPE COOLING PERSPECTIVES FOR . . . 11 V. CONCLUSION 10 Bulk niobium and Nb-Cu technologies are nowadays quite mature. We can therefore envisage an improvement in terms of design freedom and cost management for pipe cooled structures. The remark that real pipe cooled cavities are much more sensitive to any nonideality, due to their lessened heat dissipation capabilities, is generally true. Nevertheless, the cavity behavior can be tamed by properly choosing the pipes size and distribution. The performance estimations presented in this paper strongly support the call for a more experimental setup implementing pipe cooling. β α 0 γ Q 083201 (2003) 10 10 9 10 0 20 40 60 peak B surf 80 100 120 [mT] FIG. 15. (Color) 2 GHz double spherical cell cavity. Curve (): 2 mm bulk Nb, bath cooled. Curve ( ): 2 mm bulk Nb, pipe cooled with 2 pipes ; 3 cm, cavity surface coverage 18%. Curve (%): Nb on Cu (5 mm thick), pipe cooled with the same configuration as in ( ). TlHe 1:8 K for all curves. example, we report the use of superconducting cavities for gravitational waves detection [19]. This detector uses two nearly spherical cells coupled through a variable length tube and operated on the TE011 mode. For this purpose, in contrast to accelerator applications, the spherical cells need to have a very high mechanical quality factor Qmech , at least for one of the quadrupolar mechanical modes of resonance. Since initial estimates suggest that bath cooling may impair the mode’s Qmech , pipes can be electron beam welded on the cell in such a way as to minimally perturb the quadrupolar mode oscillation. That is, pipes should run on the nodal lines of the mechanical resonant mode. For this particular application, we have calculated the Q0 estimates considering purely thermal dissipations, that is, with no surface defects and no electron emission—the latter assumption is justified by the TE011 mode of resonance. The results are sketched in Fig. 15 and show a quite dramatic decrease in the theoretical performance (compared to the bath cooled case). This is due to the nonoptimal positioning of the pipes with respect to the e.m. field distribution and to the use of only two small pipes, both characteristics chosen to cope with the quadrupolar nodal lines. As shown in the same figure, performances can be considerably improved using Nb on Cu cavities with a Cu thickness of approximately 5 mm. 083201-8 [1] J. Susta, P. Kneisel, and M. Wiseman, in Proceedings of the International Conference on Particle Accelerator, Washington, DC, 1993 (IEEE, Piscataway, NJ, 1993), p. 1060. [2] H. Vogel, DESY Laboratory Report No. DESY M-87-04, 1987. [3] P. Fernandes and R. Parodi, Cryogenics 11, 433 (1984). [4] R. L. Geng, in Proceedings of the International Conference on RF Superconductivity, 2001, Tsukuba (proc. ref. 010212-01). [5] C. Benvenuti et al., Physica (Amsterdam) 316C, 153–188 (1999). [6] P. Fernandes and R. Parodi, in Proceedings of the LINAC 86, Stanford, CA (SLAC Report No. 303, 1986), p. 330. [7] P. B. Wilson, SLAC Laboratory Report No. SLAC-TN70-35, 1970. [8] H. Padamsee, J. Knobloch, and T. Hays, Rf Superconductivity for Accelerators (Wiley, New York, 1998). [9] NBS monograph. [10] M.W. Zemansky, Heat and Thermodynamics (McGrawHill, New York, 1968), p. 94. [11] K. Mittag, Cryogenics 13, 94 (1973). [12] A. Boucheffa and M. X. Francois, in Proceedings of the Seventh Workshop on RF Superconductivity, Gif sur Yvette, France, 1995, pp. 659–669. [13] C. Johannes, in Proceedings of the International Conference on Cryogenic Engineering, ICEC 3, Berlin, Germany, 1970, p. 97. [14] D. N. Lyon, Int. Adv. Cryog. Eng. 14, 159 (1969). [15] http://trasco.lnl.infn.it/ [16] D. Barni, in Proceedings of the International Conference on RF Superconductivity, 2001, Tsukuba (JP) (proc. ref. TA009). [17] R. Parodi, in Proceedings of the International Conference on RF Superconductivity, 2001, Tsukuba (JP) (proc. ref. PR005). [18] H. Safa, in Proceedings of the International Conference on RF Superconductivity, 2001, Tsukuba (JP) (proc. ref. MA008). [19] A. Chincarini et al., Classical Quantum Gravity 20, 3505–3522 (2003). 083201-8
W2751461606.txt	https://www.ajol.info/index.php/tjpr/article/download/160737/150306	en		<i>Caulis Lonicerae</i> Japonicae extract shows protective effect on osteoporosis in rats	Tropical journal of pharmaceutical research	2,017	cc-by	3,457	Li & Mu Tropical Journal of Pharmaceutical Research August 2017; 16 (8): 1881-1886 ISSN: 1596-5996 (print); 1596-9827 (electronic) © Pharmacotherapy Group, Faculty of Pharmacy, University of Benin, Benin City, 300001 Nigeria. All rights reserved. Available online at http://www.tjpr.org http://dx.doi.org/10.4314/tjpr.v16i8.18 Original Research Article Caulis Lonicerae Japonicae extract shows protective effect on osteoporosis in rats Bao-cheng Li and Wei-dong Mu* Department of Traumatic Orthopaedics, Shandong Provincial Hospital Affiliated to Shandong University, Jinan 250021, Shandong Province, China For correspondence: Email: muweidong133@126.com; Tel: +860531 68773195 Sent for review: 18 February 2017 Revised accepted: 12 July 2017 Abstract Purpose: To investigate the effect of Caulis lonicerae Japonicae extract (CLJE) on ovariectomyinduced osteoporosis in rats. Methods: Female Sprague-Dawley rats were randomly assigned to a control group (normal rats) and five ovariectomy (OVX) subgroups: OVX with vehicle (OVX), OVX with 17ß-estradiol (E2, 25µg/kg/day), and OVX with CLJE doses (150, 300, and 600 mg/kg/day). Daily oral administration of E2 or CLJE started 4 weeks after OVX and lasted for 16 weeks. The bone mineral density (BMD) of L4 vertebrae and right femurs was determined. The length of each femur was measured with a micrometer, and the center of the diaphysis was determined. Three representative L4 vertebrae were selected to evaluate trabecular microarchitecture. Serum alkaline phosphatase (ALP), urinary calcium (U-Ca), urinary phosphorus (U-P), urinary creatinine (Cr) and osteocalcin (OC) levels were measured using a diagnostic reagent kit. Result: The results show that a high-dose of CLJE (600 mg/kg) significantly inhibited bone mineral density (BMD) reduction of L4 vertebrae (0.24 ± 0.02, p < 0.05) and femur (0.24 ± 0.03, p < 0.05) caused by OVX, and prevented the deterioration of trabecular microarchitecture (p < 0.05). High-dose CLJE also improved morphometric parameters, viz, trabecular number (Tb-N) (4.6 ± 0.3, p < 0.05), trabecular thickness (Tb-Th, 0.084 ± 0.012, p < 0.05) and trabecular separation (Tb-Sp, 0.14 ± 0.02, p < 0.05) in L4 vertebrae significantly. Conclusion: The results indicate that CLJE prevents OVX-induced osteoporosis in rats. Thus, CLJE is a potential natural alternative for the treatment of postmenopausal osteoporosis in future. Keywords: Caulis Lonicerae Japonicae, Post-menopausal osteoporosis, Ovariectomy, Bone mineral density, Trabecular microarchitecture, Diaphysis Tropical Journal of Pharmaceutical Research is indexed by Science Citation Index (SciSearch), Scopus, International Pharmaceutical Abstract, Chemical Abstracts, Embase, Index Copernicus, EBSCO, African Index Medicus, JournalSeek, Journal Citation Reports/Science Edition, Directory of Open Access Journals (DOAJ), African Journal Online, Bioline International, Open-J-Gate and Pharmacy Abstracts INTRODUCTION Osteoporosis is a systemic skeletal disease characterized by reduced bone mass and microarchitectural deterioration of bone tissue with a consequent increase in bone fragility and susceptibility to fractures [1]. According to data released by the World Health Organization (WHO), osteoporosis affects approximately a million people throughout Europe, the USA, and Japan [2]. The incidence of osteoporosis increases dramatically with life expectancy. Accordingly, the risk of osteoporotic fractures and their associated costs is rising rapidly due to aging population [3]. In the elderly, hip fractures are closely associated with mortality [4]. Hormone deficiency is known to impair cancellous metaphyseal bone and reduce BMD in humans and animals. Therefore, estrogen Trop J Pharm Res, August 2017; 16(8): 1881 Li & Mu deficiency in post-menopausal women has been regarded as a critical cause of this population’s susceptibility to osteoporosis [5]. Osteoporosis is twice as common in women as in men [6], and approximately one in three women over 50 years old experiences an osteoporotic fracture in her lifetime [7]. Clinically, hormone replacement therapy (HRT) has been a popular therapeutic strategy designed for postmenopausal osteoporosis [8,9]. However, the long-term application of HRT has potential malignant effects on reproductive tissues [10-13]. Other medicines that stimulate bone formation (e.g. growth hormone, sodium fluoride, and parathyroid hormone) or inhibit bone resorption (e.g. bisphosphonates and calcitonin) may prevent bone loss progression in established osteoporosis. However, these medications are not effective for a large proportion of the world population, especially in developing countries, and these medicine have side effects, such as gastrointestinal reactions, cancers, osteonecrosis of the jaw, and reduced skeletal strength [14,15]. Consequently, to substitute or reduce the medicines used currently, there are efforts to study new drugs with improved therapeutic efficacy, fewer undesirable side effects, and lower price. Caulis lonicerae has been widely used as a kidney-tonifying and anti-osteoporosis herb for the treatment of nephrasthenia syndrome [16], osteoporosis [17] and bone fracture [18] for thousands of years in China. The aim of the present study was to systematically evaluate the effect of CLJE on osteoporosis induced by OVX in rats. and freeze-dried to obtain CLJE [19]. The yield was 58.82 %. Animals and treatments Healthy three-month-old female Sprague-Dawley rats (weight, 220 ± 10 g) provided by the Experimental Animal Center of Shandong Province (Certificate no. SYXK 2004 - 0003). The animals had free access to feed and water, and were allowed to acclimatize for at least one week before use. The rat experiment was approved by the Animal Care and Use Committee of Shandong University of Traditional Chinese Medicine (approval ref no. 20130709) and was carried out in compliance with the Directive 2010/63/EU on the handling of animals used for scientific purposes [20,21]. Sixty rats were randomly divided into six groups of ten individuals: a sham-operated group (control) and five ovariectomy (OVX) subgroups, that is, OVX with vehicle (OVX), OVX with 17ßestradiol (E2, 25 mg/kg/day), and OVX with CLJE doses (150, 300 and 600 mg/kg/day). Daily oral administration of E2 or CLJE started 4 weeks after OVX and lasted for 16 weeks. Bone mineral density measurement The BMD of the L4 vertebrae and right femurs was estimated using dual-energy X-ray absorptiometry scanning (DEXA, GE Healthcare, USA) with small animal measurement. The measurements were expressed as grams of mineral contents per cm2 of surface area. Scans were performed by the same blinded technician. Three-point bending test EXPERIMENTAL Preparation of Caulis lonicerae extract The herbal samples of Caulis lonicerae Japonicae were collected from Zhangjiajie City, Hunan Province in China in May 2016. Taxonomic identification of the plant was performed by Professor WeiWang of Shandong University of Traditional Chinese Medicine, in China. A voucher specimen (no. CLJE 201605023) was assigned and the specimen deposited in the herbarium of Shandong University of Traditional Chinese Medicine, China for future reference. One batch (100 g) of the herbal material Caulis lonicerae was dried in an oven. Aqueous extract of CLJE was obtained by steeping the dried Caulis lonicerae in water at 60 ℃ three times for 1 h each. The liquid extract was dried in an oven Before mechanical testing, the rats were sacrificed by cervical spondylosis. Then the left femurs were slowly thawed at room temperature. The length of each femur (distance from the intermalleolar to the intercondylar region) was measured with a micrometer, and the center of the diaphysis was determined. Micro-CT analysis Based on the BMD, three representatives L4 vertebrae from each group were selected to evaluate the trabecular microarchitecture using eXplore Locus SP preclinical specimen microcomputed tomography (MicroCT, GE Healthcare, USA). Before the scans, the bones were positioned with gauze in the sample holder and allowed to reach room temperature. The L4 vertebrae were scanned from the anterior endplate in the posterior endplate direction (22 Trop J Pharm Res, August 2017; 16(8): 1882 Li & Mu um/slice). The isotropic voxel resolution of bone was 22 um3. The volume of interest (VOI) was selected as a region 25 slices away from the anterior endplate to the posterior endplate, ranging to 125 slices. The three-dimensional images were reconstructed with the purpose of visualization and display. After analyzing the VOI, morphometric bone parameters, including trabecular number (Tb-N), trabecular separation (Tb-Sp), trabecular thickness (Tb-Th) were obtained. The VOI analysis was performed blindly by the same operator. Biochemical analysis of serum and urine specimens The serum of rats were collected by picking eyeball, and the urine of rats were collected by stimulating back. The levels of serum alkaline phosphatase (ALP), urinary calcium (U-Ca), urinary phosphorus (U-P), and urinary creatinine (Cr) were measured on an automatic analyzer (Ciba-Corning 550, USA) using a diagnostic reagent kit. Serum osteocalcin (OC) concentration was determined using a rat OC ELISA kit (San Clemente, CA, USA). Statistical analysis Data are expressed as mean ± SD. Statistical analysis was performed using one-way ANOVA combined with Bonferroni’s multiple comparison test using SPSS 16.0. Differences were considered statistically significant at p < 0.05. RESULTS BMD of L4 vertebrae and femurs The BMD of the L4 vertebrae and femurs presented in Table 1. These results demonstrate that OVX significantly decreased the BMD in the L4 vertebrae and femurs compared to the sham group (p < 0.05). Compared to the OVX group, CLJE treatment significantly prevented the BMD decrease in OVX-induced L4 vertebrae and femurs (all p < 0.05) in a dose-dependent manner. E2 also significantly increased the BMD of the L4 vertebrae and femurs (both p < 0.05), which was similar to that observed in the H-CLJE group (p > 0.05). Mechanical characteristics of femur The results of the mechanical testing of femur are presented in Table 2. Compared with the sham group, 16 weeks of estrogen deficiency significantly decreased the maximum load and maximum stress (both p < 0.05). Meanwhile, higher dosage of CLJE treatments (300 or 600 mg/kg/day) markedly prevented the OVXinduced tendency to decrease these parameters (p < 0.05). E2 also increased these biomechanical parameters, which were significantly higher than those of the OVX group (p < 0.05). It is worth noting that the increase in maximum load observed for the H-CLJE group was similar to that of E2 (p > 0.05). Table 1: Effect of CLJE on BMD of L4 vertebrae and femur (n = 10) Group Dosage(mg/kg) BMD of vertebra(g/cm2) BMD of femur(g/cm2) * Control 0.28±0.03 0.27±0.03 OVX 0.13±0.02 0.13±0.03 E2 0.025 0.24±0.03* 0.17±0.04* L-CLJE 150 0.15±0.02* 0.15±0.03* * M-CLJE 300 0.19±0.02 0.17±0.02* * H-CLJE 600 0.24±0.02 0.24±0.03* * ** P < 0.05 and p < 0.01 versus OVX group. L-CLJE: low dose CLJE, M-CLJE: medium dose CLJE, HCLJE: high dose CLJE Table 2: Effect of CLJE on mechanical characteristics of femur (n = 10) Group Dose (mg/kg) Maximum load(N) Maximum stress(MPa) * Control 132.5±5.3 203.7±6.1* OVX 95.6±4.3 148.3±5.6 * * E2 0.025 121.4±5.0 181.4±5.4 L-CLJE 150 92.3±4.5 149.3±4.6 * * M-CLJE 300 102.4±5.2 170.5±5.1 * H-CLJE 600 117.1±4.7 176.3±5.3* * ** P < 0.05 and p < 0.01 versus OVX group. L-CLJE: low dose CLJE, M-CLJE: medium dose CLJE, HCLJE: high dose CLJE Trop J Pharm Res, August 2017; 16(8): 1883 Li & Mu Table 3: Effect of CLJE on morphometric parameters of L4 vertebrae (n = 10) Group Dose (mg/kg) Tb-N(1/mm) Tb-Th(mm) Tb-Sp(mm) Control 5.8±0.3* 0.098±0.013* 0.11±0.02* OVX 2.4±0.2 0.068±0.014 0.46±0.03 * * * E2 0.025 4.3±0.3 0.093±0.011 0.24±0.02 L-CLJE 150 2.7±0.3 0.053±0.015 0.38±0.03 M-CLJE 300 3.5±0.3* 0.085±0.014* 0.26±0.03* H-CLJE 600 4.6±0.3* 0.084±0.012* 0.14±0.02* * ** P < 0.05 and p < 0.01 versus OVX group. L-CLJE: low dose CLJE, M-CLJE: medium dose CLJE, HCLJE: high dose CLJE Table 4: Effect of CLJE on biochemical profile of rat serum and urine (n = 10) Group Dose (mg/kg) U-Ca/Cr (mmol/mmol) U-P/Cr (mmol/mmol) ALP (U/L) OC (mmol/L) Control - 0.14±0.04* 3.3±0.3* 102.5±11.6* 6.9±0.4* OVX 0.53±0.03 * E2 0.025 0.31±0.03 L-CLJE 150 0.35±0.02* * M-CLJE 300 0.33±0.02 H-CLJE 600 0.25±0.03* * ** P < 0.05and p < 0.01 versus OVX group. L-CLJE: CLJE: high dose CLJE 6.7±0.2 244.2±27.5 16.5±0.3 * * * 4.1±0.3 132.6±15.4 12.6±0.3 * 5.6±0.3 172.4±17.1 13.8±0.4 * * * 4.2±0.2 166.2±15.3 12.1±0.3 * * * 3.4±0.3 135.4±16.4 8.6±0.3 low dose CLJE, M-CLJE: medium dose CLJE, H- Microarchitecture of L4 vertebrae DISCUSSION Analysis of the representative samples (Table 3) indicate that OVX resulted in the deterioration of the trabecular bone microarchitecture, as demonstrated by the reduced Tb-N and Tb-Th compared with the sham group (both p < 0.05). In contrast, Tb-Sp was significantly increased in response to OVX compared to the sham group (p < 0.05). Higher dosage of CLJE treatment (300 or 600 mg/kg/day) significantly improved the microarchitecture deterioration mentioned above (p < 0.05), while E2 also reversed these parameters to the similar degree as that in the HCLJE group (p > 0.05). High incidence, serious complications, financial burden and dramatically decreased living quality, characterize the severity of osteoporosis in humans. Despite the pharmacological and clinical advantages of HRT as a widely accepted therapeutic strategy for osteoporosis, serious side effects of long-term application have also been reported. Therefore, the development of new preventive and therapeutic drugs for osteoporosis is urgently needed. In recent decades, Chinese medicinal herbals have been extensively investigated for their pharmacological effects related to bone protection. Biochemical profile of rat serum and urine Bone remodeling is the biologic process that mediates changes in the traits that influence bone strength [1]. Any reasons such as menopause will disturb the balance between formation and resorption and cause bone mass loss [22]. Therefore, we used OVX rats as animal model for human osteoporosis in vivo experiments. It has been reported that statistically significant bone loss can be seen after 30 days [23], so treatment was initiated 4 weeks after OVX. Consistent with other studies, OVX caused significantly higher body weights in our present study, which may be attributed to fat deposition caused by the lack of estrogen [24]. Previous studies suggest that estrogen plays an important role in stimulating the differentiation of progenitor cells through the osteoblast lineage but not the adipocyte lineage [25]. Data presented in Table 4 show the effects of CLJE on biochemical parameters in the serum and urine of OVX rats. Serum U-Ca/Cr, U-P/Cr, ALP, and OC levels were significantly increased in the OVX group compared to the sham group (p < 0.05). All three CLJE doses significantly decreased the U-Ca/Cr and ALP levels (p < 0.05) in a dose-dependent manner. Higher dosage of CLJE treatment (300 or 600 mg/kg/day) decreased the U-P/Cr and OC levels (p < 0.05). E2 administration also reversed these increases which were statistically significant. It is worth noting that the reductions in U-P/Cr, ALP, and OC in the H-CLJE group were similar to those observed for E2 (all p > 0.05). Trop J Pharm Res, August 2017; 16(8): 1884 Li & Mu Contribution of Authors Decreased BMD is one of the major factors jeopardizing bone strength, resulting in increased susceptibility to fractures [26]. Thus, BMD measurement can best predict fracture risk [27]. The results in the present study showed that OVX reduced BMD in the right femurs and L4 vertebrae, which are rich in trabecular bone, while treatment with high-dose dose-dependently prevented the decreases in BMD. Although BMD is among the strongest predictors of facture resistance, both empirical observations and theoretical analyses show that the biomechanical properties of bone and trabecular microarchitecture influence trabecular bone strength as well [28-30]. Three-point bending tests of the left femurs in our study indicated that the higher crocin doses (300 or 600 mg/kg/day) prevented the OVX-induced tendency toward decreased biomechanical parameters. Moreover, measurements of structural parameters using micro-CT also showed that treatment with CLJE effectively restored the trabecular micro architectural properties compared to the OVX group. In addition, measurement of bone markers plays a role in osteoporosis diagnosis and treatment. Bone mass loss, as evidenced by enhanced levels of ALP, OC, U-Ca/Cr, and U-P/Cr, indicated upregulation of bone turnover by OVX. The bone turnover markers above were dose-dependently reversed by CLJE, indicating a reduction in bone turnover rate after treatment of CLJE. The results suggest that CLJE’s potential effectiveness in treating osteoporosis in menopausal women. CONCLUSION The findings of this study show that CLJE, at high doses over a 16-week period, prevents OVX-induced osteoporosis in rats. Thus, CLJE has the potential to be further developed as a natural alternative for postmenopausal osteoporosis management in future. The authors declare that this work was done by the authors named in this article and all liabilities pertaining to claims relating to the content of this article will be borne by them. Open Access This is an Open Access article that uses a funding model which does not charge readers or their institutions for access and distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/ 4.0) and the Budapest Open Access Initiative (http://www.budapestopenaccessinitiative.org/rea d), which permit unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. REFERENCES 1. Genant HK, Cooper C, Poor G, Reid I. Interim report and recommendations of the World Health Organization task-force for osteoporosis. Osteoporos Int. 1999; 10: 259–264. 2. 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Clinical use of bone densitometry: scientific review. J Amer Med Asso 2002; 288: 1889-1897. 28. Garnero P, Vergnaud P, Hoyle N. Evaluation of a fully automated serum assay for total N-terminal propeptide of type I collagen in postmenopausal osteoporosis. Clin Chem. 2008; 54: 188–96. 29. Keaveny TM, Morgan EF, Niebur GL. Biomechanics of trabecular bone. Anua Rev Biomed Eng. 2001; 3: 307333. Directive 2010/63/EU on the protection of animals used 30. Delmas P, Eastell R, Garnero P, Seibel M. The use of for scientific purposes [cited 2013 Jan 16]. Available biochemical markers of bone turnover in osteoporosis. from: Osteoporos Int. 2000; 11: S2–S17. http://ec.europa.eu/environment/chemicals/lab_animals/l egislation_en.htm. Trop J Pharm Res, August 2017; 16(8): 1886
https://openalex.org/W4361264138	https://figshare.com/articles/journal_contribution/Supplementary_Table_3_from_Methylation_Silencing_of_Transforming_Growth_Factor-_Receptor_Type_II_in_Rat_Prostate_Cancers/22370813/1/files/39815885.pdf	English	null	Supplementary Table 5 from Methylation Silencing of Transforming Growth Factor-β Receptor Type II in Rat Prostate Cancers	null	2,023	cc-by	116	Supplementary Table 3 Numbers of up-regulated genes by 5-aza-dC treatment Supplementary Table 3 Numbers of up-regulated genes by 5-aza-dC treatment PLS10 PLS20 PLS30 ≥ 4-fold (probe sets) 341 126 132 ≥ 8-fold (probe sets) 192 71 67 ≥ 16-fold (probe sets) 69 33 24 ≥ 16-fold (genes and ESTs) 69 32 22 ≥ 16-fold genes and ESTs 69 32 22 ESTs 22 19 12 Annotated genes 47 13 10 Named genes 47 13 10 ≥ 500 bp promoter CGI (Takai and Jones, strict) 8 1 0 ≥ 300 bp promoter CGI (moderate) 2 2 1 ≥ 200 bp promoter CGI (Gardiner-Garden, relaxed) 8 3 1 Without promoter CGI 27 6 6 Location unknown 2 1 2
https://openalex.org/W3195598287	http://jumdc.com/index.php/jumdc/article/download/549/489	English	null	VARIETY OF DRUGS SOLD WITHOUT PRESCRIPTION FOR COMMONLY PRESENTING COMPLAINTS IN RURAL AND URBAN PHARMACIES OF FAISALABAD DISTRICT	Journal of University Medical & Dental College	2,021	cc-by	4,010	VARIETY OF DRUGS SOLD WITHOUT PRESCRIPTION FOR COMMONLY PRESENTING COMPLAINTS IN RURAL AND URBAN PHARMACIES OF FAISALABAD DISTRICT Muhammad Hamza Ranaa, Muhammad Husnaina, Muhammad Hamza Iqbala, Noor-i-Kiran Naeemb, Muhammad Usmanc, Yasir Yaqoobd 4th Year MBBS student, Aziz Fatima Medical and Dental College, Faisalabad. a4th Year MBBS student, Aziz Fatima Medical and Dental College, Faisalabad. bAssistant Professor, Department of Medical Education, Aziz Fatima Medical and Dental College, Faisalabad. bAssistant Professor, Department of Medical Education, Aziz Fatima Medical and Dental College, Faisalabad. cAssociate Professor, Department of Pathology, Aziz Fatimah Medical and Dental College, Faisalabad. dSenior Registrar, District Headquarter (DHQ) Hospital, Faisalabad. ABSTRACT: BACKGROUND & OBJECTIVE: Pharmacies play an important role in provision of health care to the community. The objective of the study was to explore the reasons for a variety of drugs sold without prescription for commonly presenting complaints in Rural and Urban Pharmacies of Faisalabad District. METHODOLOGY: This explanatory sequential mixed method design involved workers from twenty- five pharmacies from urban and rural areas of Faisalabad from February to July 2020. After obtaining informed consent, fifty pharmacy workers filled a pre-designed questionnaire (followed by twenty- five semi-structured, individual, face-to-face interviews. Quantitative data was analyzed via SPSS software and transcribed interviews were organized manually for data analysis. RESULTS: Response rate was 76.2%. As reported by the pharmacists,40% and 90% of urban and rural population respectively came to pharmacies for over the counter drugs. Fifty percent belonged to middle class among urban and 70% belonged to rural population. Data analysis led to formation of 36 codes, 6 sub themes and 3 themes. Out of the four reasons quoted by the pharmacists (time constraints, lack of basic facility locally, financial constraints, and myths/fear of going to doctor), there was a statistically significant difference for rural population going directly to pharmacies because of financial constraints. BACKGROUND & OBJECTIVE: Pharmacies play an important role in provision of health care to the community. The objective of the study was to explore the reasons for a variety of drugs sold without prescription for commonly presenting complaints in Rural and Urban Pharmacies of Faisalabad District. METHODOLOGY: This explanatory sequential mixed method design involved workers from twenty- five pharmacies from urban and rural areas of Faisalabad from February to July 2020. After obtaining informed consent, fifty pharmacy workers filled a pre-designed questionnaire (followed by twenty- five semi-structured, individual, face-to-face interviews. Quantitative data was analyzed via SPSS software and transcribed interviews were organized manually for data analysis. RESULTS: Response rate was 76.2%. As reported by the pharmacists,40% and 90% of urban and rural population respectively came to pharmacies for over the counter drugs. Fifty percent belonged to middle class among urban and 70% belonged to rural population. Data analysis led to formation of 36 codes, 6 sub themes and 3 themes. Original Article Original Article INTRODUCTION: pharmaceutical care, the only void in the policy is the shortage of pharmacists and concern of objection in the separation of medical practitioners[13,14]. The African nation also faces scarce availability of pharmacists, with a total of only 619 pharmacists serving the mass population of 2.9 million[15]. World Health Organization (WHO) defines health as a state of complete physical, mental and social well-being and not merely disease or infirmity[1]. Studies have revealed that the multidisciplinary approach and expertise are key factors for achieving ultimate population health goals[2]. In order to achieve the aim of health for all, it is optimum for different health professionals to work in collaboration for fulfilling the health needs of the patient[3]. Over the last few decades, pharmacies are have been evolving to promote pharmaceutical care and standard provision of care for patients[4]. In essence, the concept of pharmaceutical care has revolutionized and enabled accountability in inpatient care to achieve optimum results with drug therapy[5]. Pharmacists are playing a significant role in dispensing better drugs to provide provisional health care services, especially in developing countries[6]. Corresponding Author: Dr. Noor-i-Kiran Naeem Assistant Professor Department of Medical Education, Aziz Fatima Medical and Dental College Faisalabad Email: noorikiran@yahoo.com In Pakistan, more than 8100 pharmacists available, with 2836 working in the public sector and 5023 working in private settings. The total number of pharmacists in Pakistan, 55% are engaged in the production of pharmaceuticals – 15% of them working at the federal and provincial drug control authority and hospital pharmacy level – with another 15% in sales and marketing of medicines, 10% in community pharmacy, and the rest 5% in teaching and research[16].In contrast to the developed countries, there is no proper recognition and drug regulatory authority in Pakistan. The reasons for these instabilities include the lack of pharmacies and pharmacists in public health services, which leads to the lack of community-pharmacist interaction[17]. Over the past few years, WHO has played a vital role in encouraging pharmacists globally[7].Moreover, WHO has also ensured and emphasized on the quality assurance and constructive implication of drugs[8].The international pharmaceutical federation and WHO have developed the "Seven-star pharmacists" guide. This guide focuses on better decision making, caregiving, active communication, life long learning and good managing skills, posing good leadership qualities with the ability to be a teacher and researcher[9]. ABSTRACT: Out of the four reasons quoted by the pharmacists (time constraints, lack of basic facility locally, financial constraints, and myths/fear of going to doctor), there was a statistically significant difference for rural population going directly to pharmacies because of financial constraints. i CONCLUSION: Lack of education and financial constraints are the leading influential factors for people taking over-the-counter drugs in both rural and urban population, with time constraints being at the top list among urban population. Robust policies and public health care programs can lead to public awareness at large and can help in creating an environment of health care provision with MINIMAL RISKS. KEYWORDS: Over-the-counter drugs, Pharmacy, Community, Urban, Rural. S: Over-the-counter drugs, Pharmacy, Community, Urban, Rural. https://doi.org/10.37723/jumdc.v12i3.549 Rana MH, Husnain M, Iqbal MH, Naeem NK, Usman M, Yaqoob Y. Variety of Drugs sold without prescription for commonly presenting complaints in rural and urban pharmacies of Faisalabad district. Journal of University Medical & Dental College. 2021;12(3):204-210.https://doi.org/10.37723/ jumdc.v12i3.549 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. JUMDC Vol. 12, Issue 3, July–September, 2021 204 Rana MH, Husnain M, Iqbal MH, et al., DRUGS SOLD WITHOUT PRESCRIPTION IN PHARMACIES DRUGS SOLD WITHOUT PRESCRIPTION IN PHARMACIES INTRODUCTION: With the rising interest and contribution to the pharmacy profession, the World Health Organization suggests the ratio of one drug for each 2000 population to provide better health care services. y p Lack of updated reforms have led to a gap where the patients have been reportedly going directly to the pharmacists to get several over the counter drugs as well as other drugs for various reasons instead of reporting to a physician first for self-medication [18]. Over the counter drugs include traditional preparations deregulated from the previous state of "Prescription drugs," also named On the counter drugs. Policies on dispensing and consumption of over the counter drugs vary globally. In Europen nations, only pharmacies distribute over the counter drugs, but in the United stated, over-the-counter drugs are sold on general retail outlets[19]. In Pakistan, a significant surge in the consumption of over-the- counter drug sales has raised alarming concerns to the local governing bodies[20]. The profession of pharmacy and pharmacists varies significantly different from one country to another. Each country posses its own risk and barriers for the development of better pharmaceutical care facilities such as qualified pharmacists or no implication of pharmaceutical health policy[10].One of the most promising developing economically growing countries, Malaysia, also enlist an acute shortage of pharmacists with a ratio of only 1:6207[11,12]. There is no such distinction between a doctor and pharmacist, as the doctor himself prescribes and dispenses medicine to the patient. Although the legislation was raised 20 years ago for g g According to a study, more than 90% of the adults consuming over-the-counter drugs for 205 205 JUMDC Vol. 12, Issue 3, July–September, 2021 Rana MH, Husnain M, Iqbal MH, et al., DRUGS SOLD WITHOUT PRESCRIPTION IN PHARMACIES DRUGS SOLD WITHOUT PRESCRIPTION IN PHARMACIES various ailments were unaware of potential side effects[21]. The aim of this study was to explore the reasons for variety of drugs sold without a prescription for commonly presenting complaints in rural and urban pharmacies of Faisalabad District. various ailments were unaware of potential side effects[21]. The aim of this study was to explore the reasons for variety of drugs sold without a prescription for commonly presenting complaints in rural and urban pharmacies of Faisalabad District. to miss any information. Data analysis was done through manual coding with the formation of open codes learning to a generation of sub-themes and themes. INTRODUCTION: The credibility of the study was ensured by doing member checking, intercoder reliability, and using the technique of reframing questions. Transferability, dependability, and conformability were done for quality assurance of the study.Test of significance was applied to the two groups of pharmacy locations (urban and rural) for the rated reasons. JUMDC Vol. 12, Issue 3, July–September, 2021 METHODOLOGY: This study was conducted using an explanatory sequential mixed method design in a two-phase scheme from February to July 2020.In the first phase, we gathered quantitative data and analyzed the findings,and in the second, we used the results to design(or build onto) the qualitative part. The general purpose of this project was to collect qualitative data that would help explain in more detail the initial quantitative results and explore them in more depth. Ethical approval was taken vide reference number dme/889-20 dated 17th February 2020 from Aziz Fatimah Medical and Dental College, Faisalabad. RESULTS: A t t l f A total of sixty-five participants were invited for filling the questionnaire, but fifty returned, giving a response rate of 76.9%. Descriptive statistics were used to analyze demographic variables and determine the reasons for patients getting over-the-counter drugs from pharmacies and not attending a clinic as perceived by the participants. The mean score was calculated for each of the categories of reasons. Table-I shows participants' characteristics in the study with equal representation of rural and urban pharmacies. All workers were male in the study. The proforma inquired about demographic details of pharmacies, drugs sold over the counter, common symptoms with which the people presented to these pharmacies, and the reason behind going there. The participants were asked to rank the above reasons according to importance on a five-point Likert scale. Continuous data were represented by means and standard deviation, and categorical data were represented by frequencies and proportions. A student t-test of significance was applied, taking a p<0.005 significant between the stated reasons in urban and rural pharmacies.i Characteristic Frequency Percentage Age group 25-40 20 40% 40-60 30 60% Location of Pharmacies Urban 27 54% Rural 23 46% Table-I: Demographic data of Participants. p This was followed by twenty-five individual face to face interviews to explore in depth the reasons and factors behind patients taking over the counter drugs from pharmacies in bother urban- rural settings. Out of these, fifteen were from urban pharmacies, and ten were conducted from rural pharmacies. Following the guidelines of a qualitative study, the numbers of interviews were done until the data saturation was observed. Table-II shows the reported percentages of patients coming to pharmacies to for purchasing drugs as perceived by the workers and owners of the pharmacies. To maintain the quality of the study, the interviews were recorded on two different devices. The recordings were transcribed completely so as not 206 Rana MH, Husnain M, Iqbal MH, et al., DRUGS SOLD WITHOUT PRESCRIPTION IN PHARMACIES Table-II: Reported percentage of patients coming to Pharmacies. which were quoted by the participants in greater depth. A 36 codes were got leading to 6 sub themes and 3 themes below: which were quoted by the participants in greater depth. Rana MH, Husnain M, Iqbal MH, et al., DRUGS SOLD WITHOUT PRESCRIPTION IN PHARMACIES DRUGS SOLD WITHOUT PRESCRIPTION IN PHARMACIES Rana MH, Husnain M, Iqbal MH, et al., DRUGS SOLD WITHOUT PRESCRIPTION IN PHARMACIES Interviewees had a range of impressions about factors influencing patients to buy drugs directly from the pharmacies; few of the quotations are as followed. Interview R-10 explained financial status as being most common reason which prevented patients attending doctor and then coming to pharmacies to buy his prescribed medicine. R-10 said: "…… mostly they avoid spending extra money on a doctor's visit and prefer coming here directly as they can then drugs directly. This saves money and time". areas are illiterate and practice superstitions. Lack of necessary health care resources in Rural health care settings also creates a significant difference between urban environments. [21] the socioeconomic background was seen to have a significant influence on the mass population visiting pharmacies without a prescription. Most of the people in rural areas visiting directly to the pharmacies without prescription belonged to low socioeconomic backgrounds. This was because of the availability of limited resources and health care facilities in rural settings. They consider this most cost-effective to visit the pharmacy while visiting the doctor first. Pharmacies in rural settings are non-prolific and don't follow the standards to dispense medications [22]. The time constraints factor was also reported by various workers from urban pharmacies as well. Few of them keenly explained about the lack of health facility and local doctor availability in their region. Interviewee U-9, one worker from urban pharmacy narrated: "People do not prefer local doctor in nearby Basic health clinic, they would want either a very Hi-Fi hospital where they can go or they would come directly to us to buy basic medicine." Interviewee R-14 explained,"… we have a local clinic, but the doctor is hardly there, so people do not go there…" The study also evaluates the main reason behind visiting the pharmacies without a prescription as financial constraints. Results reveal that in the rural population (4.24 + 0.51), people tend to visit pharmacies without visiting the doctor and opt for self medications without knowing the potential outcomes of the drugs. Lack of awareness and education forces them not to see the doctor as this will cause them to pay extra charges for the provision of health care. Contrary to that, people living in rural settings also believe and practice various myths and superstitions. Rana MH, Husnain M, Iqbal MH, et al., DRUGS SOLD WITHOUT PRESCRIPTION IN PHARMACIES Another study supports these barriers elaborates that rural people consider sadness and happiness to be the cause of mental disorders and a prevailing or life-threatening disease is due to God's punishment[23]. However, all these superstitons are baseless according to scientific knowledge, as science provides a reason for every happening. Much stress was made by workers from rural pharmacies about the beliefs of people coming to rural pharmacies about doctor's visits. Interviewee R-3 said, "Mostly they think that their case would be worsened if the patient is taken to a doctor, that she would turn a normal delivery case into an operation one…." Last, people preferred going directly to the pharmacies to get drugs and were into the habit of self-medication. DISCUSSION: Th l f h The results of this study show that the frequency of people visiting health care pharmacies in urban pharmacies (F= 27, 54%) is slightly more than in rural areas (F=23,46%). People with age groups ranging from 40 to 60 years visit more frequently than those of the age group 25-40. Similarly, Table II illustrates the percentage of people visiting pharmacies with prescription (60%) is more in an urban area while in rural settings (10%). Over 90 percent of people in rural pharmacies visit without prescription. This shows the concern of people living in urban areas seeking health care services. People living in urban areas are more literate, and the urban population is more affluent [19]. However, a significant proportion of people living in rural The scarcity of primary health care facilities and doctors available is one of the predisposing factors compelling the residents of rural areas to move towards pharmacies. Not much attention is paid for the provision of better health care facilities. Due to the high population and inadequate resources available, there is a lack of necessary health care facilities in rural areas[5]. RESULTS: A t t l f A 36 codes were got leading to 6 sub themes and 3 themes below: Characteristic Urban Rural On basis of doctor's prescription With Prescription 60% 10% Without Prescription 40% 90% Gender Male 55% 54% Female 45% 46% Socioeconomic status Lower 25% 70% Middle 50% 15% Upper 25% 15% 1. Personal Factors a. Self-medication b. Myths and fears about going to a doctor c. Time constraints 2. External Factors a. Lack of local health Center b. Lack of local doctor 3. Financial Factors a. Economic class a. Lack of local health Center b. Lack of local doctor 3. Financial Factors Table-III depicts various subthemes got from a thematic analysis of the interview and workers' rating of those reasons for drug purchase. It can be seen that workers from rural pharmacies ranked "financial constraints" as the topmost influencing factor for requesting drugs without prescription, whereas those from urban pharmacies ranked 'Self-medication" as the topmost reason. There were significant results among the urban and rural data set. During phase two, twenty participants underwent individual, face-to-face interviews (15 from urban pharmacies and 10 from rural pharmacies). The aim of interviews was to explore the reasons Table-III: Reasons for requesting pharmacies for drug purchases without doctors' prescriptions Characteristic Urban Pharmacy (n=27) Rural Pharmacy (n=23) p-value Self-Medication 4.51 + 0.49 4.05 + 0.53 0.003 Lack of basic facility locally 3.38 + 0.57 4.46 + 0.552 0.000 Financial Constraints 3.88 + 0.506 4.24 + 0.51 0.016 Myths and fears of going to doctor 3.93 + 0.59 4.31 + 0.62 0.032 Table-IV shows the themes and subthemes found in the study regarding perceived reasons for patients reporting directly to the pharmacies for buying medicines. 207 JUMDC Vol. 12, Issue 3, July–September, 2021 Table-IV: : Breakup of perceived reasons for buying medicine without prescription. Themes Sub Themes No. of Codes (125) Total Urban pharmacy workers Rural Pharmacy workers Personal Factors Self- medication 25 17(68.0) 8(32.0) Myths and fears about going to doctor 16 2(12.5) 14(87.5) Time Constraints 23 15(65.2) 8(34.8) External factors Lack of local health facility 13 5(38.5) 8(61.5) Socioeconomic factors Economic class 25 10(40.0) 15(60.0) eakup of perceived reasons for buying medicine without prescription. e-IV: : Breakup of perceived reasons for buying medicine without presc 207 DISCLOSURE: 9. Malau-Aduli BS, Alele FO, Heggarty P, Reeve C, Teague PA. Key elements of effective postgraduate GP educational environments: a mixed methods study. BMJ open. 2021;11(2):e041110. None. REFERENCES. 1. McCartney G, Popham F, McMaster R, Cumbers A. Defining health and health inequalities. Public health. 2019;172:22-30. Doi:10.1016/j.puhe.2019.03.023 10. Nisa ZU, Zafar A, Sher F. Assessment of knowledge, attitude and practice of adverse drug reaction reporting among healthcare professionals in secondary and tertiary hospitals in the capital of Pakistan. Saudi Pharmaceutical Journal. 2018;26(4):453- 461. Doi:10.1016/j.jsps.2018.02.014 2. Dalton K, Byrne S. Role of the pharmacist in reducing healthcare costs: current insights. Integrated Pharmacy Research & Practice. 2017;6:37-46. Doi: 10.2147/IPRP.S108047 11. Javeed A, Mahmood KT. Community pharmacy practice in Pakistan: from past to present-a review. Journal of Pharmaceutical Sciences and Research. 2012;4(2):1703-1708. 3. Okai GA, Abekah-Nkrumah G, Asuming PO. Determinants of community pharmacy utilization in Ghana. Journal of Pharmaceutical Health Services Research. 2020;11(2):159- 165. Doi:10.1111/jphs.12338 3. Okai GA, Abekah-Nkrumah G, Asuming PO. Determinants of community pharmacy utilization in Ghana. Journal of Pharmaceutical Health Services Research. 2020;11(2):159- 165. Doi:10.1111/jphs.12338 12. 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Clinical Pharmacy in Pakistan. Pharmaceutical Drug Regulatory Affairs 209 JUMDC Vol. CONCLUSION: Lack of education and financial constraints are the leading influential factors for people taking over the counter drugs in both rural and urban population with time constraints being at the top list among urban population. Robust poilicies and JUMDC Vol. 12, Issue 3, July–September, 2021 208 CONFLICT OF INTEREST: None. CONFLICT OF INTEREST: None. CONFLICT OF INTEREST: None. GRANT SUPPORT & FINANCIAL DRUGS SOLD WITHOUT PRESCRIPTION IN PHARMACIES DRUGS SOLD WITHOUT PRESCRIPTION IN PHARMACIES Rana MH, Husnain M, Iqbal MH, et al., public health care programs can lead to public awareness at large and can help in creating a an environment of health care provision with minimal risks. 8. Asayut N, Sookaneknun P, Chaiyasong S, Saramunee K. Outcomes, costs and stakeholders' perspectives associated with the incorporation of community pharmacy services into the National Health Insurance System in Thailand: a systematic review. International Journal of Pharmacy Practice. 2018;26(1):16-27. 8. Asayut N, Sookaneknun P, Chaiyasong S, Saramunee K. Outcomes, costs and stakeholders' perspectives associated with the incorporation of community pharmacy services into the National Health Insurance System in Thailand: a systematic review. International Journal of Pharmacy Practice. 2018;26(1):16-27. ACKNOWLEDGEMENT: None. DRUGS SOLD WITHOUT PRESCRIPTION IN PHARMACIES DRUGS SOLD WITHOUT PRESCRIPTION IN PHARMACIES Journal. 2019;2(1): 000111. Journal. 2019;2(1): 000111. JUMDC Vol. 12, Issue 3, July–September, 2021 REFERENCES. 12, Issue 3, July–September, 2021 209 Authors Contribution: 17. Khan AA. 15th International Pharmacy Conference and Exhibition. Lahore Pakistan Pharmacy Association. 2009. Muhammad Hamza Rana: Idea conception, data collection, data analysis, and manuscript Writing. 18. Ahmad A, Patel I, Mohanta GP, Balkrishnan R. Evaluation of self medication practices in rural area of town Sahaswan at Northern India. Annals of Medical and Health Sciences Research. 2014;4(8):73-78.Doi: 10.4103/2141-9248.138012 Muhammad Husnain: Data collection, design of the work and acquisition. Muhammad Hamza Iqbal: Correction and revision Muhammad Hamza Iqbal: Correction and revision 19. Marathe PA, Kamat SK, Tripathi RK, Raut SB, Khatri NP. Over-the-counter medicines: Global perspective and Indian scenario. Journal of Postgraduate Medicine. 2020;66(1):28-34. Doi :10.4103/jpgm.JPGM_381_19 Noor-i-Kiran Naeem: Idea conception, data analysis and manuscript Writing.i Noor-i-Kiran Naeem: Idea conception, data analysis and manuscript Writing.i Muhammad Usman: Data analysis, final approval of the manuscript to be published. Muhammad Usman: Data analysis, final approval of the manuscript to be published. Yasir Yaqoob: Data compilation and critical analysis. 20. Maqbool S. Ban on over-the-counter sale of antibiotics. https://www.thenews.com.pk/ print/464111-ban-on-over-the-counter-sale- of-antibiotics. Accessed September 18, 2020. Submitted for Publication: 26-01-2021 Accepted After revision : 15-06-2021 21. Brandão GR, Teixeira L, Araújo L, Paúl C, Ribeiro O. Self-medication in older european adults: Prevalence and predictive factors. Archives of Gerontology and Geriatrics. 2020;91:104189. Doi:10.1016/j.archger. 2020.104189 22. Burns A, Goodlet KJ, Chapman A, Roberts EP. A case report of self-medication with over-the-counter fish antibiotic: Implications for pharmacists. Journal of the American Pharmacists Association. 2020;60(4):e121- 3. Doi:10.1016/j.japh.2019.12.020 23. Nations T. 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https://openalex.org/W4389061560	https://maut.journals.ekb.eg/article_327097_0658f38e76334daec51bdaa49e9ca681.pdf	Arabic	null	"إعلانات التوعية ودورها في تنمية المجتمع. "	Mağallaẗ Al-Funūn wa Al-ʿulūm Al-Ṭaṭbīqiyyaẗ	2,023	cc-by	8,834	المجلد العاشر- العدد ال رابع– أكتوبر2023 من هنا جاء البحث الذي يهدف إلى إ براز دور إعالنات التوعية ودورها في تنمية المجتمع وتغيير ثقافته نحو األفضل، وذلك من خالل رصد وتحليل أنواع إعالنات التوعية المختلفة، ودور إعالنات التوعية في المجتمع ومن ثم إجراء إستبيان بأخذ آراء مجموعة من متلقي اإلعالنات .للتوصل من خاللها إلى النتائج وقد ت:وصل البحث إلى - .قبول الفرض األول وهو إعالنات التوعية لها تأثير على المتلقين المستهدفين - قبول الفرض الثاني وهو إعالنات التوعية تعمل على إقناع المتلقين بأن دور اإلعالن يتعدى جذب اإلنتباه أو تسهيل فهم موضوع لكنه يجعل المتلقين يتبنوا سلوكيات وإتجاهات هادفه رغبةً . المجلد العاشر- العدد ال رابع– أكتوبر2023 منهم في التقدم أاإ أ ع يا "Awareness Advertisements and their Role in Community Development" محمد محمود أحمد شحاته هبه عبد المهيمن عوض أستاذ تصميم األغلفة المتفرغ أستاذ التصميم المساعد ً ورئيس قسم اإلعالن سابقا ورئيس قسم اإلعالن كلية الفنو ن التطبيقية- .جامعة حلوان كلية الفنون التطبيقية– جامعة .دمياط نهلة محمد رضا عبد الحكم قطامش باحثة ماجستير قسم اإلعالن– كلية الفنون التطبيقية– .جامعة دمياط "Awareness Advertisements and their Role in Community Development" محمد محمود أحمد شحاته هبه عبد المهيمن عوض أستاذ تصميم األغلفة المتفرغ أستاذ التصميم المساعد ً ورئيس قسم اإلعالن سابقا ورئيس قسم اإلعالن كلية الفنو ن التطبيقية- .جامعة حلوان كلية الفنون التطبيقية– جامعة .دمياط نهلة محمد رضا عبد الحكم قطامش باحثة ماجستير قسم اإلعالن– كلية الفنون التطبيقية– .جامعة دمياط Awareness Advertisements and their Role in Community Development :ملخص البحث ،تعد إعالنات التوعية أحد الوسائل اإلعالنية الهامة في المجتمع لما لها من دور فعال وتأثير كبير في التوعية المجتمعية حيث تستخدم إعالنات التوعية لخدمة قضايا أفراد المجتمع بمختلف الفئات ونشر التوعية ودعم اإليجابيات التي تعزز سير المجتمع نحو التطور، وتوفير مادة إعالنية إبداعية قادرة على توجيه أفراد المجتمع .نحو األفضل فهي إعالنات تهدف إلى إقناع المتلقين بالمشاركة أو الحذر أو تغيير النمط المعيشي لما فيه من وضع أفضل لهم، فنجد أن إعالنات التوعية تجعل األفراد قادرون على م عرفة األمور والقضايا والمعلومات وإدراكها بطريقة جيدة من جوانبها المتعددة ،بحيث تخلق لديهم إحساسا ً بأهمية هذه القضايا واألمور مما تجعلهم قادرون على إتخاذ قرار بشأنها أو سلوك تجاهها ومع تطور القضايا التي تمس المجتمع تم اإلحتياج إلى التوجه إلعالنات التوعية و تجديدها، حيث أن إعالنات التوعية تحتاج إلى اإلبداع واإلبتكار في تصميمها وتنفيذها وأن يعتمد على أسس فنية جيدة وذلك ليحقق اإلعالن الهدف المرجو منه بفعالية ونجاح ويحقق أكبر قدر من اإلستفادة والوصول إلى المتلقين المستهدفين؛ وتنقسم إعالنات التوعية إلى عدة أنواع هي إعالنات توعية تبعا ً للنطاق الجغرافي (إعالنات توعية محلية وقومية ودولية) وكذلك إعالنات توعية حسب الوسيلة المستخدمة في اإلعالن (إعالنات توعية مقروءة كالصحف والمجالت وإعالنات الطرق وإعالنات مسموعة ومرئية مثل الراديو والتلفزيون والسينما واإلنترنت) كما تنقس م إعالنات التوعية حسب القضية المطروحة (إعالنات :توعية إجتماعية وبيئية وصحية وسياسية وتعليمية)، حيث ظهرت مشكلة البحث والتي تتلخص في السؤال التالي هل تحقق إعالنات التوعية التغطية المطلوبة والهدف المنشود من الرسالة اإلعالنية؟ . المجلد العاشر- العدد ال رابع– أكتوبر2023 المجلد العاشر- العدد ال رابع– أكتوبر2023 المجلد العاشر- العدد ال رابع– أكتوبر2023 " .إعالنات التوعية ودورها في تنمية المجتمع" "Awareness Advertisements and their Role in Community Development" محمد محمود أحمد شحاته هبه عبد المهيمن عوض أستاذ تصميم األغلفة المتفرغ أستاذ التصميم المساعد ً ورئيس قسم اإلعالن سابقا ورئيس قسم اإلعالن كلية الفنو ن التطبيقية- .جامعة حلوان كلية الفنون التطبيقية– جامعة .دمياط نهلة محمد رضا عبد الحكم قطامش باحثة ماجستير قسم اإلعالن– كلية الفنون التطبيقية– .جامعة دمياط :ملخص البحث ،تعد إعالنات التوعية أحد الوسائل اإلعالنية الهامة في المجتمع لما لها من دور فعال وتأثير كبير في التوعية المجتمعية حيث تستخدم إعالنات التوعية لخدمة قضايا أفراد المجتمع بمختلف الفئات ونشر التوعية ودعم اإليجابيات التي تعزز سير المجتمع نحو التطور، وتوفير مادة إعالنية إبداعية قادرة على توجيه أفراد المجتمع .نحو األفضل فهي إعالنات تهدف إلى إقناع المتلقين بالمشاركة أو الحذر أو تغيير النمط المعيشي لما فيه من وضع أفضل لهم، فنجد أن إعالنات التوعية تجعل األفراد قادرون على م عرفة األمور والقضايا والمعلومات وإدراكها بطريقة جيدة من جوانبها المتعددة ،بحيث تخلق لديهم إحساسا ً بأهمية هذه القضايا واألمور مما تجعلهم قادرون على إتخاذ قرار بشأنها أو سلوك تجاهها ومع تطور القضايا التي تمس المجتمع تم اإلحتياج إلى التوجه إلعالنات التوعية و تجديدها، حيث أن إعالنات التوعية تحتاج إلى اإلبداع واإلبتكار في تصميمها وتنفيذها وأن يعتمد على أسس فنية جيدة وذلك ليحقق اإلعالن الهدف المرجو منه بفعالية ونجاح ويحقق أكبر قدر من اإلستفادة والوصول إلى المتلقين المستهدفين؛ وتنقسم إعالنات التوعية إلى عدة أنواع هي إعالنات توعية تبعا ً للنطاق الجغرافي (إعالنات توعية محلية وقومية ودولية) وكذلك إعالنات توعية حسب الوسيلة المستخدمة في اإلعالن (إعالنات توعية مقروءة كالصحف والمجالت وإعالنات الطرق وإعالنات مسموعة ومرئية مثل الراديو والتلفزيون والسينما واإلنترنت) كما تنقس م إعالنات التوعية حسب القضية المطروحة (إعالنات :توعية إجتماعية وبيئية وصحية وسياسية وتعليمية)، حيث ظهرت مشكلة البحث والتي تتلخص في السؤال التالي هل تحقق إعالنات التوعية التغطية المطلوبة والهدف المنشود من الرسالة اإلعالنية؟ . Journal of Applied Art and Science - International Periodical Scientific Peer Reviewed - Issued By Faculty of Applied Arts - Damietta Univ. - Egypt Journal of Applied Art and Science - International Periodical Scientific Peer Reviewed - Issued By Faculty of Applied Arts - Damietta Univ. - Egypt 255 (ISSN 2537-1061) (Print) (ISSN 2537 107X) (Online) المجلد العاشر- العدد ال رابع– أكتوبر2023 من هنا جاء البحث الذي يهدف إلى إ براز دور إعالنات التوعية ودورها في تنمية المجتمع وتغيير ثقافته نحو األفضل، وذلك من خالل رصد وتحليل أنواع إعالنات التوعية المختلفة، ودور إعالنات التوعية في المجتمع ومن ثم إجراء إستبيان بأخذ آراء مجموعة من متلقي اإلعالنات .للتوصل من خاللها إلى النتائج - .قبول الفرض األول وهو إعالنات التوعية لها تأثير على المتلقين المستهدفين - قبول الفرض الثاني وهو إعالنات التوعية تعمل على إقناع المتلقين بأن دور اإلعالن يتعدى جذب اإلنتباه أو تسهيل فهم موضوع لكنه يجعل المتلقين يتبنوا سلوكيات وإتجاهات هادفه رغبةً . منهم في التقدم - .قبول الفرض الثالث وهو أن اإلبتكار في تصميم إعالنات التوعية له تأثير إيجابي على المتلقين المستهدفين :كلمات مفتاحية .إعالنات التوعية، دورإعالنات التوعية في تنمية المجتمع 255 (ISSN 2537-107X) (Online) المجلد العاشر- العدد ال رابع– أكتوبر2023 المجلد العاشر- العدد ال رابع– أكتوبر2023 ف3 اإلبتكار في تصميم إعالنات التوعية له تأثير إيجابي على المتلقين المستهدفين ويساعدهم على الشعور باإلنجذاب تجاه اإلعالن ويساعدهم على سهولة تذكره مر ة .آخرى :امقدمة :منهجية البحث .يتبع البحث المنهج الوصفي تم التحليلي :إعالنات التوعية اإلعالن الذي يستهدف التنمية اإلجتماعية في كافة المجاالت والذي يتم تخطيطه ضمن حمله مكثفه من جانب هيئات او مؤسسات ال تهدف إلى تحقيق الربح و لكن تهدف إلى اإلرتقاء بالمجتمع وتحسين ظرو فه وتنميته و كذلك رفع مستوى الوعي لدى األفراد2 . ويتميز عن باقي األنواع بأنه أكثرها موضوعية وأنه يقدم للمشارك في اإلتصال الحقائق والمعلومات واألخبار حول الموضوعات واألمور الهامة واألحداث التي ترتبط به بهدف تكوين أفكار صحيحة أو تعديل أفكار قديمة خاطئة وهذا ا لنوع من اإلعالنات يخضع غالبا ً إلشراف .الحكومات3 خ إن مهمة اإلعالن التوعوي هي التأثير على التفكير وجذب األشخاص وترك إنطباع إيجابي وتغيير سلوك المتلقي1 . Journal of Applied Art and Science - International Periodical Scientific Peer Reviewed - Issued By Faculty of Applied Arts - Damietta Univ. - Egypt المجلد العاشر- العدد ال رابع– أكتوبر2023 حيث أن الهدف األساسي منه هو تغيير إتجاه المتلقي ثم تغيير سلوكه وذلك عن طريق توظيف كافة اإلست راتيجيات اإلقناعية الالزمة للتأثير في المتلقي وبناء صورة ذهنية جيدة في عقل المتلقي المشكالت والقضايا المختلفة التي .تواجه المجتمع :مشكلة البحث :سؤالين وهما يمكن تحديد مشكلة البحث ف :أنواع إعالنات التوعية : أوال ً: إعالنات التوعية تبعا للنطاق الجغرافي تنقسم إعالنات التوعية وفقا للنطاق الجغرافي إلى ثالثة : أنواع رئيسة لإلعالن هما4 اإ ع 1 - اإلعالن المحلي : ينحصر هذا النوع من اإلعالن على منطقة جغرافية محدده ويعتمد هذ النوع على إستخدام وسائل نشر محلية كاإلذاعة المحلية و الصحف و المجالت و الملصقات وكذلك النشرات و المطويات التي يتم توزيعها داخل هذه المنطقه المحدده و يعد هذا متخصصا ً بتوجيه رساله إعالنية معينه إ لى منطقه محدده مهتمة بقضية معينه5 ، مثل حملة " 2 كفاية" التي أطلقتها وزارة التضامن اإلجتماعي للتوعية بخطورة الزيادة السكانية حيث تم توجيه هذه الحملة إلى محافظات الصعيد األكثر فقرا ً واألكثر إنجابا ً كما في ( شكل1 .) ي - هل تنجح إعالنات التوعية في التأثير اإليجابي .على المتلقين المستهدفين؟ - هل تحقق إعالنات التوعية التغطية المطلوبة .والهدف المنشود من الرسالة اإلعالنية؟ :هدف البحث إبراز دور إعالنات التوعية ودورها الكبير في التأثير على .المتلقين المستهدفين وتغيير ثقافتهم نحو األفضل :أهمية البحث تكمن أهمية البحث في كونه يتطرق لنوع مهم من اإلعالن وهو إعالنات التوعية ويوضح الب ح ث أنواعه وأشكاله ويمكن من خالل هذا البحث التوصل إلى مدى تأثير .إعالنات التوعية في المتلقي ( ) ( شكل1 " ) يوضح إعالن2 "كفاية. 26 :فروض البحث ف1 إعالنات التوعية لها تأثير على المتلقين المستهدفين وتعمل على تغيير ثقافتهم تجاه القضايا والمشكالت .المختلفة ف2 دور اإلعالن يت عدى جذب اإلنتباه أو تسهيل فهم موضوع لكنه يجعل المتلقين يتبنوا سلوكيات وإتجاهات .هادفه رغبة ً منهم في التقدم ( شكل1 " ) يوضح إعالن2 "كفاية. 26 (ISSN 2537-1061) (Print) 256 (ISSN 2537-107X) (Online) المجلد العاشر- العدد ال رابع– أكتوبر2023 المجلد العاشر- العدد ال رابع– أكتوبر2023 "رحلة المخدرات ممكن تبسطك في أولها بس أكيد هضيعك في آخرها" ، "المخدرات رحلتها "صغيره متسافرهاش..أنت أقوى من المخدرات وقد تم اإلستعانه ببعض الشخصيات المؤثرة في الشباب الفترة الماضية للمشاركة في حمالت الت وعية ضد خطر إدمان المخدرات بإعتبار قضية مكافحة تعاطى المخدرات قضية قومية لحماية الشباب من الوقوع فى براثن اإلدمان ( كما في شكل2 ) . Journal of Applied Art and Science - International Periodical Scientific Peer Reviewed - Issued By Faculty of Applied Arts - Damietta Univ. - Egypt المجلد العاشر- العدد ال رابع– أكتوبر2023  توجد ايضا ً وسائل إعالنية آخرى ورقية مطبوعة تستخدم في إعالنات التوعية حيث أنها تتميز بدرجة كبيرة من التنوع في شكلها و حجمها و تظهر هذه المطبوعات في أشكال : مختلفة كاآلتي 1 - .نشرات 2 - .مطويات 3 - .كتيبات 4 - .تقويمات 1 - : النشرات شكل من أشكال اإلعالنات الورقية معدة للتوزيع الواسع وعادة ما يتم نشرها أو توزيعها في أماكن عامة و تعددت أنواع المنشورات من منشورات منسوخة غير مكلفة إلى منشورات دورية ملونة مكلفة12 ، وللنشرات عدة أنواع مختلفه منها النشرات الدورية والغير دورية والنشرات الداخلية والخارجية وكذلك ونشرات والمصورة اإلخبارية النشرات المعلومات العامة والمعلومات الخاصة مثل النشرات المستخدمة في حمالت التوعية القومية ضد مرض شلل األطفال وأهمية التطعيم بالنسبة ( لألطفال كما في شكل4 ) ويمثل الشكل نشرة اعالنية توعوية عن التطعيم ضد مرض شلل . االطفال ( شكل4) يوضح نشرة إعالنية عن الحملة القومية للتطعيم ضد مرض شلل األطفا.ل29 2 - :المطويات عبارة عن فرخ من الورق يتم طيه بطر ق مختلفه .وتعتبر المطويات أحد األشكال التي يصدر بها إعالنات التوعية حيث تتميز المطويات بأنها صغيرة الحجم وإمكانية توزيعها في سهولة ويسر كما أنها وسيلة إعالنية ملموسة قريبة من يد القارئ حيث يتم تنسيق المعلومات والصور والرسوم فيها بشكل منظم13 ( ، كما في شكل5 ) ويمثل مطوية إعال نية عن التوعية المرورية تم .إصدارها من خالل إدارة مرور اإلسكندرية ويجعله يتجه نحو الفكرة أو الموضوع و يتخذ القرار باإلستفادة منه ا9. إ  إعالن: المجلة هي من الوسائل اإلعالنية المطبوعة التي تحظي بإهتمام لدى جمهور معين من القراء حيث يختلف هذا الجمهور بإختالف نوع المجلة وما تحتويه من موضوعات والفئة الموجهة إليها وتحتوي المجالت على العديد من الموضوعات المصورة والتحقيقات واألخبار بهدف تقديم تحليل تفص يلي لكل الموضوعات10 .  إعالن: المجلة هي من الوسائل اإلعالنية المطبوعة التي تحظي بإهتمام لدى جمهور معين من القراء حيث يختلف هذا الجمهور بإختالف نوع المجلة وما تحتويه من موضوعات والفئة الموجهة إليها وتحتوي المجالت على العديد من الموضوعات المصورة والتحقيقات واألخبار بهدف تقديم تحليل تفص يلي لكل الموضوعات10 . Journal of Applied Art and Science - International Periodical Scientific Peer Reviewed - Issued By Faculty of Applied Arts - Damietta Univ. - Egypt 258 (ISSN 2537-1061) (Print) Journal of Applied Art and Science - International Periodical Scientific Peer Reviewed - Issued By Faculty of Applied Arts - Damietta Univ. - Egypt 258 (ISSN 2537-1061) (Print) (ISSN 2537-107X) (Online) المجلد العاشر- العدد ال رابع– أكتوبر2023 1 - : النشرات شكل من أشكال اإلعالنات الورقية معدة للتوزيع الواسع وعادة ما يتم نشرها أو توزيعها في أماكن عامة و تعددت أنواع المنشورات من منشورات منسوخة غير مكلفة إلى منشورات دورية ملونة مكلفة12 ، وللنشرات عدة أنواع مختلفه منها النشرات الدورية والغير دورية والنشرات الداخلية والخارجية وكذلك ونشرات والمصورة اإلخبارية النشرات المعلومات العامة والمعلومات الخاصة مثل النشرات المستخدمة في حمالت التوعية القومية ضد مرض شلل األطفال وأهمية التطعيم بالنسبة ( لألطفال كما في شكل4 ) ويمثل الشكل نشرة اعالنية توعوية عن التطعيم ضد مرض شلل . االطفال المجلد العاشر- العدد ال رابع– أكتوبر2023 2 - اإلعالن: القومي هو اإلعالن الذي يغطي الدولة ككل و يعتمد على إستخدام الوسائل العامة في النشر كالمجالت و الصحف القومية و محطات اإلذاعة و القنوات التي يغطي بثها كافة أرجاء الدولة6 ، والمثال على ذلك الحملة التي أطلقها صندوق مكافحة وعالج اإلدمان التابع لوزارة التضامن ا إلجتماعي لتوعية الشباب بخطورة تعاطي المخدرات تحت شعار ( شكل2 .") يوضح إعالن "أنت أقوى من المخدرات27 ( شكل2 .") يوضح إعالن "أنت أقوى من المخدرات27 3 - : اإلعالن الدولي وهو اإلعالن الذي يغطي أكثر من دولة على مستوى العالم أي الذي يتعدى حدود الدولة األم مصدر اإلعالن ويعتمد على وسائل النشر المختلفة في هذه الدول مثل واإلذاعة والتلفزيون الصحف والمجالت وإعالنات اإلنترنت وتقوم به المنظمات العالمية والدولية7 ، مثل إعالنات التوعية التي تقوم بها منظمة الصحة العالمية للتوعية من خطر إنتشار بعض األمراض واألوبئة والفيروسات ومثال على ذلك حملة التوعية التي قامت بها منظمة الصحة العالمية للتوعية من خطر فيروس كورونا المستجد كما في ( شكل3 .) ( شكل3 .) شكل ( 3 .) يوضح إعالن توعية لتجنب العدوى بفيروس كورونا28 ثانيا ً : إعالنات التوعية تبعا ً للوسيلة المستخدمة في :اإلعالن أ: : إعالنات الوسائل المقروءة كالصحف و المجالت و الملصقات اإلعالنية الداخلية و الكتيبات وإعالنات الطرق واإلعالنات المضيئة وإعالنات وسائل النقل8.  : إعالنات الصحف هي نشاط صحفي مدفوع األجر يلتزم بشروط و قواعد فنية و شكلية ويعرض خصائص الخدمة المراد التوعية والتعريف بها وهو نشاط يؤثر في المتلقي ( ل.) شكل ( 3 .) يوضح إعالن توعية لتجنب العدوى بفيروس كورونا28 اا Journal of Applied Art and Science - International Periodical Scientific Peer Reviewed - Issued By Faculty of Applied Arts - Damietta Univ. - Egypt 257 المجلد العاشر- العدد ال رابع– أكتوبر2023 المجلد العاشر- العدد ال رابع– أكتوبر2023 ويجعله يتجه نحو الفكرة أو الموضوع و يتخذ القرار باإلستفادة منه ا9.  إعالن: المجلة هي من الوسائل اإلعالنية المطبوعة التي تحظي بإهتمام لدى جمهور معين من القراء حيث يختلف هذا الجمهور بإختالف نوع المجلة وما تحتويه من موضوعات والفئة الموجهة إليها وتحتوي المجالت على العديد من الموضوعات المصورة والتحقيقات واألخبار بهدف تقديم تحليل تفص يلي لكل الموضوعات10 .  الملصقات اإلعالنية الداخلية" In door " : يعتبر الملصق اإلعالني وسيلة هامة من وسائل اإلعالن خاصة ً في مجال التوعية لما تتصف به من إمكانية التكرار واإلتصال مع الفئة المستهدفة كما أنه يجمع مؤثرات بصرية مركزة ومختصرة ولكنها ذات تأثير مباشر وذات قدرة على جذب اإلنتباه1 1.  الملصقات اإلعالنية الداخلية" In door " : .وتتخذ الكتيبات أشكاال ومقاسات متنوعة - تحتوي الكتيبات على شرح القضية المطروحة وأسبابها وطرق عالجها لذلك تعتبر الكتيبات من الوسائل اإلعالنية اإلقناعية الهامة وتحتاج الكتيبات إلى إهتمام خاص أثناء التصميم نظرا ً إلحتوائه على العديد من الصور والرسوم التوضيحية باإلضافة إلحتواءه على كمية كبيرة من المعلومات والبيانات14 ، ( كما في شكل6 .) ( شكل6 .) يوضح كتيب عن حقوق الطفل31 4 - :التقويمات األيام لتنظيم وسيلة 5 - واألسابيع 6 - والشهور ألهداف إجتماعية أو إدارية أو دينية ويتم ذلك بتحديد األيام وإعطائها توقيت محدد .مثل نتائج الحائط والجيب والمكتب  إعالنات التوعية في الطرق ووسائل نقل الركاب" Out door " : :تنقسم هذه اإلعالنات إلى ثالثة أنواع رئيسة متجاورة على تركيبات خشبية أو معدنية معدة .خصيصا ً لذلك أو على وسائل النقل المختلفة15 2 - :اللوحات المنقوشة أو المرسومة تعد خصيصا لتصميم اإلعالن المطلوب عرضه ويتم رسم اإلعالن عليها باأللوان وتستخدم لهذا الغرض الجدران الجانبية للمباني أو القمم العالية للمنازل ( شكل5 .) يوضح مطوية إعالنية عن التوعية المرورية أصدرتها إدارة مرور اإلسكندرية30 3 - : الكتيبات عبارة عن مطبوعات صغيرة الحجم تضم عدد من الصفحات حوالي8 صفحات قد يزيد عن ذلك ويكون بالطول أو العرض ويحتوي على غالف . .وتتخذ الكتيبات أشكاال ومقاسات متنوعة - تحتوي الكتيبات على شرح القضية المطروحة الهامة وتحتاج الكتيبات إلى إهتمام خاص أثناء التصميم نظرا ً إلحتوائه على العديد من الصور والرسوم التوضيحية باإلضافة إلحتواءه على كمية كبيرة من المعلومات والبيانات14 ، ( كما في شكل6 .) (شكل5درتها إدارة ر ر اإل كندرية ) ي ضح ط ية إعالنية عن الت عية ال ر رية أ30 ( شكل5 .) يوضح مطوية إعالنية عن التوعية المرورية أصدرتها إدارة مرور اإلسكندرية30 3 - : الكتيبات عبارة عن مطبوعات صغيرة الحجم تضم عدد من الصفحات حوالي8 صفحات قد يزيد عن ذلك ويكون بالطول أو العرض ويحتوي على غالف .  الملصقات اإلعالنية الداخلية" In door " :  الملصقات اإلعالنية الداخليةIn door : يعتبر الملصق اإلعالني وسيلة هامة من وسائل اإلعالن خاصة ً في مجال التوعية لما تتصف به من إمكانية التكرار واإلتصال مع الفئة المستهدفة كما أنه يجمع مؤثرات بصرية مركزة ومختصرة ولكنها ذات تأثير مباشر وذات قدرة على جذب اإلنتباه1 1.  توجد ايضا ً وسائل إعالنية آخرى ورقية مطبوعة تستخدم في إعالنات التوعية حيث أنها تتميز بدرجة كبيرة من التنوع في شكلها و حجمها و تظهر هذه المطبوعات في أشكال : مختلفة كاآلتي ( شكل4) يوضح نشرة إعالنية عن الحملة القومية للتطعيم ضد مرض شلل األطفا.ل29 ( شكل4) يوضح نشرة إعالنية عن الحملة القومية للتطعيم ضد مرض شلل األطفا.ل29 ( شكل4) يوضح نشرة إعالنية عن الحملة القومية للتطعيم ضد مرض شلل األطفا.ل29 ( شكل4) يوضح نشرة إعالنية عن الحملة القومية للتطعيم ضد مرض شلل األطفا.ل 2 - :المطويات عبارة عن فرخ من الورق يتم طيه بطر ق مختلفه .وتعتبر المطويات أحد األشكال التي يصدر بها إعالنات التوعية حيث تتميز المطويات بأنها صغيرة الحجم وإمكانية توزيعها في سهولة ويسر كما أنها وسيلة إعالنية ملموسة قريبة من يد القارئ حيث يتم تنسيق المعلومات والصور والرسوم فيها بشكل منظم13 ( ، كما في شكل5 ) ويمثل مطوية إعال نية عن التوعية المرورية تم .إصدارها من خالل إدارة مرور اإلسكندرية من يد القارئ حيث يتم تنسيق المعلومات والصور والرسوم فيها بشكل منظم13 ( ، كما في شكل5 ) ويمثل مطوية إعال نية عن التوعية المرورية تم .إصدارها من خالل إدارة مرور اإلسكندرية 2 - :المطويات عبارة عن فرخ من الورق يتم طيه بطر ق مختلفه .وتعتبر المطويات أحد األشكال التي يصدر بها إعالنات التوعية حيث تتميز المطويات بأنها صغيرة الحجم وإمكانية توزيعها في سهولة ويسر كما أنها وسيلة إعالنية ملموسة قريبة من يد القارئ حيث يتم تنسيق المعلومات والصور والرسوم فيها بشكل منظم13 ( ، كما في شكل5 ) ويمثل مطوية إعال نية عن التوعية المرورية تم .إصدارها من خالل إدارة مرور اإلسكندرية 258 المجلد العاشر- العدد ال رابع– أكتوبر2023 ( شكل5 .) يوضح مطوية إعالنية عن التوعية المرورية أصدرتها إدارة مرور اإلسكندرية30 3 - : الكتيبات عبارة عن مطبوعات صغيرة الحجم تضم عدد من الصفحات حوالي8 صفحات قد يزيد عن ذلك ويكون بالطول أو العرض ويحتوي على غالف .  الملصقات اإلعالنية الداخلية" In door " : - Egypt (ISSN 2537-1061) (Print) 259 (ISSN 2537-107X) (Online) المجلد العاشر- العدد ال رابع– أكتوبر2023 المجلد العاشر- العدد ال رابع– أكتوبر2023  : السينما تعتبر السينما من الوسائل اإلعالنية ،أيضا وهي تشترك مع التلفزيون في مميزاته اإلعالنية كالصورة و الصوت و الحركة وتضيف إليها إ مكانية استخدام األلوان واتساع الشاشة وبالتالي كبر حجم الصورة المعروضة مما .يجعلها وسيلة جيدة من وسائل إعالنات التوعية وتتعدد أنواع إعالنات التوعية في السينما فمنها اإلعالنات الثابته التي تحتوي صورة معينة تعرض على شاشة السينما ومنها إعالنات ثابته ناطقه التي تحتوي صورة معينة يصاحبها تعليق ناطق ومنها اإلعالنات المتحركة الناطقة التي تحتوي على أشخاص ومناظر بصورتها الطبيعية19 . وتتميز إعالنات السينما بأنها تعرض اإلعالن بألوانه الطبيعية وبحج م كبير20 .  : اإلذاعة أو الراديو يعد الراديو وسيلة إعالمية إعالنية تقوم بحمل وعرض الرسالة اإلعالنية للجمهور . وتطورت اإلذاعة تبعا للتطور التكنولوجي والتقدم التقني في جميع المجاالت وأصبحت موجودة في جميع المجتمعات في كل وقت حيث نجدها منتشرة بكثرة في المنازل وأثناء قيادة األشخاص لسياراتهم ي نصتون لها بكل إهتمام؛ لهذا تعتبر اإلذاعة من الوسائل الهامة لنشر إعالنات التوعية مثل إعالنات التوعية الخاصة بتنظيم األسرة ومواعيد تطعيمات األطفال17 .  : التلفزيون يعتبر التلفزيون من الوسائل اإلعالمية اإلعالنية الهامة، وذلك نظرا لما يقدمه للمعلن من إمكانات وتكنولوجيا فنية . اليمكن تواجدها في الوسائل اإلعالنية اآلخرى فهو يمتاز بأن له تأثير فعال على الجمهور المشاهد بمختلف فئات األسر فنجدهم جميعا يشاهدون التلفزيون ويتف اعلون معه ويتأثرون بما يقدمه من موضوعات ترفيهية وثقافية ،وتعليمية وإجتماعية مختلفة وبالتالي تتسلل اإلعالنات من خالل هذه المواد إلى الفرد إضافة إلى ذلك تأثيره ،السريع والمباشر فإن التلفزيون يغطي السوق المحلية ،كلها وبالتالي فالمعلن يستطيع الوصول إلى كل فئات الجمهور الذي يرغب ،في الوصول إليهم وعن طريق إعادة بث ،اإلعالن يضمن المعلن درجة كبيرة من إثارة اإلنتباه و التذكر لدى األفراد لإلعالن .ولذلك يعد التليفزيون من أفضل وسائل نشر إعالنات التوعية نظرا ً ألنه يجمع بين كافة الخصائص اإلعالنية الموجودة في الوسائل اآلخرى فمن حيث الرؤية (الصحف والمجالت) ومن حيث الصوت والمؤثرات الصوتية (االذاعة) عالوة على ذلك استخدام .صور طبيعية متحركة18  :شبكة اإلنترنت هي شبكة إتصال عالمية عبر الحواسيب واألجهزة اللوحية والهواتف النقالة تسمح بنقل المعلومات إلكترونيا عبرهذه الشبكة، ولقد إزداد إستخدام اإلنترنت بظهور العنكبوتية الشبكة(World Wide Web) والتي تعد خطوة تمهيدية في مجال اإلعالن .  الملصقات اإلعالنية الداخلية" In door " : .وتتخذ الكتيبات أشكاال ومقاسات متنوعة - اتحتوي الكتيبات على شرح القضية المطروحة الهامة وتحتاج الكتيبات إلى إهتمام خاص أثناء التصميم نظرا ً إلحتوائه على العديد من الصور والرسوم التوضيحية باإلضافة إلحتواءه على كمية كبيرة من المعلومات والبيانات14 ، ( كما في شكل6 .) .رية أصدرتها إدارة مرور اإلسكندرية30 الهامة وتحتاج الكتيبات إلى إهتمام خاص أثناء التصميم نظرا ً إلحتوائه على العديد من الصور والرسوم التوضيحية باإلضافة إلحتواءه على كمية كبيرة من المعلومات والبيانات14 ، ( كما في شكل6 .) ( شكل5) يوضح مطوية إعالنية عن التوع 3 - : الكتيبات عبارة عن مطبوعات صغيرة الحجم تضم عدد من الصفحات حوالي8 صفحات قد يزيد عن ذلك ويكون بالطول أو العرض ويحتوي على غالف . .وتتخذ الكتيبات أشكاال ومقاسات متنوعة - تحتوي الكتيبات على شرح القضية المطروحة وأسبابها وطرق عالجها لذلك تعتبر الكتيبات من الوسائل اإلعالنية اإلقناعية ( شكل6 .) يوضح كتيب عن حقوق الطفل31 4 - :التقويمات األيام لتنظيم وسيلة 5 - أواألسابيع 4 - :التقويمات األيام لتنظيم وسيلة 5 - أواألسابيع متجاورة على تركيبات خشبية أو معدنية معدة .خصيصا ً لذلك أو على وسائل النقل المختلفة15 6 - والشهور ألهداف إجتماعية أو إدارية أو دينية ويتم ذلك بتحديد األيام وإعطائها توقيت محدد .مثل نتائج الحائط والجيب والمكتب 2 - :اللوحات المنقوشة أو المرسومة تعد خصيصا لتصميم اإلعالن المطلوب عرضه ويتم رسم اإلعالن عليها باأللوان وتستخدم لهذا الغرض الجدران الجانبية للمباني أو القمم العالية للمنازل . التي يمكن رؤيتها من مسافات بعيدة  إعالنات التوعية في الطرق ووسائل نقل الركاب" Out door " : ااإ :قسم هذه اإلعالنات إلى ثالثة أنواع رئيسة 1 - :الملصقات تعتبر الملصقات من أقدم الطرق التي إستخدمها المعلنون في مصر وذلك إلنخفاض تكلفتها وسهولة تغييرها بعد أسبوع أو بضعة أسابيع قليلة حيث يتم طبع اإلعالن ع لى فرخ واحد من الورق أو أكثر ثم لصقها 3 - اللوحات المضيئة : هي اإلعالنات التي تكون على شكل الفتات مضيئة بالطرق ويشع منها ال ضوء وتستخدم بصفة أساسية في الليل حيث يتم تثبيتها على أعمدة اإلنارة بالشوارع أو فوق أسطح المنازل وتستخدم اإلضاءة المبهرة 3 - اللوحات المضيئة : هي اإلعالنات التي تكون على شكل الفتات مضيئة بالطرق ويشع منها ال ضوء وتستخدم بصفة أساسية في الليل حيث يتم تثبيتها على أعمدة اإلنارة بالشوارع أو فوق أسطح المنازل وتستخدم اإلضاءة المبهرة Journal of Applied Art and Science - International Periodical Scientific Peer Reviewed - Issued By Faculty of Applied Arts - Damietta Univ. Journal of Applied Art and Science - International Periodical Scientific Peer Reviewed - Issued By Faculty of Applied Arts - Damietta Univ. - Egypt 260 (ISSN 2537-1061) (Print) Journal of Applied Art and Science - International Periodical Scientific Peer Reviewed - Issued By Faculty of Applied Arts - Damietta Univ. - Egypt 260 (ISSN 2537-1061) (Print) (ISSN 2537-107X) (Online) Journal of Applied Art and Science - International Periodical Scientific Peer Reviewed - Issued By Faculty of Applied Arts - Damietta Univ. - Egypt  الملصقات اإلعالنية الداخلية" In door " : إن شبكة اإلنترنت تشهد تزايد سريع وضخم في أعداد المشتركين فيها وبالتالي أصبح من السهل نشر إعالنات التوعية على شبكات اإلنترنت وتتميز إعالنات اإلنترنت بإنها ذات فعالية كبيرة وذلك نظرا ً لتوجيهها إلى عدد كبير من الجمهور على مستوى العالم كما أنها ذات تكلفه منخفضه إذا ق ورنت بوسائل اإلعالن اآلخرى21 . آ : ثالثا ً: تقسيم إعالنات التوعية تبعا للقضية المطروحة تنقسم أنواع إعالنات التوعية إلى (توعية إجتماعية– توعية سياسية– توعية بيئية– توعية صحية– توعية . )تعليمية إااإ :أ: إعالنات التوعية اإلجتماعية هي اإلعالنات التي تهدف الى حل مشكالت إجتماعية مثل البطالة والتدخين وإدمان المخدرات وقضايا تشغيل األطفال والرفق بالحيوان وغيرها من القضايا التي تهم ( المجتمع .كما في شكل7 ) إعالن توعية اجتماعية .عن أضرار التدخين على صحة اإلنسان المدخن Journal of Applied Art and Science - International Periodical Scientific Peer Reviewed - Issued By Faculty of Applied Arts - Damietta Univ. - Egypt urnal of Applied Art and Science - International Periodical Scientific Peer Reviewed - Issued By Faculty of Applied Arts - Damietta Univ. - Eg 260 (ISSN 2537-107X) (Online) المجلد العاشر- العدد ال رابع– أكتوبر2023 (شكل7ض إ الن ت ) ( شكل7) يوضح إعالن توعية عن أضرار التدخين على صحة اإلنسان. 32 ( شكل7) يوضح إعالن توعية عن أضرار التدخين على صحة اإلنسان. 32 :ب: إعالنات التوعية السياسية هي اإلعالنات التي تلعب دورا ً مهما ًفي تنمية الوعي السياسي لدى األفراد وتثقيفهم من الناحية السياسية ويتعلق مفهوم الوعي السياسي باألفراد والمنظمات والمجتمعات على حد سواء مثل اإلنتخابات والقيام بالمظاهرات والثورات22 . ( كما في شكل8 ) إعالن توعية سياسية عن أهمية المشاركة في اإلنتخابات .وإبداء الرأي ( شكل8) يوضح إعالن توعية سياسية. 33 :ج: إعالنات التوعية البيئية وهي تلك اإلعالنات( شكل9 إأ) إعالن توعية يحث على الحفاظ على ( 7) يوضح إعالن توعية عن أضرار التدخين على صحة اإلنسان. 32 : سية هي اإلعالنات تنمية الوعي السياسي احية السياسية ويتعلق باألفراد والمنظمات والمجتمعات على حد سواء مثل اإلنتخابات والقي بالمظاهرات والثورات22 . ( كما في شكل8) إعال توعية سياسية عن أهمية المشاركة في اإلنتخابا .وإبداء الرأي ( شكل8) يوضح إعالن توعية سياسية. 33 ( شكل7) يوضح إعالن توعية عن أضرار التدخين على صحة اإلنسان. 32 : سياسية هي اإلعالنات ًفي تنمية الوعي السياسي ن الناحية السياسية ويتعلق سي باألفراد والمنظمات والمجتمعات على حد س بالمظاهرات والثورات2 توعية سياسية عن أهمي .وإبداء الرأي ( شكل8) يوضح إعالن توعية سياسية. 33 ( شكل7) يوضح إعالن توعية عن أضرار التدخين على صحة اإلنسان.  الملصقات اإلعالنية الداخلية" In door " : 32 ( شكل7) يوضح إعالن توعية عن أضرار التدخين على صحة اإلنسان. 32 :ب: إعالنات التوعية السياسية هي اإلعالنات التي تلعب دورا ً مهما ًفي تنمية الوعي السياسي لدى األفراد وتثقيفهم من الناحية السياسية ويتعلق مفهوم الوعي السياسي باألفراد والمنظمات والمجتمعات على حد سواء مثل اإلنتخابات والقيام بالمظاهرات والثورات22 . ( كما في شكل8 ) إعالن توعية سياسية عن أهمية المشاركة في اإلنتخابات .وإبداء الرأي ( شكل8) يوضح إعالن توعية سياسية. 33 ( شكل8) يوضح إعالن توعية سياسية. 33 :ج: إعالنات التوعية البيئية وهي تلك اإلعالنات التي تهتم بالقضايا البيئية وتهدف إلى رفع الوعي البيئي لدى األفراد ليحافظوا على بيئتهم من التلوث مثل الحفاظ على مياه النيل من التلوث والحفاظ على الهواء من التلوث باألدخنه الضارة ويمثل ( شكل9 ) إعالن توعية يحث على الحفاظ على البيئة من التلو ث واإلهتمام بزراعة األشجار وزيادة المساحات الخضراء لما لها من تأثير .إيجابي على الفرد والمجتمع ككل ر وي ل أ ب ( شكل9) يوضح إعالن توعية بيئية. 34 أر وي ب ( شكل9) يوضح إعالن توعية بيئية. 34 Applied Art and Science - International Periodical Scientific Peer Reviewed - Issued By Faculty of Applied Arts - Damietta Univ. - Egypt of Applied Art and Science - International Periodical Scientific Peer Reviewed - Issued By Faculty of Applied Arts - Damietta Univ. - Egypt 261 (ISSN 2537-107X) (Online) المجلد العاشر- العدد ال رابع– أكتوبر2023 ( والتوعية بمرض األنفلونزا .ويمثل شكل10 ) إعالن توعية صحية بمرض سرطان الثدي بضرورة الكشف المبكر لتجنب أي إصابا ت . وإكتشافها مبكرا ً وعالجها في أسرع وقت ( شكل10 .) يمثل إعالن توعية صحية35 ( شكل10 .) يمثل إعالن توعية صحية35 ( شكل10 .) يمثل إعالن توعية صحية35 :ه: إعالنات التوعية التعليمية هي تلك اإلعالنات التي تهدف إلى زيادة الوعي لدى األفراد بأهمية التعليم في حياة كل فرد مثل إعالنات محو األمية ( .كما في شكل11 ) إعالن توعية بحق كل فرد في التعليم وأهمية التعليم بالنسبة للمرأة وذلك لمسؤليتها عن تعليم وتربية أبنائها فهم أجيال .المستقبل 4 - التعريف بالمؤسسات والمنظمات الخدمية والغير هادفة للربح وتعريف المتلقيين بأهمية هذه المؤسسات وأهمية دورها الفعال في المجتمع وذلك لخلق روابط إجتماعية بين . الجمهور والجهة المعلنه 5 - الوصول إلى أكبر قدر من الجمهور وذلك من خالل اإلنتشار عبر الوسائل اإلعالنية وتحقيق . Journal of Applied Art and Science - International Periodical Scientific Peer Reviewed - Issued By Faculty of Applied Arts - Damietta Univ. - Egypt Journal of Applied Art and Science - International Periodical Scientific Peer Reviewed - Issued By Faculty of Applied Arts - Damietta Univ. - Egypt  الملصقات اإلعالنية الداخلية" In door " : الهدف المنشود والتغطية المطلوبة شكل( 11 .) يمثل إعالن توعية تعليمية36 ا 6 - تعريف الجمهور بموضوع الرسالة اإلعالنية وذلك عن طريق توفير كافة المعلومات المطلوبة وتجنيب المتلقي تعب و عناء ومجهود البحث عنها إلتخاذ القرار المالئم .له 7 - اإلقناع بأن دور اإلعالن ال يتوقف فقط عند جذب اإلنتباه أو تسهيل فهم موضوع اإلعالن لكنه يتعدى ذلك ليجعل المتلقي يتبنى سلوكيات وإتجاهات هادفه والتخلي عن السلوكيات . السلبية رغبة منه في التغيير لألفضل شكل( 11 .) يمثل إعالن توعية تعليمية36 : دور إعالن التوعية في المجتمع23إا 8 - إضفاء الق يمة واألهمية على مضمون اإلعالن من خالل تنبيه الجمهورإلى قيمة المادة . اإلعالنية إلعالنات التوعية عدة أهداف ووظائف تسعى من : خاللها إلى تغيير سلوك األفراد لألفضل كاآلتي إلعالنات التوعية عدة أهداف ووظائف تسعى من : خاللها إلى تغيير سلوك األفراد لألفضل كاآلتي 1 - خلق الوعي أو اإلدراك وذلك من خالل التعريف الدقيق والمفصل بالفكرة محور اإلعالن حيث نجد أن الجمهور يتجنب الموضوعات واألفكار الغريبه الغير منسجمه . مع معتقداته الشخصية 1 - خلق الوعي أو اإلدراك وذلك من خالل التعريف الدقيق والمفصل بالفكرة محور اإلعالن حيث نجد أن الجمهور يتجنب الموضوعات واألفكار الغريبه الغير منسجمه . مع معتقداته الشخصية 9 - التذكير وتتمثل الوظيفة التذكيرية لإلعالن في الحفاظ على الفكرة أو الموضوع في ذهن المتلقي وذلك من خالل التكرار المستمر .للرسالة اإلعالنية 10 - يقوم إعالن التوعية بسؤولية إجتماعية وهي التي تعنى بالقضايا اإلجتماعية كالصحة والبيئة واإلقتصاد والتعليم24 ، وآخرى أخالقية وهي التي تهتم بالمبادئ كتحقيق العدالة وعدم التمييز وعدم إيذاء الغير قوال ً أو فعال ً وعدم الخداع25 . 2 - التأثير في اإلتجاهات حيث أن الهدف األساسي من إعالنات التوعية هو تغيير اإلتجاهات ومن ثم تغيي ر السلوك وذلك عن . طريق توظيف اإلستراتيجيات اإلقناعية إإ 3 - بناء صورة ذهنية جيدة عن القضايا . والموضوعات في عقول المتلقيين Journal of Applied Art and Science - International Periodical Scientific Peer Reviewed - Issued By Faculty of Applied Arts - Damietta Univ. - Egypt (ISSN 2537-1061) (Print) 262 (ISSN 2537-107X) (Online) المجلد العاشر- العدد ال رابع– أكتوبر2023 :دراسة إستبيانية :إجراءات اإلستبيان - تم إجراء دراسة إستبيانية بأخذ آراء مجمو عة من وغير المتخصصين اإلعالنات متلقي المتخصصين من فئات عمرية مختلفة عن إعالنات التوعية ودورها ومدى تأثيرها على المتلقين ،وذلك من خالل إجراء استبيان إلكتروني بإستخدام أحد تطبيقاتGoogle "نماذج "جوجل"Google forms" إلستطالع اآلراء ً وجمع البيانات تلقائيا. Journal of Applied Art and Science - International Periodical Scientific Peer Reviewed - Issued By Faculty of Applied Arts - Damietta Univ. - Egypt 263 (ISSN 2537-1061) (Print)  الملصقات اإلعالنية الداخلية" In door " : - بعد اإلنتهاء من جمع البيانات تم إدخال البيانات .ً وتحليلها إحصائيا :نموذج اإلستبيان السؤال نعم ال ً أحيانا هل إعالنات التوعية ت جعل المتلقين يتبنوا سلوكيات وإتجاهات هادفه؟ هل تهتم بإعالنات التوعية وما تحتويه من رسائل توعوية هامة؟ هل تعد إعالنات التوعية وسيلة القضايا مواجهة في هامة واألزمات المختلفة التي تواجه المجتمع؟ هل تخلق إعالنات التوعية الوعي واإلدراك لدى الجمهور المتلقي وتساعد في تثقيفه؟ هل ترى أن إعالنات التوعية تحقق المتلقين توعية في أهدافها المستهدفين؟ أكثر التوعبة إعالنات هل اإلعالنات من تأثيرا ًوجذبا ً التجارية؟ هل ترى أن األبتكار في تصميم إعالنات التوعية له تأثير إيجابي في وصول الرسالة اإلعالنية للجمهور المتلقي؟ :نتيجة اإلستبيان - تم أخذ آراء مجموعة من متلقي اإلعالنات المتخصصين وغير المتخصصين من فئات عمرية مختلفة عن إعالنات التوعية ودورها ومدى تأثيرها على المتلقين وكان عدد األفراد المستجيبين161 فرد، مثلت فئة الشباب النسبة ( األكبر في المرحلة العمرية من20 – 35 ) عاما ً بنسبة تصل إلى95.8 % من إجمالي المستجيبين، ومثل حوالي 4 % منهم كانت ( أعمارهم تتراوح ما بين36 – 50 فأكثر)، كما كانت نسبة اإلناث في اإلجابة عن اإلستبيان ال تقل عن65 % من العينة بينما كانت نسبة الذكور ال تتعدى35 % . - كانت نسبة الموافقة بنعم للسؤال األول64.6 % من العينة على أن إعالنات التوعية تجعل المتلقين يتبنوا سلوكيات وإتجاهات هادفة بينما ً كانت نسبة أحيانا32.9 % ، أما نسبة الرفض بال2.5 % . المتلقين ،وذلك من خالل إجراء استبيان إلكتروني بإستخدام أحد تطبيقاتGoogle "نماذج "جوجل"Google forms" إلستطالع اآلراء ً وجمع البيانات تلقائيا.  الملصقات اإلعالنية الداخلية" In door " : - بعد اإلنتهاء من جمع البيانات تم إدخال البيانات .ً وتحليلها إحصائيا :نموذج اإلستبيان السؤال نعم ال ً أحيانا هل إعالنات التوعية ت جعل المتلقين يتبنوا سلوكيات وإتجاهات هادفه؟ هل تهتم بإعالنات التوعية وما تحتويه من رسائل توعوية هامة؟ هل تعد إعالنات التوعية وسيلة القضايا مواجهة في هامة واألزمات المختلفة التي تواجه المجتمع؟ هل تخلق إعالنات التوعية الوعي واإلدراك لدى الجمهور المتلقي وتساعد في تثقيفه؟ هل ترى أن إعالنات التوعية تحقق المتلقين توعية في أهدافها المستهدفين؟ أكثر التوعبة إعالنات هل اإلعالنات من تأثيرا ًوجذبا ً التجارية؟ هل ترى أن األبتكار في تصميم إعالنات التوعية له تأثير إيجابي في وصول الرسالة اإلعالنية للجمهور المتلقي؟ :نتيجة اإلستبيان - تم أخذ آراء مجموعة من متلقي اإلعالنات المتخصصين وغير المتخصصين من فئات عمرية مختلفة عن إعالنات التوعية ودورها ومدى تأثيرها على المتلقين وكان عدد األفراد المستجيبين161 فرد، مثلت فئة الشباب النسبة ( األكبر في المرحلة العمرية من20 – 35 ) عاما ً بنسبة تصل إلى95.8 % من إجمالي المستجيبين، ومثل حوالي 4 % منهم كانت ( أعمارهم تتراوح ما بين36 – 50 فأكثر)، كما كانت نسبة اإلناث في اإلجابة عن اإلستبيان ال تقل عن65 % من العينة بينما كانت نسبة الذكور ال تتعدى35 % . - كانت نسبة الموافقة بنعم للسؤال األول64.6 % من العينة على أن إعالنات التوعية تجعل المتلقين يتبنوا سلوكيات وإتجاهات هادفة بينما ً كانت نسبة أحيانا32.9 % ، أما نسبة الرفض بال2.5 % . :دراسة إستبيانية :إجراءات اإلستبيان - تم إجراء دراسة إستبيانية بأخذ آراء مجمو عة من وغير المتخصصين اإلعالنات متلقي المتخصصين من فئات عمرية مختلفة عن إعالنات التوعية ودورها ومدى تأثيرها على المتلقين ،وذلك من خالل إجراء استبيان إلكتروني بإستخدام أحد تطبيقاتGoogle "نماذج "جوجل"Google forms" إلستطالع اآلراء ً وجمع البيانات تلقائيا. Journal of Applied Art and Science - International Periodical Scientific Peer Reviewed - Issued By Faculty of Applied Arts - Damietta Univ. - Egypt 263 (ISSN 2537-1061) (Print) (ISSN 2537-107X) (Online)  الملصقات اإلعالنية الداخلية" In door " : بعد اإلنتهاء من جمع البيانات تم إدخال البيانات .ً وتحليلها إحصائيا السؤال نعم ال ً أحيانا هل إعالنات التوعية ت جعل المتلقين يتبنوا سلوكيات وإتجاهات هادفه؟ هل تهتم بإعالنات التوعية وما تحتويه من رسائل توعوية هامة؟ هل تعد إعالنات التوعية وسيلة القضايا مواجهة في هامة واألزمات المختلفة التي تواجه المجتمع؟ هل تخلق إعالنات التوعية الوعي واإلدراك لدى الجمهور المتلقي وتساعد في تثقيفه؟ هل ترى أن إعالنات التوعية تحقق المتلقين توعية في أهدافها المستهدفين؟ أكثر التوعبة إعالنات هل اإلعالنات من تأثيرا ًوجذبا ً التجارية؟ هل ترى أن األبتكار في تصميم إعالنات التوعية له تأثير إيجابي في وصول الرسالة اإلعالنية للجمهور المتلقي؟ :نتيجة اإلستبيان - تم أخذ آراء مجموعة من متلقي اإلعالنات المتخصصين وغير المتخصصين من فئات عمرية مختلفة عن إعالنات التوعية ودورها ومدى تأثيرها على المتلقين وكان عدد األفراد المستجيبين161 فرد، مثلت فئة الشباب النسبة ( األكبر في المرحلة العمرية من20 – 35 ) عاما ً بنسبة تصل إلى95.8 % من إجمالي المستجيبين، ومثل حوالي 4 % منهم كانت (أعمارهم تتراوح ما بين36 50فأكثر)، كما كانت نسبة اإلناث في اإلجابة عن اإلستبيان ال تقل عن65 % من العينة بينما كانت نسبة الذكور ال تتعدى35 % . - كانت نسبة الموافقة بنعم للسؤال األول64.6 % من العينة على أن إعالنات التوعية تجعل المتلقين يتبنوا سلوكيات وإتجاهات هادفة بينما ً كانت نسبة أحيانا32.9 % ، أما نسبة الرفض بال2.5 % . :نتيجة اإلستبيان :نتيجة اإلستبيان :نتيجة اإلستبيان - تم أخذ آراء مجموعة من متلقي اإلعالنات المتخصصين وغير المتخصصين من فئات عمرية مختلفة عن إعالنات التوعية ودورها ومدى تأثيرها على المتلقين وكان عدد األفراد المستجيبين161 فرد، مثلت فئة الشباب النسبة ( األكبر في المرحلة العمرية من20 – 35 ) عاما ً بنسبة تصل إلى95.8 % من إجمالي المستجيبين، ومثل حوالي 4 % منهم كانت ( أعمارهم تتراوح ما بين36 – 50 فأكثر)، كما - كانت نسبة الموافقة بنعم للسؤال األول64.6 % من العينة على أن إعالنات التوعية تجعل المتلقين يتبنوا سلوكيات وإتجاهات هادفة بينما ً كانت نسبة أحيانا32.9 % ، أما نسبة الرفض بال2.5 % . - كانت نسبة الموافقة بنعم للسؤال األول64.6 % من العينة على أن إعالنات التوعية تجعل المتلقين يتبنوا سلوكيات وإتجاهات هادفة بينما ً كانت نسبة أحيانا32.9 % ، أما نسبة الرفض بال2.5 % . Journal of Applied Art and Science - International Periodical Scientific Peer Reviewed - Issued By Faculty of Applied Arts - Damietta Univ.  الملصقات اإلعالنية الداخلية" In door " : - Egypt 263 (ISSN 2537-107X) (Online) المجلد العاشر- العدد ال رابع– أكتوبر2023 المجلد العاشر- العدد ال رابع– أكتوبر2023 - كانت نسبة الموافقة بنعم للسؤال الثاني74.7 % من العينة على أن لديهم إهتمام بإعالنات التوعية وما تحويه من رسائل توعوية هامة، بينما كانت ً نسبة أحيانا22.4 % ، أما نسبة الرفض بال 3.2 % . Journal of Applied Art and Science - International Periodical Scientific Peer Reviewed - Issued By Faculty of Applied Arts - Damietta Univ. - Egypt 264 (ISSN 2537-1061) (Print) (ISSN 2537-107X) (Online) - كانت نسبة الموافقة بنعم للسؤال الثاني74.7 % من العينة على أن لديهم إهتمام بإعالنات التوعية وما تحويه من رسائل توعوية هامة، بينما كانت ً نسبة أحيانا22.4 % ، أما نسبة الرفض بال 3.2 % . - كانت نسبة الموافقة بنعم للسؤال الثالث79.5 % على أن تعد إعالنات التوعية وسيلة هامة في مواجهة القضايا واألزمات المختلفة التي تواجه ً المجتمع، بينما كانت نسبة أحيانا19.3 % ، أما كانت بال الرفض نسبة1.2 % . - كانت نسبة الموافقة بنعم للسؤال الرابع66.5 % على أن تخلق إعالنات التوعية الوعي واإلدراك لدى الجمهور المتلقي وتساعد في تثقيفه، بينما كانت نسبة أحيانا31.7 % ، أما نسبة الرفض بال كانت1.8 % . Applied Art and Science - International Periodical Scientific Peer Reviewed - Issued By Faculty of Applied Arts - Damietta Univ. - Egypt Journal of Applied Art and Science - International Periodical Scientific Peer Reviewed - Issued By Faculty of Applied Arts - Damietta Univ. - Egypt 264 (ISSN 2537-107X) (Online) المجلد العاشر- العدد ال رابع– أكتوبر2023 Journal of Applied Art and Science - International Periodical Scientific Peer Reviewed - Issued By Faculty of Applied Arts - Damietta Univ. - Egypt - كانت نسبة الموافقه بنعم للسؤال الخامس 57.7 % على أن إعالنات التوعية تحقق أهدافها في توعية المتلقين المستهدفين، بينما كانت نسبة ً أحيانا40 % ، أما نسبة الرفض بال فكانت 2.3 % . - كانت نسبة الموافقة بنعم للسؤال السادس 54.5 % ً على أن إعالنات التوعية أكثر تأثيرا وجذبا ً من اإلعالنات التجارية، بينما كانت نسبة ً أحيانا25.5 % ، أما نسبة الرفض بال كانت 20 % . ومما سبق يتبين من خالل تحليل نتائج اإلستبيان ما - ضرورة إهتمام الحكومات ب إعالنات التوعية نظرا ً لتأثيرها الفعال في تحقيق أهدافها في توعية أفراد المجتمع إإا - ضرورة إهتمام الحكومات ب إعالنات التوعية نظرا ً لتأثيرها الفعال في تحقيق أهدافها في توعية أفراد المجتمع - اإلهتمام باإلبتكار في تصميم إعالنات التوعية حتى تحقق التغطية المطلوبة والهدف المنشود .من الرسالة اإلعالنية :يلي - أهمية إعالنات التوعية بالنسبة للمجتمع في مواجهة القضايا واألزمات المختلفة،حيث أكدت نسبة ال تقل عن79.5 % على أهمية إعالنات التوعية، كما تساعد إعالنات التوعية على إتباع السلوكيات السليمة تجاه المواقف المختلفة وكذلك تساعد على خلق الوعي لدى ال.جمهور المتلقى - اإلهتمام باإلبتكار في تصميم إعالنات التوعية حتى تحقق التغطية المطلوبة والهدف المنشود .من الرسالة اإلعالنية  الملصقات اإلعالنية الداخلية" In door " : - كانت نسبة الموافقة بنعم للسؤال السا بع91.9 % على أن اإلبتكار في تصميم إعالنات التوعية له تأثير إيجابي في وصول الرسالة اإلعالنية ً للجمهور المتلقي، بينما كانت نسبة أحيانا7 % ، أما نسبة الرفض بال كانت1.1 % - كانت نسبة الموافقه بنعم للسؤال الخامس 57.7 % على أن إعالنات التوعية تحقق أهدافها في توعية المتلقين المستهدفين، بينما كانت نسبة ً أحيانا40 % ، أما نسبة الرفض بال فكانت 2.3 % . - كانت نسبة الموافقة بنعم للسؤال السادس 54.5 % ً على أن إعالنات التوعية أكثر تأثيرا وجذبا ً من اإلعالنات التجارية، بينما كانت نسبة ً أحيانا25.5 % ، أما نسبة الرفض بال كانت 20 % . ً أحيانا25.5 % ، أما نسبة الرفض بال كانت 20 % . - كانت نسبة الموافقة بنعم للسؤال السا بع91.9 % على أن اإلبتكار في تصميم إعالنات التوعية له تأثير إيجابي في وصول الرسالة اإلعالنية ً للجمهور المتلقي، بينما كانت نسبة أحيانا7 % ، أما نسبة الرفض بال كانت1.1 % Journal of Applied Art and Science - International Periodical Scientific Peer Reviewed - Issued By Faculty of Applied Arts - Damietta Univ. - Egypt Journal of Applied Art and Science - International Periodical Scientific Peer Reviewed - Issued By Faculty of Applied Arts - Damietta Univ. - Egypt 265 (ISSN 2537-107X) (Online) (ISSN 2537-107X) (Online) المجلد العاشر- العدد ال رابع– أكتوبر2023 :المراجع العربية - ضرورة اإلبتكار في تصميم إعالنات التوعية حيث أكدت نسبة ال تقل عن91.9 % على أن اإلبتكار في تصميم إعالنات التوعية له تأثير إيجابي في وصول الرسالة اإلعالنية للجمهور .المتلقي - ضرورة اإلبتكار في تصميم إعالنات التوعية حيث أكدت نسبة ال تقل عن91.9 % على أن اإلبتكار في تصميم إعالنات التوعية له تأثير إيجابي في وصول الرسالة اإلعالنية للجمهور .المتلقي - ضرورة اإلبتكار في تصميم إعالنات التوعية حيث أكدت نسبة ال تقل عن91.9 % على أن اإلبتكار في تصميم إعالنات التوعية له تأثير إيجابي في وصول الرسالة اإلعالنية للجمهور .المتلقي 1 - أسماء محمد زكي الدحدوح : "الدور الجمالي لإلتصال في اإلعالن التوعوي اإللكتروني في "صحافة اإلنفوجرافيك– مجلة الفنون والعلوم التطبيقية– المجلد الثامن– العدد الثاني– ابريل2021 .م Journal of Applied Art and Science - International Periodical Scientific Peer Reviewed - Issued By Faculty of Applied Arts - Damietta Univ. - Egypt المجلد العاشر- العدد ال رابع– أكتوبر2023 :المراجع األجنبية 11 - ": مي رضوان صيوح استراتيجية تصميمية لتحقيق الهوية البصرية العالنات التوعية المطبوعة و تاثيرها على "المتلقي– دكتوراه 11 - ": مي رضوان صيوح استراتيجية تصميمية لتحقيق الهوية البصرية العالنات التوعية المطبوعة و تاثيرها على "المتلقي– دكتوراه 11 - ": مي رضوان صيوح استراتيجية تصميمية لتحقيق الهوية البصرية العالنات التوعية المطبوعة و تاثيرها على "المتلقي– دكتوراه – كلية الفنون التطبيقية– جامعة حلوان– 2013 .م 24- Conrad Berenson – Henry Elibrt – 24- Conrad Berenson – Henry Elibrt – 24- Conrad Berenson – Henry Elibrt – The Social Dynamics Of Marketing – Random House – U.S.A – 2017. 25- George Zinkan – Advertising Ethics: Emerging Methods and Trends – American Academy of Advertising – . 1994 – U.S.A The Social Dynamics Of Marketing – Random House – U.S.A – 2017. – كلية الفنون التطبيقية– جامعة حلوان– 2013 .م 25- George Zinkan – Advertising Ethics: Emerging Methods and Trends – American Academy of Advertising – . 1994 – U.S.A 12 - المطبوعات :"تصميم صالح أشرف "اإلعالمية– دار النهضة العربية– القاهرة – 1999 .م 13 - صفوت محمد العالم :" دراسات في اإلعالم "المروري– دار الثقافة العربية– القاهرة– 1999 .م :انتائج البحث 11 - ": مي رضوان صيوح استراتيجية تصميمية لتحقيق الهوية البصرية العالنات التوعية المطبوعة و تاثيرها على "المتلقي– دكتوراه – كلية الفنون التطبيقية– جامعة حلوان– 2013 .م 12 - المطبوعات :"تصميم صالح أشرف "اإلعالمية– دار النهضة العربية– القاهرة – 1999 .م 13 - صفوت محمد العالم :" دراسات في اإلعالم "المروري– دار الثقافة العربية– القاهرة– 1999 .م 14 - ": مي رضوان صيوح است راتيجية تصميمية لتحقيق الهوية البصرية العالنات التوعية "المطبوعة و تاثيرها على المتلقي– مرجع .سابق 15 - صفوت محمد العالم :" دراسات في اإلعالم "المروري– .مرجع سابق 16 - حسام فتحي ابوطعيمة:" اإلعالن و سلوك "المستهلك– .مرجع سابق 17 - شريف مراد :" دور بحوث التسويق في 19 - حسام فتحي ابوطعيمة:" اإلعالن و سلوك "المستهلك– .مرجع سابق 20 - شريف مراد :" دور بحوث التسويق في تفعيل اإلعالن في المؤسسة "االقتصادية – .مرجع سابق 21 - حسام فتحي ابوطعيمة:" اإلعالن و سلوك "المستهلك– .مرجع سابق 22 - عبير عبد المنعم :"علم اإلجتماع وتنمية المحلية بالمتغيرات اإلجتماعي الوعي "والعالمية– المكتبة العصرية– 2009 .م 23 - شدوان علي شيبة :"االعالن المدخل والنظرية" – دار المعرفه الجامعيه– قسم االعالم– كلية االداب– جامعة االسكندرية– 2008 .م :المراجع األجنبية 6 - "سامي عبد العزيز :" مقدمة في اإلعالن– مركز التعليم المفتوح كلية اإلعالم– جامعة القاهرة– مصر– 2004م. 6 - "سامي عبد العزيز :" مقدمة في اإلعالن– مركز التعليم المفتوح كلية اإلعالم– جامعة القاهرة– مصر– 2004م. 7 - :عصام الدين امين ابو علفة : " الترويج المفاهيم- االستراتيجيات- : العمليات النظرية والتطبيق " – مؤسسة حورس الدولية : طيبة للنشر والتوزيع– االسكندرية– مصر – 2002 .م اإ 21 - حسام فتحي ابوطعيمة:" اإلعالن و سلوك "المستهلك– .مرجع سابق إ 22 - عبير عبد المنعم :"علم اإلجتماع وتنمية المحلية بالمتغيرات اإلجتماعي الوعي "والعالمية– المكتبة العصرية– 2009 .م 22 - عبير عبد المنعم :"علم اإلجتماع وتنمية المحلية بالمتغيرات اإلجتماعي الوعي "والعالمية– المكتبة العصرية– 2009 .م 8 - حسام فتحي ابوطعيمة:" اإلعالن و سلوك "المستهلك– .مرجع سابق اا 23 - شدوان علي شيبة :"االعالن المدخل والنظرية" – دار المعرفه الجامعيه– قسم االعالم– كلية االداب– جامعة االسكندرية– 2008 .م :المراجع األجنبية 9 - صفوت العالم– ": نهله الحفناوي فن االعالن "الصحفي– الدار العربية للنشر والتوزيع– كلية االعالم– جامعة القاهرة– 2006 .م 10 - "محمد فريد الصحن :" االعالن– الدار الجامعية– االسكندرية– 1988م. Journal of Applied Art and Science - International Periodical Scientific Peer Reviewed - Issued By Faculty of Applied Arts - Damietta Univ. - Egypt :انتائج البحث 2 - رشا محمود السيد محمود :"تحقيق التكامل بين تنمية الوعي المجتمعي و العدالة االجتماعية من خالل تفعيل دور اعالنات "التوعية– بحث – كلية الخدمة االجتماعية– جامعة حلوان– القاهرة– 2011 . م - إعالنات التوعية لها تأثير على المتلقين المستهدفين وتعمل على تغيير ثقافتهم تجاه .القضايا والمشكالت المختلفة ا - دور اإلعالن يتعدى جذب اإلنتباه أو تسهيل فهم موضوع لكنه يجعل المتلقين يتبنوا سلوكيات وإتجاهات هادفه رغبة ً منهم في .التقدم 3 - رحمه مجدي خالد الدعدع :"اإلعالنات المجسمة (ثالثية األبعاد) كشكل من أشكال "إعالنات الطريق– مجلة الفنون والعلوم التطبيقية– المجلد التاسع– العدد األول– يناير2022 .م م - اإلبتكار في تصميم إعالنات التوعية له تأثير إيجابي على المتلقين المستهدفين ويساعدهم على الشعور باإلنجذاب تجاه اإلعالن .ويساعدهم على سهولة تذكره مرة آخرى م - اإلبتكار في تصميم إعالنات التوعية له تأثير إيجابي على المتلقين المستهدفين ويساعدهم على الشعور باإلنجذاب تجاه اإلعالن .ويساعدهم على سهولة تذكره مرة آخرى 4 - حسام فتحي ابو طعيمة :"االعالن و سلوك "المستهلك– دار الفاروق للنشر– الطبعة االولى– االردن– 2008 .م 5 - عبد السالم ابو قحف :"محاضرات في هندسة "االعالن– الدار الجامعية– لبنان– 1995 .م 5 - عبد السالم ابو قحف :"محاضرات في هندسة "االعالن– الدار الجامعية– لبنان– 1995 .م - ضرورة اإلهتمام بإستخدام إعالنات التوعية في مواجهة القضايا واألزمات التي تواجه المجتمع لما لها من تأثير إيجابي على أفراد .المجتمع وتغير ثقافتهم نحو األفضل - ضرورة اإلهتمام بإستخدام إعالنات التوعية في مواجهة القضايا واألزمات التي تواجه المجتمع لما لها من تأثير إيجابي على أفراد .المجتمع وتغير ثقافتهم نحو األفضل (ISSN 2537-1061) (Print) 266 (ISSN 2537-107X) (Online) المجلد العاشر- العدد ال رابع– أكتوبر2023 المجلد العاشر- العدد ال رابع– أكتوبر2023 6 - "سامي عبد العزيز :" مقدمة في اإلعالن– مركز التعليم المفتوح كلية اإلعالم– جامعة القاهرة– مصر– 2004م. 7 - :عصام الدين امين ابو علفة : " الترويج المفاهيم- االستراتيجيات- : العمليات النظرية والتطبيق " – مؤسسة حورس الدولية : طيبة للنشر والتوزيع– االسكندرية– مصر – 2002 .م 8 - حسام فتحي ابوطعيمة:" اإلعالن و سلوك "المستهلك– .مرجع سابق 9 - صفوت العالم– ": نهله الحفناوي فن االعالن "الصحفي– الدار العربية للنشر والتوزيع– كلية االعالم– جامعة القاهرة– 2006 .م 10 - "محمد فريد الصحن :" االعالن– الدار الجامعية– االسكندرية– 1988م. Journal of Applied Art and Science - International Periodical Scientific Peer Reviewed - Issued By Faculty of Applied Arts - Damietta Univ. - Egypt Journal of Applied Art and Science - International Periodical Scientific Peer Reviewed - Issued By Faculty of Applied Arts - Damietta Univ. - Egypt 268 (ISSN 2537-1061) (Print) Journal of Applied Art and Science - International Periodical Scientific Peer Reviewed - Issued By Faculty of Applied Arts - Damietta Univ. - Egypt 268 (ISSN 2537-1061) (Print) (ISSN 2537-107X) (Online) Abstract: Awareness advertisements are one of the important advertising media in community because of their effective role and the great impact in community awareness. Awareness advertisements are used to serve the issues of community members in various groups, spread awareness and support the positives that enhance the community’s progress towards the development, and provide a creative advertising material capable of guiding community members for the better. They are advertisements aimed at persuading recipients to participate to be cautious, or to change the style of life in a style that would be better for them. We find that awareness advertisements make individuals able to know and understand things, issues and information in a good way from their many aspects so that it creates a sense of the importance of these issues and matters, which makes them able to make a decision about them or to behavior towards them, with the development of issues affecting the community, there was a need to direct to awareness advertisements and to renew them, as awareness advertisements need creativity and innovation in their design and implementation, and to rely on good technical foundations in order for the advertisement to achieve the desired goal effectively and successfully and achieve the greatest benefit and reach the target recipients. Awareness advertisements are divided into several types: awareness advertisements according to the geographical scope (local, national and international awareness advertisements), as well as awareness advertisements according to the medium used in the advertisement (readable awareness advertisements such as newspapers, magazines, road advertisements, audio and visual advertisements such as radio, television, cinema and the Internet). Awareness advertisements are also divided according to The issue at hand (social, environmental, health, political and educational awareness advertisements), where the research problem emerged, which is summarized in the following question: Do awareness advertisements achieve the required coverage and the desired goal of the advertising message? From here came the research, which aims to highlight the role of awareness advertisements and their role in the development of society and change its culture for the better, by monitoring and analyzing the different types of awareness advertisements, and the role of awareness advertisements in the community and then conducting a questionnaire by taking the opinions of a group of advertisement recipients to reach the results: Awareness Advertisements and their Role in Community Development Awareness Advertisements and their Role in Community Development Awareness Advertisements and their Role in Community Developmen Journal of Applied Art and Science - International Periodical Scientific Peer Reviewed - Issued By Faculty of Applied Arts - Damietta Univ. - Egypt :المواقع اإللكترونية 26- https://aawsat.com/home/article/1638486/ %D8%AD%D9%85%D9%84%D8%A9- %D9%85%D8%B5%D8%B1%D9%8A% D8%A9%D9%84%D8%AA%D9%86%D 8%B8%D9%8A%D9%85%D8%A7%D9 %84%D8%A3%D8%B3%D8%B1%D8% A9%D8%AA%D8%AB%D9%8A%D8% B1%D8%A7%D9%86%D8%AA%D9%8 2%D8%A7%D8%AF%D8%A7%D8%A A%D8%A3%D9%87%D9%84%D8%A7 %D9%84%D8%B5%D8%B9%D9%8A% D8%AF, Acceseed:August,19,2022. 27- 14 - ": مي رضوان صيوح است راتيجية تصميمية لتحقيق الهوية البصرية العالنات التوعية "المطبوعة و تاثيرها على المتلقي– مرجع .سابق إا 15 - صفوت محمد العالم :" دراسات في اإلعالم "المروري– .مرجع سابق إا 15 - صفوت محمد العالم :" دراسات في اإلعالم "المروري– .مرجع سابق إا 16 - حسام فتحي ابوطعيمة:" اإلعالن و سلوك "المستهلك– .مرجع سابق 17 - شريف مراد :" دور بحوث التسويق في تفعيل اإلعالن في المؤسسة "االقتصادية – ماجستير– كلية العلوم االقتصادية والعلوم التجارية– جامعة المسيلة– الجزائر– 2006 .م م 18 - " :"االعالن السلمي علي– دارغريب للطباعة والنشر– القاهرة– مصر– 1997م. https://www.shorouknews.com/news/view .aspx?cdate=14052019&id=9ea8d9ee- Journal of Applied Art and Science - International Periodical Scientific Peer Reviewed - Issued By Faculty of Applied Arts - Damietta Univ. - Egypt 267 (ISSN 2537-1061) (Print) (ISSN 2537-107X) (Online) Journal of Applied Art and Science - International Periodical Scientific Peer Reviewed - Issued By Faculty of Applied Arts - Damietta Univ. - Egypt (ISSN 2537-1061) (Print) 267 (ISSN 2537-107X) (Online) المجلد العاشر- العدد ال رابع– أكتوبر2023 32-https://www.youm7.com/story/2016/1 /2561127,Acceseed:August,19,2022. 33- https://m.facebook.com/elections.eg/photo s/a-/1007716929271103- ,Acceseed:August,19,2022. 34- https://eg.mostaql.com/portfolio/570909, Acceseed:August,19,2022. 35- https://www.akhbaralaan.net/health/2016/ 11/22,Acceseed:August,19,2022. 36- https://twitter.com/moe_jdh_20_1003/stat us/103840414394428211-,Acceseed- :August,19,2022 a95c-4c91-9fe3-a733e6afd0a5, a95c-4c91-9fe3-a733e6afd0a5, Acceseed:August,19,2022. 28- https://www.ei- ie.org/en/item/23259:world-health- organisation,Acceseed: August,19,2022. 29- ie.org/en/item/23259:world-health- organisation,Acceseed: August,19,2022. 29- https://m.facebook.com/elections.eg/photo s/a-/1007716929271103- https://gate.ahram.org.eg/News/2113073.a https://gate.ahram.org.eg/News/2113073.a spx,Acceseed:August,19,2022. 30- https://www.masrawy.com/news/news_re gions/details/2017/1/10/1011503- ,Acceseed:August,19,2022. 31- https://mawdoo3.com/%D8%A8%D9%86 -%D9%-88%D8%AF_%D8%A7- 8%A7%D9%84%D8%B7%D9%81%D9 %84,Acceseed:August,19,2022. spx,Acceseed:August,19,2022. 30- https://www.masrawy.com/news/news_re gions/details/2017/1/10/1011503- 268 (ISSN 2537-107X) (Online) المجلد العاشر- العدد ال رابع– أكتوبر2023 The research found: - Acceptance of the first hypothesis, which is awareness advertisements have an impact on the target recipients. - Acceptance of the first hypothesis, which is awareness advertisements have an impact on the target recipients. - Acceptance of the second hypothesis, which is awareness advertisements that work to convince the recipients that the role of the advertisement goes beyond attracting attention or facilitating understanding a topic, but it makes the recipients adopt targeted behaviors and trends in their desire to progress. - Acceptance of the second hypothesis, which is awareness advertisements that work to convince the recipients that the role of the advertisement goes beyond attracting attention or facilitating understanding a topic, but it makes the recipients adopt targeted - Acceptance of the third hypothesis, which is that innovation in the design of awareness advertisements has a positive impact on the intended recipients. - Acceptance of the third hypothesis, which is that innovation in the design of awareness advertisements has a positive impact on the intended recipients. (ISSN 2537-1061) (Print) 269
https://openalex.org/W2727642337	https://europepmc.org/articles/pmc5489147?pdf=render	English	null	The association between levels of alcohol consumption and mental health problems and academic performance among young university students	PloS one	2,017	cc-by	7,083	RESEARCH ARTICLE The association between levels of alcohol consumption and mental health problems and academic performance among young university students Chimwemwe Tembo1, Sharyn Burns2, Fatch Kalembo3 1 Saint. John of God Hospitaller Services, Lilongwe, Malawi, 2 School of Public Health, Curtin University, Perth, Western Australia, 3 Faculty of Health Sciences, Mzuzu University, Mzuzu, Malawi 1 Saint. John of God Hospitaller Services, Lilongwe, Malawi, 2 School of Public Health, Curtin University, Perth, Western Australia, 3 Faculty of Health Sciences, Mzuzu University, Mzuzu, Malawi chimweptembo@yahoo.co.uk a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Tembo C, Burns S, Kalembo F (2017) The association between levels of alcohol consumption and mental health problems and academic performance among young university students. PLoS ONE 12(6): e0178142. https://doi.org/ 10.1371/journal.pone.0178142 Methods This study used a quantitative cross-sectional design using data that were collected in 2014 as part of the Youth Alcohol Project (YAP). Participants were randomly drawn from a cross sectional sample of 6000 undergraduate students. Included in the study were only students who were within the age of 18–24, undergraduate, and internally enrolled at the main cam- pus. A total of 2518 undergraduate students aged 18 to 24 years who were enrolled inter- nally at Curtin University Bentley campus were randomly recruited. Data were collected through an online survey. Students were invited to participate in the study through their stu- dent email address. The email invitations coincided with the release of semester results to increase the likelihood of students accessing their emails. A further 628 students were ran- domly recruited through face to face intercept survey during the campus market days. Data were collected by trained research assistants. Validated instruments were used to collected data on levels of alcohol consumption, mental health, and academic performance. Copyright: © 2017 Tembo 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: Data are hosted by Curtin University survey office through The Human research ethics committee for researchers who meet the criteria for access to confidential data. Interested researchers may reach out to the Human Research Ethics Committee (hre@curtin. edu.au), or the chair person Human Research ethics Committee, Professor Peter O’ Leary (phone: +61892661855; email peter.oleary@curtin. edu.au). Purpose Mental health problems and harmful alcohol consumption have been found to be high among young university students compared to the general population in Australia. This research aimed to investigate the association between levels of drinking and mental health problems and academic performance among university students aged 18 to 24 years. OPEN ACCESS Citation: Tembo C, Burns S, Kalembo F (2017) The association between levels of alcohol consumption and mental health problems and academic performance among young university students. PLoS ONE 12(6): e0178142. https://doi.org/ 10.1371/journal.pone.0178142 Editor: Andrew R. Dalby, University of Westminster, UNITED KINGDOM Received: December 19, 2016 Accepted: May 8, 2017 Published: June 28, 2017 Copyright: © 2017 Tembo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Introduction Alcohol-related harm and mental health problems are among the leading public health issues in today’s society [1, 2]. For many young Australians, alcohol plays an important role in their social life [3,4]. Twenty-one percent of young Australians aged 18–24 have been found to engage in risky drinking [5] and this behaviour puts them at greater risk of short and long- term harms associated with alcohol consumption [3]. Young university students have been found to experience greater levels of alcohol-related harm compared to their non-university attending peers [6, 7]. The university environment provides a unique social context that often promotes excessive drinking [8]. Additionally, young students may be influenced by a range of developmental, environmental, and lifestyle changes which may be associated with ‘rites of passage’ [7]. The environment, both psychological and physical, also influences alcohol con- sumption [9]. For example, alcohol advertising, alcohol related events at universities and pres- sure to belong compounded by new independence are some of the attributing factors to risky drinking behaviours [9]. The Tertiary Health Research Intervention study (THRIVE) found 90% of university students (17–24 years old) consumed alcohol in the last 12 months of the study with average volumes for a typical drinking session being 5.09 standard drinks for females and 8.68 for males [8]. In addition, 48% of students aged 17–24 years exceeded the threshold for acute alcohol-related harm at least once in the last four weeks of the survey [8]. Similarly, another Australian university found that approximately 50% of young students drank to intoxication on one or more days per week [10]. Harmful levels of alcohol consump- tion are associated with increased risk of both long and short-term health effects [5]. Short- term harms are found to be high among young university students as a result of episodic drinking [11]. The most common harms experienced by students are vomiting, aggression, missing classes, underachievement, financial problems, and memory loss [6]. Additionally, there is substantial research that demonstrates that harmful and hazardous alcohol consump- tion also contributes to arange of mental health problems and disorders including depression, anxiety, and stress or psychological distress [12,13]. Studies in Europe, the USA and Australia haveshown a high prevalence of alcohol use disorders, alcohol dependence, stress, anxiety,eat- ing disorders and depression among university students [14–17] compared to their non-uni- versity peers [18]. Conclusion The study shows that a considerable proportion of undergraduate students at university con- sume alcohol at hazardous or harmful levels. In addition, high levels of alcohol consumption are associated with poor academic performance and mental health outcomes among stu- dents. The results of the study warrant multi-strategy interventions that focus on policy, organisational, educational, environmental and economic strategies that will help to reduce alcohol related harms among university students. Levels of drinking and mental health problems among university students assignments (aOR = 3.5, 95% CI 2.0–6.0) independently predicted for moderate or hazard- ous alcohol consumption. Funding: The authors received no specific funding for this work. Funding: The authors received no specific funding for this work. Competing interests: The authors have declared that no competing interest exist. Competing interests: The authors have declared that no competing interest exist. Results A considerable proportion of participants (44%) reported consuming alcohol at hazardous or harmful levels. Multiple logistic regression analysis showed that students who were consum- ing alcohol at hazardous levels were 1.2 times more likely to report psychological distress than those with lower levels of alcohol consumption (aOR 1.2, 95% CI: 1.1–1.5). In addition, being late for class (aOR 1.7, 95% CI:1.1–2.4), missing classes (aOR = 2.6, 95% CI: 1.9– 2.6), inability to concentrate in class (aOR = 2.6, 95% CI: 1.9–3.4), and inability to complete PLOS ONE \| https://doi.org/10.1371/journal.pone.0178142 June 28, 2017 1 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0178142 June 28, 2017 Study design and sample size Cross-sectional data were collected from a random sample of undergraduate students as part of the Youth Alcohol Project (YAP) [11]. The YAP aimed to promote a culture within the uni- versity that supports responsible levels of alcohol consumption. The sample included under- graduate students aged 18 to 24 years who were enrolled internally at Curtin University Bentley campus. Participants were randomly recruited through the University Survey’s Office and via a face to face intercept survey. An online questionnaire was emailed randomly to a total of 6000 stu- dent through their university email address. Two follow-up emails were sent to maximise par- ticipation rates. A total of 1930 students completed the survey online (response rate 32.2%), and an additional 628 students were recruited via face to face intercept survey during campus market day. The total sample of students recruited that provided completed questionnaires was 2518. Levels of drinking and mental health problems among university students that mental health problems contribute to the seven top ten barriers to academic performance [17,20]. A range of interventions that are universal, selective, or indicated are recommended to address alcohol-related problemsand mental health [21]. For example instance,university- based early intervention programs have shown to effectively intervene with heavier users of alcohol [21]. In addition, mental health prevention, promotion, and early intervention strate- gies demonstrate positive outcomes when dealing with mental health problems. Such interven- tion should include mental health literacy (knowledge and beliefs about mental disorders that aid their recognition management and prevention) [22,23]. For example, a strategy of the Youth Alcohol Project was Mental Health First Aid (MHFA) training to increase mental health literacy among nursing students [24,25]. The intervention aimed to train participants to con- sider signs and symptoms of mental health problems and to enable them to provide an appro- priate response to someone experiencing a mental health problem or crisis until professional help was available [25]. The study found significant improvements in knowledge scores, confi- dence in helping, mental health first aid intentions, stigma and social distance for the interven- tion compared to control group participants [26]. Literature provides evidence that the university setting offers an environment that promotes excessive drinking [9] and young uni- versity students are at increased risk of mental health related issues. Therefore, there is a need to explore the extent of the burden of mental health problems in relation to the level of alcohol consumption and identify the predictors to hazardous drinking so that it guides interventions that decrease risk and improve psychological wellbeing of stu- dents in universities. The aim of this paper is to describe the association between levels of alco- hol consumption and mental health problems among students aged between 18–24. PLOS ONE \| https://doi.org/10.1371/journal.pone.0178142 June 28, 2017 Introduction The association between alcohol and mental health is bi-directional with studies suggesting that individuals who are predisposed to harmfulalcohol consumption are prone to episodes of stress, depression, and anxiety [13]. Poor mental health among university students has been associated with academic pressure and irregular sleep patterns [7] and has a negative effect on academic outcomes [19]. A study conducted in Canada and the USA found PLOS ONE \| https://doi.org/10.1371/journal.pone.0178142 June 28, 2017 2 / 13 Instrumentation The survey collected demographic, levels of alcohol consumption, mental health, and aca- demic performance data. Demographic data were collected using a general questionnaire that included the following information age, gender, Faculty, residence, employment status, and domestic or international student status. Levels of alcohol consumption were assessed using the Alcohol Use Disorders Identification Test (AUDIT) [27, 28]. The AUDIT has been described as an accurate tool to detect alcohol dependence among university students [29, 30]. Cross-national standardisation of the AUDIT was validated in primary health care settings in six countries (Cronbach alpha 0.87) [29]. 3 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0178142 June 28, 2017 Levels of drinking and mental health problems among university students AUDIT is a 10 item questionnaire that uses an overall harm score to categorise drinking levels into four risk levels namely: 0–7 low risk, 8–15 risky or hazardous, 16–19 r harmful and 20 and high risk [27]. Consistent with other studies, this study used the 10 item AUDIT and computed to binary variables: low-risk levels of consumption < 8 or hazardous levels of alco- hol consumption 8 [11, 31]. Mental health was assessed using the Kessler psychological dis- tress scale (K10) [32, 33]. The K10 is a ten-item questionnaire asking questions about anxiety and depressive symptoms that an individual experienced in the past 30-days period. The scores range from 10 to 50 [34]. Consistent with other studies, psychological distress was categorised into four categories: 10–15 no to low psychological distress, 16–21 moderate distress, 22–29 high distress and 30–50 very high distress [17, 35]. A binary variable of low and moderate/ high/very high categories was created from the four categories of psychological distress. Low psychological distress category from the four psychological distress categories formed the first category of the binary variable while moderate, high and very high distress categories were combined to form a moderate/high and very high category. The K10 instrument was vali- dated in previous studies and has been reported to have good internal reliability with Cron- bach alpha score of 0.84 [36,37]. Academic problems were measured using the Academic Role Expectation and Alcohol Scale (AREAS) [38]. The four items in this scale addressed the num- ber of times the student had been ‘late to class’, ‘missed class’, was ‘unable to concentrate’ and ‘failed to complete assignment’ as a result of their alcohol consumption for a reference period of twelve months (score range 0–16) [38]. Instrumentation The responses included ‘not at all’, ‘once’, ‘twice’, ‘three times’ and ‘four times or more’. The academic problem scale is a validated tool that has been used by a number of researchers to assess academic problems, and consistency was tested using coefficient alpha. The coefficient alpha was 76 [38, 39]. Data analysis Data were cleaned and analysed using IBM SPSS version 22.0. The general characteristics of the study participants were analysed using descriptive statistics. The dependent variable was a binary score of low risk and hazardous drinking. Chi-square was used to find the association between binary score AUDIT and independent variables of interest (psychological distress and academic performance). Using binary logistic regression, a full model including demographic variables that were significant in Chi-square analysis and the independent variables of interest were computed in one model. Odds ratios were used to determine the relative risk as well as the strength of association between the dependent and explanatory variables. All analyses were two-sided and the p-value was considered highly significant at <0.001 and moderately signifi- cant at < 0.05. Ethical approval The study received ethics approval from Curtin University Human Research Ethics Commit- tee (HR 54/2013). Written and verbal consent was sought from the study participants and doc- umented. In addition, permission to conduct the study with students was obtained from the management of Curtin University. Demographic characteristics of study participants Data were collected from 2518 undergraduate students. Half of the participants (50%, n = 1208) were aged 18–20 years, and half were aged 21-25years (n = 1214). Sixty-two percent (n = 1504) were females and 37.5%, (n = 908) were males. The majority of participants (88.7%, n = 2223) 4 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0178142 June 28, 2017 Levels of drinking and mental health problems among university students were domestic students and 11.3% (n = 283) were international. Regarding the year of study, the largest proportion of students (33.7%, n = 789) were in their first year of study. The largest proportion of students (30%, n = 696) worked 1-11hours a week and 28.6% (n = 661), worked 20 hours or more a week whilst 26% (n = 573) were not in any employment. In addition, the majority of participants lived with their parents (60.3% n = 1418) (see Table 1). PLOS ONE \| https://doi.org/10.1371/journal.pone.0178142 June 28, 2017 https://doi.org/10.1371/journal.pone.0178142.t001 Descriptive statistics of alcohol consumption, mental health status, and academic performance During the past 12 months, (16.3%, n = 303) of respondents reported being late at least once, (22.8%, n = 422) had missed a class, (25.2%, n = 466) reported to be unable to concentrate in class, and (6.8%, n = 127) had failed to complete an assignment on time as a result of alcohol consumption. while 44% (n = 761) reported consuming alcohol at hazardous and harmful levels. As regards to mental health status, 58.1% (n = 1276) reported low levels of distress, 28.9% (n = 636) while 44% (n 761) reported consuming alcohol at hazardous and harmful levels. As regards to mental health status, 58.1% (n = 1276) reported low levels of distress, 28.9% (n = 636) reported moderate distress, and 13% reported high/ very high psychological distress. During the past 12 months, (16.3%, n = 303) of respondents reported being late at least once, (22.8%, n = 422) had missed a class, (25.2%, n = 466) reported to be unable to concentrate in class, and (6.8%, n = 127) had failed to complete an assignment on time as a result of alcohol consumption. p p g y g p y g During the past 12 months, (16.3%, n = 303) of respondents reported being late at least once, (22.8%, n = 422) had missed a class, (25.2%, n = 466) reported to be unable to concentrate in class, and (6.8%, n = 127) had failed to complete an assignment on time as a result of alcohol consumption. Demographic factors associated with moderate or hazardous levels of alcohol consumption When variables were compared age, gender, international/national, student status, residential status and hours in paid work were significantly associated with hazardous or harmful levels of alcohol consumption (p<0.05) (see Table 3). However, there was no significant association between alcohol consumption and Faculty of study. Descriptive statistics of alcohol consumption, mental health status, and academic performance Table 2 describes patterns of alcohol consumption, mental health, and academic performance. Fifty-six percent (n = 1054) of the respondents reported consuming alcohol at low-risk levels Table 1. Demographic characteristics of study participants. Variable Frequency (N) Percentage% Age 18–20 1208 49.9 21–25 1214 50.1 Gender Male 908 37.5 Female 1504 62.1 other 9 0.4 Faculty Health Sciences 876 36.2 Science and Engineering 541 22.3 Humanities 537 22.2 Business school 462 19.1 Centre for Aboriginal Studies 6 0.2 International or national Domestic 283 88.7 international 2223 11.3 Employment hours per week None 573 24.8 1–5 hours 250 10.8 6–10 hours 446 19.3 11–19 hours 661 28.6 20 + 378 16.4 Years of study One 789 33.7 Two 698 29.8 Three 536 22.9 Four or more 321 13.7 Where participants live while at University Share a ﬂat /house 590 25.1 Hall of student housing 114 4.9 Live with parents or guardians 1418 60.3 Live alone 46 2.0 Live with partner 128 5.4 Board 21 0.9 Other 33 1.4 https://doi.org/10.1371/journal.pone.0178142.t001 Table 1. Demographic characteristics of study participants. PLOS ONE \| https://doi.org/10.1371/journal.pone.0178142 June 28, 2017 5 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0178142 June 28, 2017 Levels of drinking and mental health problems among university students Table 2. Descriptive statistics alcohol consumption, mental health status and academic performance. Variable Frequency Percentage Alcohol consumption Low level 1054 55.9 hazardous Level 679 36.0 Harmful level 154 8.2 Mental health status Low psychological distress 1276 58.1 Moderate psychological distress 636 28.9 High psychological distress 226 10.3 Very high psychological distress 59 2.7 Been late for classes Not at all 1550 83.6 Ever been late 303 16.4 Missing classes Not at all 1431 77.2 Ever missed classes 422 22.8 Unable to concentrate in class Not at all 1387 74.9 Unable to concentrate 466 25.1 Failed to complete assignment Not at all 1726 93.1 Ever failed to complete 127 6.9 https://doi.org/10.1371/journal.pone.0178142.t002 Table 2. Descriptive statistics alcohol consumption, mental health status and academic performance. while 44% (n = 761) reported consuming alcohol at hazardous and harmful levels. As regards to mental health status, 58.1% (n = 1276) reported low levels of distress, 28.9% (n = 636) reported moderate distress, and 13% reported high/ very high psychological distress. Bivariate association between moderate or hazardous alcohol consumption and academic performance and mental health Psychological distress was significantly associated with hazardous or harmful levels of alcohol consumption (p< 0.001). Participants who reported moderate/ high distress were more likely to have consumed alcohol at hazardous or harmful levels. (See Table 4). All four academic areas; being late for classes (p = <0.001) missing classes (p< 0.001), unable to concentrate in class (p = < 0.001) and failure to complete the assignment (p = <0.001), were significantly associated with hazardous or harmful levels of alcohol consumption. 6 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0178142 June 28, 2017 The multivariate regression model also found that students who failed to complete assignments were 3.5 times more likely to drink alcohol at hazardous or harmful levels than those who did not (aOR = 3.5, 95% CI 2.0– Variable Low Risk N % Hazardous/harmful N % p- value Age 18–20 563 (59.0) 391 (41.0) 0.007* 21–25 607 (65.1) 326 (34.9) Faculty Health sciences 429 (62.5) 257 (37.5) 0.116 Science and Engineering 245 (59.8) 165 (40.2) Humanities 285 (66.0) 147 (34.0) Curtin Business school 210 (59.2) 145 (40.8) Centre for aboriginal studies 1 (25.0) 3 (75) Gender Males 405(57.5) 299 (42.5) 0.003* Females 761 (64.8) 413 (35.2) International/domestic <0.001 international 157 (88.2) 21 (11.8) Domestic 1013 (59.3) 696 (40.7) Residence while at university <0.001 Share a ﬂat/house 274 (56) 209 (43.3) Student housing/hall of residence 46 (51.1) 44 (48.9) Live with parents 728 (64.1) 407 (35.9) Live alone 19 (59.4) 13 (40.6) Live with partner & children 82 (76.6) 25 (23.4) Board 7 (38.9) 11 (61.1) others 14 (63.6) 8 (36.4) Hours spent on work <0.001 None 282(69.1) 126 (30.2) 1–5 hours 145 (70.4) 61 (29.6) 6–10 hours 228 (62.6) 136 (37.4) 11–19 hours 339 (59.3) 233 (40.7) 20+ 176 (52.2) 161 (47.8) p <0.001; * p <0.01 https://doi.org/10.1371/journal.pone.0178142.t003 PLOS ONE \| https://doi.org/10.1371/journal.pone.0178142 June 28, 2017 https://doi.org/10.1371/journal.pone.0178142.t003 Levels of drinking and mental health problems among university students Table 3. Demographic factors associated with moderate or hazardous levels of alcohol consumption in bivariate analysis. Variable Low Risk N % Hazardous/harmful N % p- value Age 18–20 563 (59.0) 391 (41.0) 0.007* 21–25 607 (65.1) 326 (34.9) Faculty Health sciences 429 (62.5) 257 (37.5) 0.116 Science and Engineering 245 (59.8) 165 (40.2) Humanities 285 (66.0) 147 (34.0) Curtin Business school 210 (59.2) 145 (40.8) Centre for aboriginal studies 1 (25.0) 3 (75) Gender Males 405(57.5) 299 (42.5) 0.003* Females 761 (64.8) 413 (35.2) International/domestic <0.001 international 157 (88.2) 21 (11.8) Domestic 1013 (59.3) 696 (40.7) Residence while at university <0.001 Share a ﬂat/house 274 (56) 209 (43.3) Student housing/hall of residence 46 (51.1) 44 (48.9) Live with parents 728 (64.1) 407 (35.9) Live alone 19 (59.4) 13 (40.6) Live with partner & children 82 (76.6) 25 (23.4) Board 7 (38.9) 11 (61.1) others 14 (63.6) 8 (36.4) Hours spent on work <0.001 None 282(69.1) 126 (30.2) 1–5 hours 145 (70.4) 61 (29.6) 6–10 hours 228 (62.6) 136 (37.4) 11–19 hours 339 (59.3) 233 (40.7) 20+ 176 (52.2) 161 (47.8) p <0 001; Multivariate association between moderate or hazardous alcohol consumption and mental health and academic performance When all factors were considered, being late for class, missing classes, inability to concentrate in class, inability to complete the assignment and moderate or high distress independently pre- dicted hazardous or harmful alcohol consumption. Students who were late for classes were 1.7 times more likely to consume alcohol at hazardous or harmful levels than those who did not (aOR 1.7, 95% CI: 1.1–2.4). In addition, students who missed classes were 2.6 times more likely to consume alcohol at hazardous or harmful levels than those who did not miss classes (aOR = 2.6, 95% CI: 1.9–2.6). As regards to concentration in class, students who were unable to concentrate in class were 2.6 times more likely to consume alcohol at hazardous or harmful levels than those who did not (aOR = 2.6, 95% CI: 1.9–3.4). Levels of drinking and mental health problems among university students Table 4. Bivariate association between moderate or hazardous alcohol consumption and academic performance and mental health. Academic AREAS Low Risk N (%) Hazardous/harmful N (%) p- value Been late <0.001 Not at all 1074 (69.3) 476 (30.7) Ever been late 78 (25.7) 225 (74.3) Missing classes <0.001 Not at all 1029 (71.9) 402 (28.1) Ever missed classes 123 (29.1) 299 (70.9) Unable to concentrate <0.001 Not at all 1006 (72.5) 381 (27.5) Unable to concentrate 146 (31.3) 320 (68.7) Failed to complete assignment <0.001 Not at all 1129 (65.4) 597 (34.6) Ever failed to complete 23 (18.1) 104 (81.9) K-10 (mental health) Low psychological distress 727 (65.3) 386 (34.7) 0.002* Moderate psychological distress 308 (57.4) 229 (42.6) High psychological distress 112 (59.3) 77 (40.7) Very high psychological distress 23 (47.9) 25 (52.1) K10 binary (mental health) Low psychological distress 727 (65.3%) (386)34.7% <0.001 Moderate/high/very high psychological distress 443 (57.2) 331 (42.8) p <0.001; * p <0.01 n between moderate or hazardous alcohol consumption and academic performance and mental health. 6.0). In terms of the association between mental health and hazardous or harmful levels of alcohol consumption, students who reported moderate or high distress were 1.3 times more likely to consume alcohol at hazardous or harmful levels (aOR = 1.3 95% CI: 1.1–1.7) com- pared to those with low psychological distress (see Table 5). Multivariate association between moderate or hazardous alcohol consumption and mental health and academic performance When all factors were considered, being late for class, missing classes, inability to concentrate in class, inability to complete the assignment and moderate or high distress independently pre- dicted hazardous or harmful alcohol consumption. Students who were late for classes were 1.7 times more likely to consume alcohol at hazardous or harmful levels than those who did not (aOR 1.7, 95% CI: 1.1–2.4). In addition, students who missed classes were 2.6 times more likely to consume alcohol at hazardous or harmful levels than those who did not miss classes (aOR = 2.6, 95% CI: 1.9–2.6). As regards to concentration in class, students who were unable to concentrate in class were 2.6 times more likely to consume alcohol at hazardous or harmful levels than those who did not (aOR = 2.6, 95% CI: 1.9–3.4). The multivariate regression model also found that students who failed to complete assignments were 3.5 times more likely to drink alcohol at hazardous or harmful levels than those who did not (aOR = 3.5, 95% CI 2.0– PLOS ONE \| https://doi.org/10.1371/journal.pone.0178142 June 28, 2017 7 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0178142 June 28, 2017 Levels of drinking and mental health problems among university students Table 5. Multivariate association between hazardous or harmful alcohol consumption and academic performance and mental health. Variable Unadjusted odds ratio uOR 95% CI P value Adjusted odds ratio aOR 95% CI p- value Late for classes Not at all 1.0 1.0 Been late 6.5 5.0–8.6 <0.001 1.7 1.1–2.4 0.008* Missed classes Not at all 1.0 1.0 Missed classes 6.2 4.9–8.0 <0.001 2.6 1.9–2.6 <0.001 Unable to concentrate in class Not at all 1.0 1.0 Unable to concentrate 5.8 4.6–7.3 <0.001 2.6 1.9–3.4 <0.001 Not able to complete assignment Not at all 1.0 1.0 Not completing assignment 2.7 1.8–3.9 <0.001 3.5 2.0–6.0 <0.001** K10 Binary Low distress 1.0 1.0 Moderate/high/very high distress 1.4 1.2–1.7 <0.001 1.3 1.1–1.7 0.009* ** p <0.001; * p <0.01; 1.0 = Reference group. Adjusted for all variables in the table as well as age, gender, residential status, student status (international or domestic) participation in work for 11–19 hours and participating in more than 20 hours. between hazardous or harmful alcohol consumption and academic performance and mental health. * p <0.01; 1.0 = Reference group. Adjusted for all variables in the table as well as age, gender, residential status, student status (international or domestic) participation in work for 11–19 hours and participating in more than 20 hours. https://doi.org/10.1371/journal.pone.0178142.t005 Furthermore, the findings are also consistent with a study by Stallman [17] that examined the psychological distress among university students. The results showed a significant differ- ence between levels of psychological distress and student’s Grade point average (GPA). Thus, for each increase in psychological distress, students showed lower GPA. These findings, along with those of this study suggest a need for comprehensive early intervention to address mental health among university students. A significant difference was found between the age and levels of drinking. The study found that the proportion of students who were between 18–20 years were found to drink more hazardously (41%) compared to those between the ages of 21–25 years (34.9%). Contrary to this, an earlier study at the same university found no significant difference between these same age groups and levels of consumption [11]. However, a 2012 study found young people aged 17–19 years were significantly more likely to consume alcohol at hazardous levels than those aged 20–25 years [8]. Discussion The aim of this study was to explore the association between levels of alcohol consumption and mental health problems and academic performance among university students between the age of 18–24 years. The study findings show that 38% of the participants consumed alcohol at a hazardous level. Males were more likely to drink at a hazardous level compared to females. The findings are consistent with findings from other studies conducted in the United States [40], New Zealand [38] and Australia [8] where males have reported consuming alcohol at haz- ardous levels than females. Being male was a significant predictor of hazardous drinking [11]. Slightly more participants were females which is consistent with findings from other cross-sec- tional studies previously conducted in Australia and New Zealand universities [6, 11, 38]. The results show a significant association between the levels of alcohol consumption and mental health problems and academic performance. Students who consumed alcohol at haz- ardous levels were more likely to experience moderate / high psychological distress and more likely to experience academic problems. The findings are consistent with a study by Hallett and colleagues where students who consumed alcohol at hazardous levels were more likely to experience academic problems which had a negative impact on learning [39]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0178142 June 28, 2017 8 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0178142 June 28, 2017 Hence, there is a need to design interventions that are universal to minimise stigma associated with mental health problems, selected interventions that are gender-focused, and indicated interventions that would assist students that are experiencing problems as a result of hazardous drinking. Individual oriented interventions like screening and brief moti- vational counseling will be necessary. Examples of such interventions have been implemented in some Australian Universities. For example “staying on Track” implemented in Queensland [17]. The limitations of this study should be considered when interpreting the results. Mental health problems in this study were limited to anxiety and depression symptoms and therefore did not encompass the full spectrum of mental health problems that students may experience. While the K10 is widely used to measure population-level mental health, especially anxiety and depression, it does not provide true diagnosis [17]. Respondents were more likely to be females compared to males and were more likely to be enrolled in the Health Science Faculty. This Fac- ulty has more females enrolled; females have been found to be more likely to respond to uni- versity surveys than men [44]. The cross-sectional design was not the most robust design to assess the association between mental health and levels of drinking due to the inability to establish causal relationships [11]. Additionally, the results are specific to one university and this limits generalisation of the results [11]. Thus, the results may not be comparable with other international studies, because of the different social, and institutional contexts, screening instruments, and even survey methodologies that were used [18]. This paper has described a significant association between the levels of alcohol consump- tion and mental health problems. In addition, the results also highlight that there is an associa- tion between mental health and academic problems and that students with moderate /high psychological distress were more likely tore port not completing assignments. The findings from this study are very comparable with other studies which demonstrate that students who report moderate to high psychological distress are more likely to experience academic prob- lems. Further research to explore the service utilisation and barriers to accessing mental health service is essential to address mental health issues and ensure young university students access available services. The relationship between alcohol outlets to excessive drinking and alcohol- related problems should also be further explored. These findings are similar to the findings of this study whereby younger students aged between 18 and 20 years were more likely to drink at hazardous levels compared to the older age group. This is an important finding given the evidence that drinking at a youn- ger age may lead to risky drinking behaviours in later years [41]. This calls for focused and comprehensive interventions that target higher risk younger drinkers. The majority of students in this study were domestic students. There was, however, a signif- icance difference between being an international or domestic student and level of alcohol con- sumption (p<0.001). Domestic students were found to consume alcohol more at hazardous levels (mean score 5.9, SD 5.7) compared to international students (mean score 3.4, SD 3.7). This may be because some international students may come from countries with different atti- tudes and beliefs associated with alcohol consumption [23]. However, this issue warrants fur- ther investigation [23]. When binary variables were created from Kessler10 [18], this study found 41.9% (n = 921) of participants reported moderate to high psychological distress. The PLOS ONE \| https://doi.org/10.1371/journal.pone.0178142 June 28, 2017 9 / 13 Levels of drinking and mental health problems among university students proportion of psychological distress among university students is reported to be higher (41.9%) compared to the general Australian adult population (29%) [42]. However, the preva- lence of university students with psychological distress is lower in this study compared to two other studies that were conducted in Australia using Kessler 10 (53%; n = 384) in 2008 [20] and in 2009 (45%; n = 1163) [43] but, higher than a 2010 study (29%; n = 6479) [17]. Despite these differences, all these studies suggest concern regarding mental health problems among university students. The findings of this study suggest universities, should be considered as an ideal setting for implementing mental health promotion programs that would aim to increase the awareness of mental health issues and responsible alcohol consumption. In addition, interventions should be youth friendly in order to create an environment that reduces the stigma associated with mental health problems and provide easy access to mental health services. Furthermore, the findings also highlight the need to address the mental health needs of young female students who are more likely to have a higher prevalence of mental health problems associated with haz- ardous drinking. PLOS ONE \| https://doi.org/10.1371/journal.pone.0178142 June 28, 2017 Visualization: CT SB. Writing – original draft: CT SB FK. Writing – review & editing: SB. This research has contributed to the body of knowledge related to the assessment of the prevalence of alcohol consumption in universities. While there is evidence that overall prevalence of alcohol consumption is slightly lower among young Australians [37], the proportion of hazardous drinking among university students in this study is still high. Therefore, the current results describing prevalence provide additional information supporting interventions for reducing alcohol consumption among young people. PLOS ONE \| https://doi.org/10.1371/journal.pone.0178142 June 28, 2017 10 / 13 Levels of drinking and mental health problems among university students The results also justify the need to consider the university setting as an ideal environment for health promotion interventions. Conceptualization: CT SB. Investigation: SB CT. Methodology: CT SB. Validation: SB. Validation: SB. Visualization: CT SB. Project administration: CT. Resources: CT. Resources: CT. Resources: CT. Software: CT FK SB. Software: CT FK SB. Supervision: CT SB. Supervision: CT SB. References 1. Bell S, Britton A. 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https://openalex.org/W3212302047	https://vestnik.mininuniver.ru/jour/article/download/1291/858	Russian	null	Comparative analysis of the subjective perception of oneself as a future parent among married couples with different reproductive history	Vestnik Mininskogo universiteta	2,021	cc-by	9,788	СРАВНИТЕЛЬНЫЙ АНАЛИЗ СУБЪЕКТИВНОГО ВОСПРИЯТИЯ СЕБЯ В РОЛИ БУДУЩЕГО РОДИТЕЛЯ СРЕДИ СЕМЕЙНЫХ ПАР С РАЗЛИЧНЫМ РЕПРОДУКТИВНЫМ АНАМНЕЗОМ1 В. А. Мошкивская1 1Городской перинатальный центр №1 г. Санкт-Петербург, Санкт-Петербург, Российская Федерация Общая психология, психология личности, история психологии Общая психология, психология личности, история психологии Статья/Article УДК 159.9:618.5-07 DOI: 10.26795/2307-1281-2021-9-4-8 1 Исследование выполнено при финансовой поддержке Гранта РФФИ № 20-013-00759 А. Вестник Мининского университета. 2021. Том 9, №4 General psychology, personality psychology, history of psychology Конфликт интересов: авторы декларируют отсутствие явных и потенциальных конфликтов интересов, связанных с публикацией настоящей статьи. Для цитирования: Мошкивская В.А. Сравнительный анализ субъективного восприятия себя в роли будущего родителя среди семейных пар с различным репродуктивным анамнезом // Вестник Мининского университета. 2021. Т. 9, №4. С.8. COMPARATIVE ANALYSIS OF THE SUBJECTIVE PERCEPTION OF ONESELF AS A FUTURE PARENT AMONG MARRIED COUPLES WITH DIFFERENT REPRODUCTIVE HISTORY1 V. A. Moshkivskaya1 1City Perinatal Center No. 1, Saint Petersburg, Russian Federation 1 The study was carried out with the financial support of the RFBR Grant No. 20-013-00759 A. АННОТАЦИЯ Введение. Проблема бесплодия широко известна по всему миру. По данным ВОЗ, 3% населения земли страдают бесплодием. В России проблема бесплодия крайне актуальна, число семейных пар, обращающихся за вспомогательными репродуктивными технологиями (далее – ВРТ), растет с каждым годом. Однако количество положительных ЭКО-протоколов, благополучных исходов беременности, качество жизни детей, рожденных раньше срока по- прежнему не высоко. Существует более 700 факторов, влияющих на исход беременности, большая часть из которых мало изучена, исследования в различных областях продолжаются. Помимо доказанных репродуктологами физиологических факторов, влияющих на исход беременности, остается ряд малоизученных факторов, вызывающих идиопатическое бесплодие. Материалы и методы. Авторское полуструктурированное интервью для семейной пары в период беременности. Мужской и женский вариант интервью. Повторное авторское интервью для женщины в раннем послеродовом периоде. Контент-анализ. Материалы и методы. Авторское полуструктурированное интервью для семейной пары в период беременности. Мужской и женский вариант интервью. Повторное авторское интервью для женщины в раннем послеродовом периоде. Контент-анализ. Результаты исследования. Проведен анализ полученных в ходе интервью основных результатов, таких как субъективная оценка восприятия беременности (телесные, эмоциональные переживания), мотив беременности, оценка собственной родительской готовности к воспитанию ребенка, родительские компетенции, изменение супружеских ролей, изучение образа жизни до и во время беременности, контакт с собственными родителями, поведение в родах, первичный контакт с новорожденным ребенком. Обсуждение и заключения. По результатам проведенного исследования получены выводы, указывающие на важность своевременного оказания психологической помощи женщинам, беременным в результате применения ВРТ. Женщины, беременные с помощью ВРТ, более тревожны, менее ориентированы на собственные силы, чаще имеют акушерские и неонатальные осложнения, имеют трудности с первичным психологическим контактом с новорожденным ребенком, обнаруживают сложности с грудным вскармливанием. Результаты исследования. Проведен анализ полученных в ходе интервью основных результатов, таких как субъективная оценка восприятия беременности (телесные, эмоциональные переживания), мотив беременности, оценка собственной родительской готовности к воспитанию ребенка, родительские компетенции, изменение супружеских ролей, изучение образа жизни до и во время беременности, контакт с собственными родителями, поведение в родах, первичный контакт с новорожденным ребенком. Обсуждение и заключения. По результатам проведенного исследования получены выводы, указывающие на важность своевременного оказания психологической помощи женщинам, беременным в результате применения ВРТ. Женщины, беременные с помощью ВРТ, более тревожны, менее ориентированы на собственные силы, чаще имеют акушерские и неонатальные осложнения, имеют трудности с первичным психологическим контактом с новорожденным ребенком, обнаруживают сложности с грудным вскармливанием. Ключевые слова: беременность, вспомогательные репродуктивные технологии, ЭКО, ВРТ, бесплодие, идиопатическое бесплодие, психология репродуктивной сферы, сравнительный анализ. Вестник Мининского университета. 2021. Том 9, №4 Vestnik of Minin University. 2021. Volume 9, no. 4 Введение Согласно МКБ-10, пара приобретает в семейном репродуктивном анамнезе диагноз «бесплодие» после одного года безуспешных попыток зачатия ребенка. Проблема бесплодия широко известна по всему миру. По данным ВОЗ, 3% населения земли «бесплодны» [29]. В России количество бесплодных пар, по данным Национального медицинского исследовательского центра акушерства, гинекологии и перинатологии имени академика В.И. Кулакова, составляет 15% [11]. Несмотря на пристальное внимание к данному вопросу, ситуация с репродуктивным здоровьем нации продолжает ухудшаться, а количество пар, обращающихся за ВРТ, возрастает. Так, в 2016 году, согласно официальной статистике Национального регистра ВРТ (отчет за 2016 и 2018 годы), количество начатых циклов с собственными ооцитами – 113976, родов – 24282, в 2018 году 145904 начатых цикла с использованием собственных ооцитов и 30796 родов. Из вышеупомянутых данных следует, что увеличивается количество ЭКО-циклов и вместе с ним возрастает количество родов. Однако процент пар с отрицательным результатом после применения ВРТ по-прежнему остается высоким. Целью исследования является анализ полученных в результате интервью факторов субъективного восприятия себя как будущей матери, ценность ребенка в семейной иерархии, оценка супружеских взаимоотношений. Общая психология, психология личности, история психологии Общая психология, психология личности, история психологии For citation: Moshkivskaya V.A. Comparative analysis of the subjective perception of oneself as a future parent among married couples with different reproductive history // Vestnik of Minin University. 2021. Vol. 9, no. 4. Р.8. Вестник Мининского университета. 2021. Том 9, №4 ABSTRACT Introduction. The problem of infertility is widely known around the world. According to WHO, 3% of the world's population suffers from infertility. In Russia, the problem of infertility is extremely urgent, the number of couples applying for assisted reproductive technologies is growing every year. However, the number of positive IVF protocols, successful pregnancy outcomes, and the quality of life of children born prematurely is still not high. There are more than 700 factors that affect the outcome of pregnancy, most of which are poorly understood, and research in various fields continues. In addition to the proven physiological factors that affect the outcome of pregnancy, there are a number of poorly understood factors that cause idiopathic infertility. Materials and Methods. Author's semi-structured interview for a married couple during pregnancy. Male and female version of the interview. Repeated author's interview for a woman in the early postpartum period. Content analysis. Results. The main results obtained during the interview were analyzed. Such as the subjective assessment of the perception of pregnancy (physical, emotional experiences), the motive of pregnancy, the assessment of one's own parental readiness to raise a child, parental competencies, changes in marital roles, studying the lifestyle before and during pregnancy, contact with one's own parents, behavior during childbirth, primary contact with a newborn child. Discussion and Conclusions. According to the results of the study, conclusions were obtained indicating the importance of timely provision of psychological assistance to pregnant women as a result of the use of ART. Women who are pregnant with ART are more anxious, less self-oriented, more likely to have obstetric and neonatal complications, difficulties with primary psychological contact with a newborn child, difficulties with breastfeeding. Keywords: pregnancy, assisted reproductive technologies, IVF, ART, infertility, idiopathic infertility, psychology of the reproductive sphere, comparative analysis. Conflict of interest: the authors declare the absence of obvious and potential conflicts of interest related to the publication of this article. Vestnik of Minin University. 2021. Volume 9, no. 4 Vestnik of Minin University. 2021. Volume 9, no. 4 Общая психология, психология личности, история психологии General psychology, personality psychology, history of psychology идиопатическим бесплодием. Среди возможных причин выделяют более 700 возможных психосоциальных факторов [27, 32, 37]. Наиболее исследуемыми из них являются: оценка уровня тревожности и депрессии [14, 17, 23], не диагностируемых ранее психических расстройств, психических реакций на соматические осложнения в беременности [15]. Vestnik of Minin University. 2021. Volume 9, no. 4 Материалы и методы Исследование проводилось на базе Санкт-Петербургского государственного бюджетного учреждения здравоохранения «Городской перинатальный центр №1». Первым этапом исследования являлся ретроспективный анализ медицинской документации женщин, состоящих на учете по беременности в ЖК №41 при СПб ГПЦ №1, и женщин, находящихся на стационарном лечении в дневном стационаре и отделении патологии беременности. Выборка исследования представлена 143 семейными парами (в 73 парах беременность женщины наступила с помощью ВРТ, в 70 семейных парах – физиологическая беременность) и 18 женщинами, принявшими участие в исследовании без участия партнера (8 женщин, беременных с помощью методов ВРТ, и 10 женщин с физиологической беременностью). Вся выборка была разделена на 2 группы. Первую группу составили женщины, беременные с помощью методов ВРТ (группа 1), вторую группу – физиологически беременные женщины (группа 2). Среди женщин, беременных с помощью ВРТ, 64 (79%) женщины имеют в анамнезе диагноз «бесплодие» (рисунок 1). Рисунок 1 – Распределение типов бесплодия в исследуемой выборке Figure 1 – Distribution of infertility types in the study sample 63% 11% 11% 7% 8% Бесплодие I / Infertility I Бесплодие II / Infertility II Мужское бесплодие / Male infertility Сочетанное / Combined Бесплодие неясного генеза / Infertility of unknown origin Рисунок 1 – Распределение типов бесплодия в исследуемой выборке Figure 1 – Distribution of infertility types in the study sample На рисунке 1 представлено распределение диагноза «бесплодие» в исследуемой выборке. Наиболее часто встречаемым является диагноз «бесплодие I», поставленный у 41 (63%) женщины, «бесплодие II» – у 7 (11%) женщин, «мужское бесплодие» – у 6 (11%), «сочетанное» – у 5 (8%), «бесплодие неясного генеза (идиопатическое)» – у 5 (8%). Дизайн исследования включал в себя работу с медицинской документацией беременной женщины, подходящие под задачи исследования семейные пары приглашались в исследование, подписывали информационное согласие и в удобное время приглашались для прохождения интервью. После рождения ребенка женщина проходила завершающее интервью самостоятельно, по запросу семья получала результаты анализа своего участия в исследовании. Обзор литературы Рассматривая состояние научной разработанности проблемы на данный момент, необходимо отметить, что тема материнства, вопросов беременности и психологического сопровождения раннего детства все более привлекательна для специалистов различных областей [2, 8, 18, 24, 25, 30, 34, 36, 38, 41, 40]. Различным отраслям науки известно обширное число факторов, влияющих на репродуктивное здоровье, однако еще больше факторов остаются малоизученными [22, 33]. Тем не менее выделен ряд репродуктивных факторов, наиболее часто встречающихся у пар с диагнозом «бесплодие» [39]. Такие факторы принято разделять на мужские, женские и сочетанные. К факторам, провоцирующим женское бесплодие, относят трубно-перитониальную патологию, эндометриоз, эндокринные нарушения, маточные факторы. На долю женского фактора бесплодия приходится около 40% [4]. Такое же процентное соотношение мужского бесплодия. К фактору мужского бесплодия относят генетические, средовые, а также сочетанные факторы [13, 19, 20, 35, 42]. Однако, помимо вышеперечисленных факторов, встречается бесплодие неясной этиологии (идиопатическое), при котором нет очевидных (вышеперечисленных) факторов, но беременность не наступает. Профессионалы различных специальностей продолжают исследования, пытаясь обнаружить факторы, провоцирующие данную категорию бесплодия. Со стороны психологического и психотерапевтического сообщества также активно ведутся исследования причин бесплодия в парах с Общая психология, психология личности, история психологии По результатам работы с медицинской документацией подходящие под задачи исследования женщины вместе с партнером приглашалась на консультацию, в ходе которой разъяснялась цель и методы исследования, подписывалось информированное согласие (одобренное Этическим комитетом СПБГУ №02-174). Далее проводилось авторское интервью с беременной женщиной и ее партнером, по желанию пары проходили интервью совместно или индивидуально. Продолжительность первого интервью для женщины в среднем составила 40 минут, для мужчины – 15. После рождения ребенка женщина участвовала во втором интервью. Первое интервью для женщин состояло из 67 вопросов, направленных на изучение мотива беременности, оценку уровня родительской компетентности, оценку знаний о фетальном алкогольном синдроме (ФАС), изучалась эмоциональная окраска беременности, образ жизни до и вовремя беременности, родительские ожидания и установки, субъективная оценка наличия/отсутствия тревожности, страха во время беременности, перспективы построения модели родительского поведения, ряд вопросов о воспоминаниях собственного детства и контакте со своими родителями. Интервью для партнера включало 23 вопроса, определяющих возраст партнера, род занятий, наличие детей с другим партнером, мотив отцовства, субъективную оценку семейных взаимоотношений и своей готовности к отцовству, а также ряд вопросов о здоровье и образе жизни. Второе интервью проводилось с женщиной в течение первой недели после рождения ребенка, очно (при рождении ребенка в СПб ГПЦ №1), заочно при рождении ребенка в другом медицинском учреждении. Включало в себя ряд медицинских вопросов (по возможности заполнялось исследователем согласно данным медицинской карты): способ родоразрешения, наличие/отсутствие осложнений в процессе ведения родов, состояние новорожденного ребенка в первые минуты жизни, наличие/отсутствие реанимационных мероприятий для новорожденного, – а также 12 устных вопросов о субъективном восприятии женщиной процесса родов и контакте с новорожденным ребенком в раннем послеродовом периоде. Социально-демографические особенности выборки. 96% пар состоят в зарегистрированном браке. Средний возраст женщин, беременных с помощью ВРТ, составил 34,8 лет, партнера – 36 лет. Средний возраст женщин с физиологической беременностью – 31 год, партнера – 32 года. 19% (n=15) пар из первой группы и 15% из второй (n=12) имеют детей. Вестник Мининского университета. 2021. Том 9, №4 Результаты исследования Работа с медицинской документацией позволила выявить наиболее распространённые соматические заболевания исследуемой выборки. В обеих группах наиболее часто встречающимся заболеванием является гастрит и миопия различной степени тяжести. В первой группе исследуемых женщин гастрит встречается у 8 (10%), миопия – у 17 (21%) респонденток. Во второй группе женщин как гастрит, так и миопия встречаются у 11 (14%). Другие представленные в исследуемой группе женщин соматические заболевания распространены значительно реже. Работа с медицинской документацией на втором этапе исследования (после рождения ребенка) позволила детально изучить особенности течения процесса родов и неонатальные показатели новорожденного ребенка. На 2 этапе исследования были проанализированы истории 100 родов, 49 женщин из первой группы и 51 женщина из второй. Вестник Мининского университета. 2021. Том 9, №4 Вестник Мининского университета. 2021. Том 9, №4 Вестник Мининского университета. 2021. Том 9, №4 General psychology, personality psychology, history of psychology General psychology, personality psychology, history of psychology Рисунок 2 – Распределение способа родоразрешения в исследуемой выборке Figure 2 – Distribution of the delivery method in the study sample 0 5 10 15 20 25 30 35 40 45 50 Плановое кесарево сечение / Planned caesarean section Экстренное кесарево сечение / Emergency C- section Через естественные родовые пути / Natural childbirth группа 1 / group 1 группа 2 / group 2 Рисунок 2 – Распределение способа родоразрешения в исследуемой выборке р р р р у Figure 2 – Distribution of the delivery method in the study sample р р р р у р Figure 2 – Distribution of the delivery method in the study sample Оперативным путем родоразрешены 29 (60%) женщин первой группы, среди них 17 (34%) в плановом порядке, 12 (25%) в экстренном. Во второй группе 6 (12%) женщин с физиологической беременностью были родоразрешены с помощью операции кесарева сечения. Из них 2 (4%) в плановом порядке, 4 (8%) в экстренном. Роды через естественные родовые пути произошли у 20 (40%) женщин из первой группы и 44 (88%) женщин из второй группы (рисунок 3). В двух случаях женщинам из первой группы понадобилось переливание крови. В 1 случае из первой группы и 10 (12%) случаях из второй группы женщинам была выполнена амниотомия. Работа с медицинской картой новорожденного ребенка позволила оценить состояние ребенка в первые сутки после рождения (рисунок 4). Vestnik of Minin University. 2021. Volume 9, no. 4 Результаты исследования Рисунок 3 – Неонатальная оценка новорожденного Figure 3 – Neonatal assessment of a newborn 0 5 10 15 20 25 30 35 40 0-5 баллов / scores 7-8 баллов / scores 5-9 баллов группа 1 / group 1 группа 2 / group 2 Рисунок 3 – Неонатальная оценка новорожденного Figure 3 – Neonatal assessment of a newborn 5 (10%) детей от женщин первой группы имели очень низкий (ниже 5) балл по шкале Апгар. Дети из второй группы не получали оценку ниже 5 баллов. Оценку 7-8 получили равное количество детей в двух группах – 10 (20%). В 8-9 баллов были оценены 27 (55%) детей из первой группы и 35 (70%) детей из второй группы. Данные показатели указывают на большую тяжесть состояния детей, рожденных от матерей первой группы. Vestnik of Minin University. 2021. Volume 9, no. 4 Вестник Мининского университета. 2021. Том 9, №4 General psychology, personality psychology, history of psychology Рисунок 5 – Мотив наступления беременности в группе 2 (женщины беременные в естественном цикле) Figure 5 – The motive of pregnancy in group 2 (women who are pregnant in the natural cycle) 19% 27% 24% 11% 3% 3% 3% 10% пришло время / the time has come естественное продолжение семьи / the natural continuation of the family женская реализация / female realization любовь к детям / love for children по настоянию родственников / pressure of relatives быть взрослой / being an adult продолжение себя / continuation of yourself случайная беременность / accidental pregnancy 10% Рисунок 5 – Мотив наступления беременности в группе 2 (женщины беременные в естественном цикле) Figure 5 – The motive of pregnancy in group 2 (women who are pregnant in the natural cyc Figure 5 – The motive of pregnancy in group 2 (women who are pregnant in the natural cycle) Из приведенных на рисунке 5 и 6 данных следует, что в первой группе женщин доминирующим мотивом является «собственная женская реализация себя как матери» (38 женщин, 47%). В данном случае беременность расценивается женщиной как желание реализовать одну из сторон своей личности, обрести женскую идентичность. Доминирующим мотивом во второй группе женщин является «естественное продолжение семьи» (n=21, 26%). Ребенок является желанным объектом, логичным продолжением партнерства двух любящих людей. При этом во второй группе в 8 (10%) парах беременность была незапланированной, однако окрашена положительными эмоциями. Довольно высок в обеих группах ответ «пришло время», в 1 группе – 10 (12%), во 2й – 15 (19%). Ответ «по настоянию родственников», в котором прослеживается внешнее социальное давление на женщину, чаще встречается в первой группе женщин – 9 (11%). Наиболее позитивный мотив, ориентированный на ребенка как на самостоятельную полноценную личность, звучал в ответе как «любовь к детям» у 9 (11%) женщин второй группы, что в 3 раза выше, чем в первой группе. Принявшие участие в исследовании мужчины затруднялись сформировать мотив отцовства, наиболее часто встречаемым ответом на вопрос «С чем связано желание иметь ребенка?» в обеих группах был «Так положено». Такой ответ был получен от 18 (22%) мужчин из первой группы и от 24 (28%) мужчин второй группы. Вторым по частоте встречаемости был ответ «Жена хотела ребенка», так ответили 24 (28%) мужчины в первой группе и 20 (22%) мужчин из второй группы. Вышеописанное репродуктивное поведение мужчин уводит деторождение, воспитательные родительские функции в зону компетенции женщины. Мужчины оставляют за собой пассивную воспитательскую роль, но берут на себя ответственность за финансовые отношения. Общая психология, психология личности, история психологии Общая психология, психология личности, история психологии Все компоненты интервью можно разделить на несколько основных условных блоков. Первый блок вопросов направлен на сбор социально-демографической информации. Второй позволял оценить мотив наступления беременности, субъективную оценку готовности к родительству, представления о материнстве. Третий блок оценивал супружеские взаимоотношения в паре, представления о переменах, предстоящих в семье с появлением ребенка, количество времени, направленное на уход за ребенком. Четвертый блок включал вопросе об образе жизни до беременности и после ее наступления. Одним из разделов интервью выступила субъективная оценка готовности к родительству как у женщин, так и у их партнеров. Свою готовность к материнству женщины обеих групп оценили достаточно высоко, женщины первой группы (ВРТ-беременность) в 7,77±1,86, где 10 – максимально возможный балл. Женщины второй группы (физиологическая беременность) оценили готовность к материнству в 7,55 ± 1,82 баллов. Партнёры из первой группы оценили свою готовность к родительству в 8,42±1,82, а партнеры из второй группы в 7,45±1,84. Респондентам задавался вопрос «Как давно появилось желание стать матерью?»: в первой группе женщин данное желание отмечается в течение 5 лет предшествовавших неудачным попыткам забеременеть. Женщинам из второй группы такое желание присуще в течение двух лет. Подготовка к данной беременности у женщин с ВРТ-беременностью в среднем заняла 12 месяцев, у женщин с физиологической беременностью – 3 месяца. Одним из важных исследуемых факторов психологической готовности к материнству является мотив появления ребенка в семье. На рисунке 5 представлены наиболее часто встречающиеся ответы на вопрос «С чем связано желание иметь ребенка?». Рисунок 4 – Мотив наступления беременности в группе 1 (женщины беременные с помощью методов ВРТ) Figure 4 – Motive of pregnancy in group 1 (women who are pregnant using IVF methods) 13% 12% 49% 3% 12% 6% 5% 0% пришло время / the time has come естественное продолжение семьи / the natural continuation of the family женская реализация / female realization любовь к детям / love for children давление родственников / pressure of relatives быть взрослой / being an adult продолжение себя / continuation of yourself случайная беременность / accidental pregnancy Рисунок 4 – Мотив наступления беременности в группе 1 (женщины беременные с помощью методов ВРТ) ( р ) Figure 4 – Motive of pregnancy in group 1 (women who are pregnant using IVF methods) Вестник Мининского университета. 2021. Том 9, №4 General psychology, personality psychology, history of psychology General psychology, personality psychology, history of psychology Отвечая на вопрос «Как вы готовились к появлению ребенка», большинство исследуемых мужчин выбрали ответ «Покупал необходимые для ребенка вещи». Такой ответ дали 36 (49%) партнеров женщин первой Vestnik of Minin University. 2021. Volume 9, no. 4 Общая психология, психология личности, история психологии группы и 27 (38%) партнёров женщин второй группы. По 18 (25%) мужчин выбрали ответ «Никак». «Читаю специальную литературу» и «Посещаю курсы молодых отцов» ответили 15 (21%) партнеров женщин первой группы и 10 (14%) партнёров женщин второй группы. На рисунке 6 изображено количество женщин, испытывающих напряжение перед предстоящими родами. Рисунок 6 – Субъективная оценка наличия страха перед предстоящими родами Figure 6 – Subjective assessment of the presence of fear of impending childbirth 69% 48% 0 0 группа 1 / group 1 группа 2 / group 2 группа 1 / group 1 группа 2 / group 2 Рисунок 6 – Субъективная оценка наличия страха перед предстоящими родами Figure 6 – Subjective assessment of the presence of fear of impending childbirth 39 (48%) женщин из первой группы и 55 (69%) женщин из второй группы отмечают у себя напряженное ожидание, страх перед предстоящими родами. Присутствие партнера на родах планируют 31 (39%) женщина из первой группы и 23 (29%) женщины второй группы. Представления о своих идеальных родах у респонденток двух групп схожи, однако женщины из первой группы в 19 случаях (20%) предпочли бы запланированное родоразрешение с помощью операции кесарево сечение. При этом большая часть женщин первой группы планирует родоразрешение через естественные родовые пути – 28 (35%). Женщины из второй группы более ориентированы на роды через естественные родовые пути – 48 (62%), и 7 (8%) предпочли бы программированные роды с помощью операции кесарева сечения. Проанализировав представления женщин об «идеальных родах», можно заключить, что женщины из первой группы более ориентированы на стороннюю помощь, присутствие партнера, запланированную на определенную дату операцию кесарева сечения и менее ориентированы на собственные ресурсы. По мнению ряда зарубежных авторов, важным показателем готовности к материнству является смена образа жизни на более здоровый, отказ обоих партнеров от употребления психоактивных веществ (ПАВ) на этапе подготовки к беременности. В интервью был включен ряд вопросов о сознательной подготовке к беременности, включающей в себя прием витаминов, отказ от вредных привычек, улучшение качества питания и профилактическую физическую нагрузку, а также прохождение регулярных плановых медицинских осмотров. По полученным в ходе исследования данным, плановой подготовкой к беременности занимались 79 (98%) партнеров из первой группы и 71 (88%) из второй исследуемой группы. Что указывает на 10%-ную разницу между двумя исследуемыми группами. Вестник Мининского университета. 2021. Том 9, №4 General psychology, personality psychology, history of psychology о беременности и вреде никотина для плода. 159 (99%) женщин выборки отмечают, что принимали в период беременности какие-либо лекарства, витамины, биологически активные добавки. Алкоголь вплоть до диагностирования беременности употребляли 37 (46%) женщин из первой группы и 43 (54%) из второй. В период вынашивания беременности периодически употребляли алкоголь 13 (16%) женщин из первой группы и 20 (25%) женщин второй группы. При этом 55 (69%) женщин из первой группы и 67 (84%) женщин из второй группы знают о негативном влиянии алкоголя на ребенка и о ФАС. Изучалось употребление алкоголя партнером в период беременности женщины. Так, 50 (68%) мужчин первой группы отметили, что продолжают употреблять алкоголь с прежней регулярностью. Во второй группе – 53 (72%) партнёра. Все женщины, принимающие участие в исследовании, отмечают, что имеют полноценный ночной сон не менее 8 часов, по потребности могут позволить себе отдохнуть днем. В распорядке дня регулярно присутствует легкая пешая прогулка средней продолжительностью около одного часа. Женщины из первой группы отметили изменение пищевых привычек в период беременности и указали на употребление в два раза большего числа овощей и фруктов, в среднем по 6 штук в день, женщины второй группы в среднем употребляют по 3 овоща/фрукта в день. Одной из задач интервьюирования являлось изучение взаимоотношений между супругами, оценка уровня родительской компетенции и фантазий о себе и своем партнере в роли родителя. Обоим партнерам было предложено оценить удовлетворенность отношений в браке от 0 до 10 баллов, где 10 – максимально благополучный вариант. Женщины из первой группы оценили удовлетворенность браком в 8,5 баллов, а их партнеры в 9 баллов. Женщины второй группы оценили свои супружеские взаимоотношения в 9 баллов, а партнеры в 8. Высокая субъективная оценка супружеских взаимоотношений в обеих группах не имеет значительных различий и находится в благополучном диапазоне. Посещением «Школы молодых родителей», чтением литературы, прослушиванием онлайн-семинаров, направленных на получение информации по уходу за новорожденным ребенком занимались 20 (25%) женщин первой группы, что на 16% меньше, чем женщин второй группы – 33 (41%). За профессиональной психологической помощью обратились 9 (11%) женщин из первой группы, 5 из которых – находясь в стационаре, по рекомендации лечащего врача. Темой обращения являлся страх невынашивания беременности, повышенная тревога по незначительному поводу, тонус матки, проработка собственных детских психотравм, а также отсутствие «моральной готовности к появлению ребенка», невозможность контакта с ребенком. General psychology, personality psychology, history of psychology 18 (9%) из всех исследуемых женщин сознательно отказались от курения в период планирования и вынашивания беременности, не отрицая вероятность возвращения к курению после рождения ребенка. 154 (94%) женщины всей выборки воздерживались от курения во время беременности, однако 7 (6%) продолжили курить, несмотря на знание Вестник Мининского университета. 2021. Том 9, №4 Vestnik of Minin University. 2021. Volume 9, no. 4 General psychology, personality psychology, history of psychology Во второй исследуемой группе женщин профессиональную психологическую помощь получили 2 (2,5%) женщины, обе по настоянию лечащего врача в связи со страхом родов через естественные родовые пути и непреодолимым страхом потери беременности (женщины с осложненным репродуктивным анамнезом). В декретный отпуск планируют пойти все 100% женщин из первой группы и 76 (93%) женщин второй группы. Более чем треть женщин как в группе беременных с помощью ВРТ (38 женщин, 47%), так и забеременевших физиологическим путем (30 женщин, 37,5%) планируют находиться в отпуске по уходу за ребенком до трех лет. До полутора лет 10 (12%) женщин первой группы и 24 (30%) женщины второй группы. До двухлетнего возраста ребенка планируют находится в декрете 16 (20%) женщин первой группы и 16 (21%) женщин второй группы. На рисунке 7 представлены наиболее часто встречаемые ответы на вопрос «Каким вам кажется время в декретном отпуске?». Vestnik of Minin University. 2021. Volume 9, no. 4 Vestnik of Minin University. 2021. Volume 9, no. 4 Общая психология, психология личности, история психологии Рисунок 7 – Субъективная оценка времени по уходу за ребенком Figure 7 – Subjective assessment of child care time 0 5 10 15 20 25 30 тревожным / anxious заботливым / caring интересным / interesting скучным / boring счастливым / happy группа 1 / group 1 группа 2 / group 2 Рисунок 7 – Субъективная оценка времени по уходу за ребенком Figure 7 – Subjective assessment of child care time Из приведенных на рисунке 7 анализируемых ответов очевидным является преобладание тревоги и скуки в первой группе женщин и преобладание интереса и счастья во второй группе женщин. Большая часть принявших участие в исследовании мужчин планирует ежедневно проводить время с ребенком – 61 (84%) партнёр первой группы и 55 (78%) партнеров второй группы. Ответ «Только в выходные» дали трое (4,5%) мужчин первой группы и двое (3%) мужчин второй группы. В интервью был включен вопрос о субъективном восприятии ощущения шевелений ребенка. 85 (53%) женщин всей выборки описывают его как приятный позитивный опыт контакта с ребенком – «нежность и счастье», 10 (6%) с оттенком тревоги – «если шевелиться, значит живой», 58 (36%) дают нейтральную окраску и 8 (5%) отмечают раздражение: «нет, это совсем не благость, когда он скачет по моему мочевому пузырю». Для изучения преобладающего фона настроения в первом триместре беременности женщин просили вспомнить и описать свое эмоциональное состояние в начале беременности (рисунок 8). General psychology, personality psychology, history of psychology угнетенное с периодическими приступами паники. 8 (12%) женщин первой группы и 5 (8%) женщин второй группы описали свое настроение как эйфорическое. Как крайне переменчивое: то желание «сдохнуть поскорее», то «радостно приподнятое» – описали свое настроение 8 (10%) женщин первой и 5 (8%) женщин второй группы. Финальная часть первого интервью для женщин содержала ряд вопросов о воспоминаниях собственного детства и несколько неоконченных предложений, которые женщина должна была продолжить первыми пришедшими в голову воспоминаниями. Ответы двух групп женщин схожи, наиболее встречаемым ответом, завершающим предложение «Я хочу быть матерью…» был ответ «…матерью здорового ребенка», так ответили 29 (18%) из всех исследуемых женщин, на втором месте по частоте встречаемости «…и я ей скоро буду» (n=20, 12%), третьим по частоте встречаемости был ответ «…большого количества детей» (n=15, 9%). Остальные ответы были единичны. Однако следует отметить тот факт, что 18 беременных женщин из первой группы (22%) дали ответ «Своих детей», что подчеркивает важность, не просто наличия в семье ребенка, а именно генетически своего ребенка. Очевидной разницы в продолжении предложения «Я буду очень счастлива, когда…» респондентки двух групп также не высказали. 146 (90%) участниц завершили предложение фразой «…когда родится здоровый ребенок», 6 (4%) ответили, что уже счастливы, 8 (6%) давали ответ, не связанный с беременностью, например, такой как «…когда добьюсь поставленных в карьере целей». В завершении предложения «Я опасаюсь…» только беременные женщины из группы ВРТ-беременности давали ответ «…преждевременных родов» (n=12, 15%), а также только в этой группе был дан ответ «…разочарования в материнстве» (n=8, 10%). Остальные ответы в двух группах совпадали, самым распространённым ответом был «…проблем со здоровьем у ребенка или моим собственным здоровьем» (n=50, 31%), «…последствий после родов и самих родов» (n=45, 28%), «…что воспитаю неправильно» (n=7, 5%). Часть вопросов интервью была направлена на изучение родительской семьи и своих представлений о детстве. Большая часть респонденток дает положительную окраску детскому периоду в своей жизни и оценивает его как «радостное и хорошее» – 72 (90%) женщины первой группы и 68 (85%) женщин второй группы. Присутствие элементов поощрения и наказания в детстве отмечают 31 (38%) женщина первой группы и 43 (54%) женщины второй группы. 47 (58%) женщин первой группы и 44 (55%) женщины второй группы имели сиблинга. 7 (8%) женщин с ВРТ-беременностью и 8 (9%) с физиологической беременностью воспитывались только матерью. При анализе взаимоотношений женщины с собственной матерью, теплые, близкие отношения описывают 26 (32%) респонденток из первой группы и 41 (51%) женщина второй группы. General psychology, personality psychology, history of psychology Рисунок 8 – Субъективная оценка эмоционального состояния в первом триместре беременности Figure 8 – Subjective assessment of the emotional state in the first trimester of pregnancy 0 5 10 15 20 25 30 35 40 45 50 положительное / positive нейтральное /neutral депрессивное / depressive эйфорическое / euphoric неустойчивое / unstable группа 1 / group 1 группа 2 / group 2 Рисунок 8 – Субъективная оценка эмоционального состояния в первом триместре беременности Figure 8 – Subjective assessment of the emotional state in the first trimester of pregnancy 32 (40%) женщины первой группы и 44 (54%) женщины второй группы отметили преобладание хорошего, спокойного настроения. 10 (13%) женщин первой группы и 12 (10%) женщин в группе с ВРТ-беременностью обозначили свое настроение как «нейтральное, нормальное». 24 (28%) женщины первой группы отметили у себя депрессивное, подавленное настроение, 12 (15%) женщин второй группы описали настроение как тревожное, волнительное, 32 (40%) женщины первой группы и 44 (54%) женщины второй группы отметили преобладание хорошего, спокойного настроения. 10 (13%) женщин первой группы и 12 (10%) женщин в группе с ВРТ-беременностью обозначили свое настроение как «нейтральное, нормальное». 24 (28%) женщины первой группы отметили у себя депрессивное, подавленное настроение, 12 (15%) женщин второй группы описали настроение как тревожное, волнительное, Вестник Мининского университета. 2021. Том 9, №4 Вестник Мининского университета. 2021. Том 9, №4 Vestnik of Minin University. 2021. Volume 9, no. 4 General psychology, personality psychology, history of psychology Отношения с отцом ближе у 25 (31%) женщин первой группы и 30 (37,5%) женщин второй группы. Также в интервью женщин просили описать свои взаимоотношения с матерью в детстве путем ассоциаций, используя хотя бы 5 слов, использование большего количества слов поощрялось. Эта задача более чем для половины респонденток оказалась сложной, они отказывались отвечать на этот вопрос. Те же, кто старался выполнить задание, испытывали трудности в подборе 5 слов. Только 18 (11%) женщин выполнили задание с легкостью и привели более 5 ассоциаций. Интересным явился фактор благополучия взаимоотношений с собственной матерью в детстве в группе женщин с физиологической беременностью. 63 (79%) описывали отношения как «теплые, заботливые, нежные, уважительные, доверительные, добрые», 4 (5%) дали негативную окраску взаимоотношений с матерью «ссоры, скандалы, недоверие», 3 (4%) основным критерием выделили контроль и Vestnik of Minin University. 2021. Volume 9, no. 4 Общая психология, психология личности, история психологии тревогу. Высок процент неблагополучных воспоминаний о детско-родительских отношениях с матерью в первой (ВРТ) группе женщин. 40 (50%) женщин описали взаимоотношения с матерью как «холодность, равнодушие, тревога, подчинение, строгость», 8 (10%) основным критерием выделили то, что мать много работала и редко занималась воспитанием (более близкие взаимоотношения у данных женщин были с другими членами семьи, отцом или бабушкой) и 15 (19%) описывают взаимоотношения как теплые и доверительные. Таким образом, по данному параметру две группы исследуемых женщин заметно различаются между собой. Общее число положительных оценок взаимоотношений с матерью в первой группе женщин – 15 (19%) и 63 (79%) во второй группе женщин. Данный феномен упоминается В.Е. Гавриловой [7]. Описывая свои взаимоотношения с отцом в детстве, женщины первой группы в 27 (34%) случаях оценивали их как «никакие», в 26 (32,5%) – как «любовь, спокойствие, радость, гордость, юмор, я, наверное, больше папина дочка», в 12 (15%) – как «контроль, эмоциональная дистанция, периодические наказания, отстранённость» и 16 (20%) описывали отношения как «уважительные, но он много работал». Ответы женщин второй группы можно разделить на три категории: наиболее часто встречаемый ответ – «защита, любовь, воспитание, поддержка, дружба, помощь» – 50 (62,5%), 16 (20%) женщин не могли дать какую-либо характеристику взаимоотношениям с отцом, 14 (17,5%) охарактеризовали взаимоотношения с отцом негативно – «безответственность, алкоголизм, ссоры». Из приведенных ответов можно заключить, что женщины первой исследуемой группы в два раза реже имели близкие отношения с отцом, чем женщины второй группы. Завершающее интервью с участием только женщины проводилось в первую неделю после рождения ребенка. В нем оценивалось субъективное восприятие родов и первичного контакта с ребенком. Вестник Мининского университета. 2021. Том 9, №4 General psychology, personality psychology, history of psychology На вопрос о кормлении грудью утвердительно ответили 24 (51%) женщины из первой группы и 30 (73%) женщин второй группы. В таблице 2 представлены наиболее частые ответы, описывающие субъективные ощущения женщин в период кормления грудью. Таблица 2 – Субъективная оценка эмоциональных и физических ощущений в период кормления грудью / Table 2 – Subjective assessment of emotional and physical sensations during breastfeeding Категория ощущений / Sensation category Женщины с ВРТ- беременностью pregnant / women using IVF methods (n=24) Женщины с физиологической беременностью / pregnant women in a natural cycle (n=30) Приятные, нежные чувства / Pleasant, tender feelings 10 (41%) 18 (60%) Нейтрально / Neutral 2 (5%) 6 (20%) «Пока не поняла, как мне весь этот процесс» / "Until I understood how this whole process is for me" 9 (37%) 4 (13%) Неприятные чувства и физический дискомфорт / Feelings and physical discomfort 4 (17%) 2 (7%) Субъективная оценка эмоциональных и физических ощущений в период кормления e 2 – Subjective assessment of emotional and physical sensations during breastfeeding В первой группе женщин 2 лидирующих ответа: описывающий процесс кормления как приятный – 10 (41%) и «пока не поняла, нравится ли мне этот процесс или нет» – 9 (37%). Наиболее часто встречаемым ответом во второй группе женщин является ответ, описывающий приятные, нежные ощущения контакта с ребенком – 18 (60%). Нейтральное отношение к грудному вскармливанию у 2 (5%) женщин первой группы и 6 (20%) женщин второй группы. Негативно описали контакт с новорожденным ребенком в период грудного вскармливания 4 (17%) женщины первой группы и 2 (7%) женщины второй группы. Полученные результаты указывают на то, что женщины первой группы испытывают меньше положительных откликов, нежности и теплоты по отношению к новорожденному и воспринимают кормление грудью как необходимый процесс. На вопрос «Испытываете ли вы сложности в уходе за новорожденным ребенком» утвердительно ответили 19 (40%) женщин первой группы и 4 (10%) женщины второй группы. Наличие помощи родственников отметили 15 (32%) женщин первой группы и 4 (10%) женщины второй группы. Из данных ответов можно сделать вывод о том, что женщины первой группы менее самостоятельны, нуждаются в дополнительной помощи и поддержке со стороны близких; женщины второй группы опираются на себя. Основные ответы, описывающие первую ночь наедине с новорожденным ребенком приведены в таблице 3. General psychology, personality psychology, history of psychology В данном интервью приняли участие 51 женщина из первой группы и 49 женщин второй группы. В родах женщин из первой группы сопровождали в 8 (17%) случаях, из них в 5 (11%) случаях муж, в 2 (4%) мама, в 1 (2%) сестра. Женщин из второй группы в процессе рождения ребенка сопровождали в 4 (10%) родах, из них в 3 (8%) муж, в 1 (2%) доула. 13 (28%) родов в группе женщин, беременных с помощью ВРТ, велись по индивидуальному договору с врачом, 21 (72%) по ОМС. Во второй группе женщин индивидуальный договор с врачом был заключен в 6 (17%) случаях, роды по ОМС – 35 (83%). В таблице 1 приведены варианты описания своего телесного и эмоционального состояния в первые дни после рождения ребенка. Таблица 1 – Субъективные описания женщинами своих ощущений после родов / Table 1 – Women's subjective descriptions of their feelings after childbirth Характеристика ощущений / characteristics of sensations Женщины с ВРТ- беременностью / pregnant women using IVF methods (n=49) Женщины с физиологической беременностью / pregnant women in a natural cycle (n=51) «Все хорошо, роды позади» / «everything is fine, the birth is over» 11 (23%) 18 (43%) «Чувствую радость» / «I feel joy» 3 (6%) 4 (10%) «Хочу спать» / «I want to sleep» 3 (6%) 7 (17%) «Счастлива» /"Happy" 4 (8%) 1 (2%) «Беспокоюсь и тревожусь» /"I'm worried and worried" 14 (30%) 2 (5%) «Физически вымотана» /"Physically exhausted" 7 (15%) 6 (14%) Таблица 1 – Субъективные описания женщинами своих ощущений после родов / Table subjective descriptions of their feelings after childbirth Субъективные описания женщинами своих ощущений после родов / Table 1 – Women's riptions of their feelings after childbirth Вестник Мининского университета. 2021. Том 9, №4 General psychology, personality psychology, history of psychology Таблица 3 – Субъективная оценка восприятия женщиной первой совместной ночи с ребенком / Table 3 – Subjective assessment of a woman's perception of the first night together with a child Категория описания первой ночи после рождения / Category of description of the first night after birth Женщины с ВРТ- беременностью / pregnant women using IVF methods (n=47) Женщины с физиологической беременностью / pregnant women in a natural cycle (n=41) «Спокойно переночевали, с одним кормлением в середине ночи» / "We spent the night quietly, with 16 (34%) 23 (56%) Vestnik of Minin University. 2021. Volume 9, no. 4 Общая психология, психология личности, история психологии one meal in the middle of the night." «Тревожно и ничего не понятно» / "It's alarming and nothing is clear" 17 (36%) 9 (22%) «Он плакал, я пыталась его успокоить. Всю ночь не спали» / “He was crying, I tried to calm him down. We didn’t sleep all night ” 14 (30%) 9 (22%) Из данных, представленных в таблице 3, следует, что первую ночь 16 (34%) женщин первой группы и 23 (56%) женщины второй группы провели спокойно, отдыхая и восстанавливаясь после родов. 31 (66%) женщина первой группы и 18 (44%) женщин второй группы испытывали тревогу и дискомфорт. По 15 (34%) женщин из каждой группы отмечают наличие страхов. Часть из них обоснована пребыванием ребенка в отделении патологии новорожденных, часть из них не имеет объективной причины. Женщины высказывали такие переживания, как «боюсь, что сделаю ему что-то не то»; «как буду справляться с ним дома»; «а хватает ли ему еды»; «переживаю за его здоровье»; «что плохо его воспитаю»; «вообще за все связанное с ним теперь переживаю». Приведенные данные указывают на большой процент тревожности у женщин обеих исследуемых групп. Завершающим вопросом интервью был вопрос «Что вас сейчас радует?», в таблице 4 приведены основные ответы. Вестник Мининского университета. 2021. Том 9, №4 General psychology, personality psychology, history of psychology Таблица 4 – Субъективная оценка текущего психологического ресурса / Table 4 – Subjective assessment of current psychological resource Категория описания того, что радует в данный момент / Category of description of what pleases at the moment Женщины с ВРТ- беременностью / pregnant women using IVF methods (n=47) Женщины с физиологической беременностью / pregnant women in a natural cycle (n=41) «Что все хорошо у нас обоих» / "That everything is fine with both of us" 15 (32%) 14 (34%) «Скоро выписка домой» / "Check out soon home" 12 (25%) 5 (13%) «Мы вместе» / "We are together" 0 3 (6%) «У меня есть ребенок» / "I have a child" 0 4 (10%) «Я – мама» / "I am a mother" 10 (21%) 12 (30%) «Я пережила роды» / "I survived childbirth" 6 (12%) 3 (6%) «Пока мало что» / "Not much so far" 4 (10%) 0 Субъективная оценка текущего психологического ресурса / Table 4 – Subjective current psychological resource Таблица 4 – Субъективная оценка текущего психологического ресурса / Table 4 assessment of current psychological resource Помимо основных приведенных в таблице 4 ответов, женщин из группы физиологической беременности радует сам факт рождения ребенка – 4 (10%) и то, что мать теперь может держать своего ребенка на руках – 3 (6%). 4 (10%) женщины второй группы отметили, что на данный момент «ничего не радует», такие ответы звучали от матерей тяжело рожденных детей, находящихся в отделении интенсивной терапии при перинатальном центре №1 или переведённых в детские городские больницы города в тяжелом состоянии. Вестник Мининского университета. 2021. Том 9, №4 Вестник Мининского университета. 2021. Том 9, №4 General psychology, personality psychology, history of psychology Обсуждение и заключения По результатам проведенного исследования получены следующие выводы. Средний возраст как исследуемых женщин с беременностью, наступившей в результате ВРТ, так и их партнеров выше, чем в группе женщин с физиологической беременностью. Желание осуществления родительской программы осознается женщинами в 2,5 раза более долгом временном периоде. Подготовка к данной беременности у женщин с ВРТ-беременностью занимает в 4 раза более долгий срок. Доминирующим мотивом для наступления беременности в группе женщин с ВРТ-беременностью является мотив реализации женской идентичности, в группе физиологически беременных женщин – естественное продолжение семьи. Психологически более здоровый, ориентированный на ребенка мотив встречается в группе женщин, беременных с помощью ВРТ, в 2 раза реже (18% против 37%). Партнеры женщин обеих групп в большинстве случаев давали нейтральные, эмоционально слабо окрашенные ответы, указывающие на низкую личностную заинтересованность в отцовстве, скорее, как компромисс с супругой и социумом. Женщины, беременные с помощью ВРТ, в 3 раза более ориентированы на программированное кесарево сечение (20% против 8%), поддержку партнера в родах, а также индивидуальную акушерско-гинекологическую команду. Число акушерских и неонатальных осложнений в родах женщин, беременных с помощью ВРТ, в два раза выше. Эмоциональное состояние в период рождения ребенка и ранний послеродовый период в шесть раз более окрашено тревогой и беспокойством (30% против 5%). Физиологические и психологические трудности при грудном вскармливании, уходе за новорожденным ребенком в четыре раза выше в группе женщин, беременных с помощью ВРТ (44% против 10%). Половина всех исследуемых женщин выборки отмечают тревогу и дискомфорт в первые сутки после родов, которые постепенно сглаживаются. Полученные результаты важны в понимании эмоционально-психологического состояния женщин, беременных с помощью ВРТ, и значимости оказания своевременной психологической помощи на разных этапах беременности, родов и в раннем послеродовом периоде. Общая психология, психология личности, история психологии 5. Вагапова А.Р. Формирование представлений у молодежи о материнстве и отцовстве // Известия Саратовского университета. Новая серия. Серия Философия. Психология. Педагогика. 2015. Т. 15, №2. C. 31-35. 6. Васильева Е.Н. Гендерные различия в структурных компонентах психологической готовности к родительству // Современные проблемы науки и образования. 2013. №6. С. 853. 7. Гаврилова В.Е. Психологические предпосылки бесплодия у здоровых женщин, состоящих в браке // Вестник ЮУрГУ. Серия: Психология. 2018. Т. 11, №3. С. 103-109. DOI: http://dx.doi.org/10.14529/psy180312. 8. Гроф С. Путешествие в поисках себя. М.: АСТ, 2008. 248 с. 9. Добряков И.В. Перинатальная психология. 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Caregivers of Children with Fetal Alcohol Spectrum Disorder: Psychosocial Factors and Evidence for Self-compassion as a Potential Intervention Target // Mindfulness. 2020. Vol. 11, no. 9. Pp. 2189-2198. 22. Crosignani P.G., Rubin B.L. Optimal use of infertility diagnostic tests and treatments // The ESHRE Capri Workshop Group. Hum Reprod. 2000. Vol. 15, no. 3. Pp.723-732. DOI: 10.1093/humrep/15.3.723. 23. Christian L.M., Franco A., Iams J.D., Sheridan J., Glaser R. Depressive symptoms predict exaggerated inflammatory responses to an in vivo immune challenge among pregnant women // Brain, Behavior and Immunity. 2010. Vol. 24, no. 1. Pp. 49-53. DOI: 10.1016/j.bbi.2009.05.055. 24. Gabnai-Nagy E., Bugán A., Bodnár B., Papp G., Nagy B.E. Association between Emotional State Changes in Infertile Couples and Outcome of Fertility Treatment // Geburtshilfe Frauenheilkd. 2020. Vol. 80, no.2. DOI: 10.1055/a-0854-5987. 25. Golombok S. Modern families: Parents and children in new family forms. Cambridge, UK: Cambridge University Press, 2015. Рp. 111-113. 26. Khizroeva J., Nalli C., Bitsadze V., Lojacono A., Zatti S., Andreoli L., Tincani A., Shoenfeld Y., Makatsariya A. Infertility in women with systemic autoimmune diseases // Best Practice & Research Clinical Endocrinology & Metabolism. 2019. Vol. 33, no. 6. DOI: 10.1016/j.beem.2019.101369. 27. Kleanthi G., Alexithymia M. Stress and Depression in Infertile Women: a Case Control Study // Mater Sociomed. 2021. Vol. 33, no. 1. Pp.70-74. DOI:10.5455/msm.2021.33.70-74 28. Linder R. Overcoming Somatic and Psychological Difficulties: New Experiences from an Integrated Linkage of Obstetrics and Psychotherapy // Journal of Prenatal and Perinatal Psychology and Health. 2010. Vol. 24, no. 4. Pp. 201-215. 29. Marín-Morales D., Carmona-Monge F.J. Personality Depressive symptoms during pregnancy and their influence on postnatal depression in Spanish pregnant Spanish women // Anales de Psicología. 2014. Vol. 30, no. 3. Pp. 908-915. DOI: 10.6018/analesps.30.3.153101. 30. Maroufizadeh S., Navid B., Omani-Samani R., Amini P. The effects of depression, anxiety and stress symptoms on the clinical pregnancy rate in women undergoing IVF treatment // BMC Res Notes. 2019. Vol. 12, no.1. DOI: 10.1186/s13104-019-4294-0. 31. McCrory C., McNally C. The Effect of Pregnancy Intention on Maternal Prenatal Behaviours and Parent and Child Health: Results of an Irish Cohort Study // Paediatric and Perinatal Epidemiology. 2013. Vol. 27, no. 2. Pp. 208-215. DOI: 10.1111/ppe.12027. 32. Mustafa A., Ali A., Mustafa M., A., Samarraie M. Physiological and hormonal study of women infertility // Science Archives. 2020. Vol. 1, no. 3. Pp. 160-165. DOI: 10.47587/SA.2020.1314. 33. Общая психология, психология личности, история психологии 29-41. DOI: 10.2147/IJGM.S241099. Общая психология, психология личности, история психологии 36. Standeven L.R., McEvoy K.O., Osborne L.M. Progesterone, reproduction, and psychiatric illness // Best Practice & Research: Clinical Obstetrics & Gynaecology. 2020. Vol. 69. Pp.108- 126. DOI: 10.1016/j.bpobgyn.2020.06.001. 37. Szkodziak F., Krzyżanowski J., Szkodziak P. Psychological aspects of infertility // A systematic review. 2020. Vol. 48, no. 6. DOI: 10.1177/0300060520932403. 38. Turner K.A., Rambhatla A., Schon S., Agarwal A., Krawetz S.A., Dupree J.M., Avidor-Reiss T. Male Infertility is a Women's Health Issue-Research and Clinical Evaluation of Male Infertility Is Needed // Cells. 2020. Vol. 16, no. 9. DOI: 10.3390/cells9040990. 39. Thurston L, Abbara A, Dhillo WS. Investigation and management of subfertility // Journal of Clinical Pathology. 2019. Vol. 72, no. 9. DOI: 10.1136/jclinpath-2018-205579. 40. Vander Borght M., Wyns C. Fertility and infertility: Definition and epidemiology // Clinical Biochemistry. 2018. Vol. 62, no. 2. DOI: 10.1016/j.clinbiochem.2018.03.012. 41. Wall G. Mothers’ experiences with intensive parenting and brain development discourse // Women’s Studies International Forum. 2010. Vol. 33, no. 3. Pp. 253-263. 42. Xavier M.J., Salas-Huetos A., Oud M.S. Disease gene discovery in male infertility: past, present and future // Human Genetics. 2021. Vol. 140. Pp. 7-19. DOI: 10.1007/s00439-020- 02202-x. Вестник Мининского университета. 2021. Том 9, №4 General psychology, personality psychology, history of psychology Pandruvada S, Royfman R, Shah TA, Sindhwani P, Dupree JM, Schon S, Avidor-Reiss T. Lack of trusted diagnostic tools for undetermined male infertility // Journal Assist Reprod Genet. 2021. Vol. 38, no. 2. Pp. 265-276. DOI: 10.1007/s10815-020-02037-5. 34. Saunders T.A., Lobel M., Veloso C., Meyer B.S. Prenatal maternal stress is associated with delivery analgesia and unplanned cesareans // Journal of Psychosomatic Obstetrics and Gynecology. 2006. Vol. 27, no. 3. Рp. 141-146. DOI: 10.1080/01674820500420637. 35. Stark M.A., Brinkley R.L. The relationship between perceived stress and health-promoting behaviors in high-risk pregnancy // Journal of Perinatal and Neonatal Nursing. 2007. Vol. 21, no. 4. Pp. 307-314. Vestnik of Minin University. 2021. Volume 9, no. 4 General psychology, personality psychology, history of psychology 11. Prohorenko N.F., Ginoyan A.B. Reform of the compulsory health insurance system: time is running out. Vestnik VSHOUZ, 2018, no. 4, pp. 28-52. (In Russ.) 12. Savenysheva S.S. Satisfaction with marriage and attitude to pregnancy and child in pregnant women. Vestnik SPBGU, 2016, no. 53-11, pp. 239-246. (In Russ.) 13. Safina N.YU., YAmandi T.A., CHernyh V.B., Akulenko L.V., Bogolyubov S.V., Vityazeva I.I., Ryzhkova O.P., Stepanova A.A., Adyan T.A., Bliznec E.A., Polyakov A.V. Genetic factors of male infertility, their combinations and spermiological characteristics of men with impaired fertility. Andrologiya i genital'naya hirurgiya, 2018, vol. 19, no. 2, pp. 40-51, doi: https://doi.org/10.17650/2070-9781-2018-19-2-40-51. (In Russ.) 14. Skvorcova M.YU., Priluckaya S.G., Barskaya E.S. Features of the psychoemotional state of women during pregnancy resulting from the use of assisted reproductive technologies. Doktor.Ru, 2018, no. 10(154), pp. 62-67, doi: 10.31550/1727-2378-2018-154-10-62-67. (In Russ.) 15. Ul'yanich A.L., Bohan T.G., Leshchinskaya S.B., Agarkova L.A., Kucenko I.G., Miheenko G.A. Features of the internal picture of pregnancy in women with somatic complications with the threat of termination of pregnancy. Vestnik Kemerovskogo gosudarstvennogo universiteta, 2019, vol. 21, no. 2(78), pp. 435-450, doi: http://dx.doi.org/10.21603/2078-8975-2019-21-2- 435-450. (In Russ.) 16. Hachatryan L.A. Trends in changes in the modern Russian family. Vestnik Permskogo universiteta. Filosofiya. Psihologiya. Sociologiya, 2014, no. 4(20), pp. 111-118. (In Russ.) 17. SHamilova N.V. Psychological reasons for impaired adaptation to pregnancy and motherhood. Problemy sovremennogo pedagogicheskogo obrazovaniya, 2016, no. 50-2, pp. 226-232. (In Russ.) 18. Astley S.J. Fetal alcohol syndrome prevention in Washington State: evidence of success. Paedietric and perinatal Epidemiology, 2004, vol. 18, no. 5, doi: 10.1111/j.1365- 3016.2004.00582.x 33. 19. Alahmar F., Ahmed T. Role of Oxidative Stress in Male Infertility: An Updated Review. Journal of human reproductive sciences, 2019, vol. 12, no. 1, pp. 4-18, doi: 10.4103/jhrs.JHRS_150_18. 20. Babakhanzadeh E. Some of the Factors Involved in Male Infertility: A Prospective Review. International journal of general medicine, 2020, vol. 13, pp. 29-41, doi: 10.2147/IJGM.S241099. 21. Biddle Z., O’Callaghan F.V., Finlay-Jones A.L., Reid N.E. Caregivers of Children with Fetal Alcohol Spectrum Disorder: Psychosocial Factors and Evidence for Self-compassion as a Potential Intervention Target. Mindfulness, 2020, vol. 11, no. 9, pp. 2189-2198. 22. Crosignani P.G., Rubin B.L. Optimal use of infertility diagnostic tests and treatments. The ESHRE Capri Workshop Group. Hum Reprod, 2000, vol. 15, no. 3, pp.723-732, doi: 10.1093/humrep/15.3.723. 23. Christian L.M., Franco A., Iams J.D., Sheridan J., Glaser R. Vestnik of Minin University. 2021. Volume 9, no. 4 References 1. Andreeva A.D. The image of family and parenting in modern mothers: a cross-cultural study. Teoreticheskaya i eksperimental'naya psihologiya, 2016, vol. 9, no. 2, pp. 17-30. (In Russ.) 2. Bojko E.L., Posiseeva L.V., Malyshkina A.I. Rehabilitation of married couples with early miscarriage in history. Medicinskij sovet, 2014, no. 9. Available at: https://cyberleninka.ru/article/n/reabilitatsiya-supruzheskih-par-s-nevynashivaniem- beremennosti-rannih-srokov-v-anamneze (accessed: 01.02.2021). (In Russ.) 3. Bloh M.E. Dynamics of marital relations during pregnancy. Reproduktivnoe zdorov'e sem'i v perinatal'noj psihologii: materialy mezhdunarodnoj nauchno-prakticheskoj konferencii. St. Petersburg, 2013. Pp. 37-41. (In Russ.) 4. Burina E.A., Moshkivskaya V.A., Kulieva A.K. Personal characteristics of women who are pregnant as a result of the use of assisted reproductive technologies. Anan'evskie chteniya – 2020. Materialy mezhdunarodnoj nauchnoj konferencii. St. Petersburg, SPBGU Publ., 2020. Pp. 980-981. (In Russ.) 5. Vagapova A.R. Formation of ideas among young people about motherhood and fatherhood. Izvestiya Saratovskogo universiteta. Novaya seriya. Seriya Filosofiya. Psihologiya. Pedagogika, 2015, vol. 15, no. 2, pp. 31-35. (In Russ.) 6. Vasil'eva E.N. Gender differences in the structural components of psychological readiness for parenthood. Sovremennye problemy nauki i obrazovaniya, 2013, no. 6, p. 853. (In Russ.) 7. Gavrilova V.E. Psychological preconditions of infertility in healthy married women. Vestnik YUUrGU. Seriya: Psihologiya, 2018, vol. 11, no. 3. pp. 103-109, doi: http://dx.doi.org/10.14529/psy180312. (In Russ.) 8. Grof S. Travel in search of yourself. Moscow, AST Publ., 2008. 248 p. (In Russ.) 9. Dobryakov I.V. Perinatal psychology. St. Petersburg, Piter Publ., 2010. 272 p. (In Russ.) 10. Dobryakov I.V. Features of the course of the perinatal period and the psychomotor development of an infant depending on the type of psychological component of the gestational dominant. Psihicheskoe zdorov'e, 2010, no. 12, pp. 13-18. (In Russ.) Вестник Мининского университета. 2021. Том 9, №4 General psychology, personality psychology, history of psychology Общая психология, психология личности, история психологии Общая психология, психология личности, история психологии Research Clinical Endocrinology & Metabolism, 2019, vol. 33, no. 6, doi: 10.1016/j.beem.2019.101369. Research Clinical Endocrinology & Metabolism, 2019, vol. 33, no. 6, doi: 10.1016/j.beem.2019.101369. 27. Kleanthi G, Alexithymia M. Stress and Depression in Infertile Women: a Case Control Study. Mater Sociomed, 2021, vol. 33, no. 1, pp. 70-74, doi: 10.5455/msm.2021.33.70-74 28. Linder R. Overcoming Somatic and Psychological Difficulties: New Experiences from an Integrated Linkage of Obstetrics and Psychotherapy. Journal of Prenatal and Perinatal Psychology and Health, 2010, vol. 24, no. 4, pp. 201-215. 29. Marín-Morales D., Carmona-Monge F.J. Personality Depressive symptoms during pregnancy and their influence on postnatal depression in Spanish pregnant Spanish women. Anales de Psicología, 2014, vol. 30, no. 3, pp. 908-915, doi: 10.6018/analesps.30.3.153101. 30. Maroufizadeh S., Navid B., Omani-Samani R., Amini P. The effects of depression, anxiety and stress symptoms on the clinical pregnancy rate in women undergoing IVF treatment. BMC Res Notes, 2019, vol. 12, no. 1, doi: 10.1186/s13104-019-4294-0. 31. McCrory C., McNally C. The Effect of Pregnancy Intention on Maternal Prenatal Behaviours and Parent and Child Health: Results of an Irish Cohort Study. Paediatric and Perinatal Epidemiology, 2013, vol. 27, no. 2, pp. 208-215, doi: 10.1111/ppe.12027. 32. Mustafa A., Ali A., Mustafa M., A., Samarraie M. Physiological and hormonal study of women infertility. Science Archives, 2020, vol. 1, no. 3, pp. 160-165, doi: 10.47587/SA.2020.1314. 33. Pandruvada S., Royfman R., Shah T.A., Sindhwani P., Dupree J.M., Schon S., Avidor-Reiss T. Lack of trusted diagnostic tools for undetermined male infertility. Journal Assist Reprod Genet, 2021, vol. 38, no. 2, pp. 265-276, doi: 10.1007/s10815-020-02037-5. 34. Saunders T.A., Lobel M., Veloso C., Meyer B.S. Prenatal maternal stress is associated with delivery analgesia and unplanned cesareans. Journal of Psychosomatic Obstetrics and Gynecology, 2006, vol. 27, no. 3, рp. 141-146, doi: 10.1080/01674820500420637. 35. Stark M.A., Brinkley R.L. The relationship between perceived stress and health-promoting behaviors in high-risk pregnancy. Journal of Perinatal and Neonatal Nursing, 2007, vol. 21, no. 4, pp. 307-314. 36. Standeven L.R., McEvoy K.O., Osborne L.M. Progesterone, reproduction, and psychiatric illness. Best Practice & Research: Clinical Obstetrics & Gynaecology, 2020, vol. 69, pp.108- 126, doi: 10.1016/j.bpobgyn.2020.06.001. 37. Szkodziak F., Krzyżanowski J., Szkodziak P. Psychological aspects of infertility. A systematic review, 2020, vol. 48, no. 6, doi: 10.1177/0300060520932403. 38. Turner K.A., Rambhatla A., Schon S., Agarwal A., Krawetz S.A., Dupree J.M., Avidor-Reiss T. Вестник Мининского университета. 2021. Том 9, №4 General psychology, personality psychology, history of psychology Depressive symptoms predict exaggerated inflammatory responses to an in vivo immune challenge among pregnant women. Brain, Behavior and Immunity, 2010, vol. 24, no. 1, pp. 49-53, doi: 10.1016/j.bbi.2009.05.055. 24. Gabnai-Nagy E, Bugán A, Bodnár B, Papp G, Nagy BE. Association between Emotional State Changes in Infertile Couples and Outcome of Fertility Treatment. Geburtshilfe Frauenheilkd, 2020, vol. 80, no. 2, doi: 10.1055/a-0854-5987. 25. Golombok S. Modern families: Parents and children in new family forms. Cambridge, UK, Cambridge University Press, 2015. Рp. 111-113. 26. Khizroeva J., Nalli C., Bitsadze V., Lojacono A., Zatti S., Andreoli L., Tincani A., Shoenfeld Y., Makatsariya A. Infertility in women with systemic autoimmune diseases. Best Practice & Vestnik of Minin University. 2021. Volume 9, no. 4 Информация об авторах Мошкивская Валентина Анатольевна – медицинский психолог Городского перинатального центра №1 г. Санкт-Петербург, Санкт-Петербург, Российская Федерация, ORCID ID: 0000-0002-6620-8360, SPIN‐код: 3218-8964, e-mail: moshkivskaia@gmail.com. Vestnik of Minin University. 2021. Volume 9, no. 4 Information about authors Moshkivskaya Valentina A. – medical psychologist of the City Perinatal Center No 1, Saint Petersburg, Saint Petersburg, Russian Federation, ORCID ID: 0000-0002-6620-8360, SPIN‐код: 3218-8964, e-mail: moshkivskaia@gmail.com. Поступила в редакцию: 24.08.2021 Принята к публикации: 01.10.2021 Опубликована: 12.11.2021 Принята к публикации: 01.10.2021 General psychology, personality psychology, history of psychology Информация об авторах Мошкивская Валентина Анатольевна – медицинский психолог Городского перинатального центра №1 г. Санкт-Петербург, Санкт-Петербург, Российская Федерация, ORCID ID: 0000-0002-6620-8360, SPIN‐код: 3218-8964, e-mail: moshkivskaia@gmail.com. Information about authors Moshkivskaya Valentina A. – medical psychologist of the City Perinatal Center No 1, Saint Petersburg, Saint Petersburg, Russian Federation, ORCID ID: 0000-0002-6620-8360, SPIN‐код: 3218-8964, e-mail: moshkivskaia@gmail.com. General psychology, personality psychology, history of psychology Общая психология, психология личности, история психологии Male Infertility is a Women's Health Issue-Research and Clinical Evaluation of Male Infertility Is Needed. Cells, 2020, vol. 16, no. 9, doi: 10.3390/cells9040990. 39. Thurston L, Abbara A, Dhillo WS. Investigation and management of subfertility. Journal of Clinical Pathology, 2019, vol. 72, no. 9, doi: 10.1136/jclinpath-2018-205579. 40. Vander Borght M., Wyns C. Fertility and infertility: Definition and epidemiology. Clinical Biochemistry, 2018, vol. 62, no. 2, doi: 10.1016/j.clinbiochem.2018.03.012. 41. Wall G. Mothers’ experiences with intensive parenting and brain development discourse. Women’s Studies International Forum, 2010, vol. 33, no. 3, pp. 253-263. 42. Xavier M.J., Salas-Huetos A., Oud M.S. Disease gene discovery in male infertility: past, present and future. Human Genetics, 2021, vol. 140, pp. 7-19, doi: 10.1007/s00439-020-02202-x. © Мошкивская В.А., 2021
https://openalex.org/W4206988837	https://www.cambridge.org/core/services/aop-cambridge-core/content/view/CDE65A901EF520D77A16CF9E964574C2/S1073110521000802a.pdf/div-class-title-from-the-shadows-the-public-health-implications-of-the-supreme-court-s-covid-free-exercise-cases-div.pdf	English	null	From the Shadows: The Public Health Implications of the Supreme Court’s COVID-Free Exercise Cases	The journal of law, medicine & ethics/The Journal of law, medicine & ethics	2,021	cc-by	14,361	The Journal of Law, Medicine & Ethics, 49 (2021): 564-579. © The Author(s), 2021. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. DOI: https://doi org/10 1017/jme 2021 80 From the Shadows: The Public Health Implications of the Supreme Court’s COVID- Free Exercise Cases Keywords: COVID-19, Free Exercise, Public Health Law, First Amendment Abstract: This article analyzes the Supreme Court’s “shadow docket” Free Exercise cases relat- ing to COVID-19. The paper highlights the decline of deference, the impact of exemptions, and the implications of the new doctrine for vaccine and other public health laws. T he relationship between religious liberty and public health has always been fraught. When plagues strike, societies often turn to prayer and communal worship. Frequently they also scape- goat non-believers, heretics, and members of minority faiths.1 That history should caution courts to be vigi- lant when pandemic responses target religious minor- ities and the exercise of religion. Yet, because patho- gens do not distinguish between religious and secular activities, governments cannot ignore the risks that religious activities can pose during a pandemic. Since the start of the COVID-19 pandemic, American courts have struggled to reconcile these dueling imperatives. T Wendy E. Parmet Early in the pandemic, most courts, including the Supreme Court,2 rejected challenges to public health emergency orders even when they applied to worship. Then on November 25, 2020, in Roman Catholic Dio- cese v. Cuomo,3 the Court changed course, off ering a strikingly diff erent approach that casts a far more skeptical eye on state health orders that touch upon religious practices, especially in-person worship. Although much remains unclear, the Court’s more recent decisions regarding COVID restrictions — all announced from the “shadow docket” without the benefi t of argument4 — forgo both deference to state offi cials and consideration of public health evidence in the determination of whether the state has regulated religious activities less favorably than comparable secular activities. Now almost any public health law that includes an exemption for some secular activity risks being subject to strict scrutiny in a Free Exer- cise claim. As a result, the states’ capacity to carry out essential public health functions, as well as protect their populations from COVID-19 or other, poten- tially more lethal, pandemics, is in jeopardy. To ensure that states are not left impotent to protect the public’s health, the Court needs to rethink its approach. While Wendy E. Parmet, J.D., is the Matthews University Profes- sor of Law and Professor of Public Policy and Urban Aff airs at Northeastern University School of Law in Boston, Mas- sachusetts, USA. 564 Part One: A Patchwork of Orders There is little question that the U.S. response to COVID-19 has been catastrophic. Although the U.S. does not have the highest per capita death rate in the world, more than 750,000 Americans had died from Third, was insufficient economic support to buffer the economic fallout from pandemic-control mea- sures.18 As public health scholars have noted, the pro- vision of economic (and other forms) of support can Part One briefly reviews the nation’s failed response to COVID and the state orders that have impacted worship. Part Two summarizes the application of the Free Exercise law to public health measures prior to and early in the pandemic. Part Three surveys the Supreme Court’s changing approach. Part Four interrogates the new approach, noting its most important features and highlighting areas of uncertainty. The Conclusion considers the potential impact of the COVID-cases on public health law post-pandemic. be critical to obtaining compliance with public health advice.19 People are more likely to stay home follow- ing potential exposure to a contagious disease if they do not have to worry about losing their job. Likewise, businesses are more likely to support public health measures if they know they can avoid economic catas- trophe. During a pandemic, economic relief can be a critical tool for disease mitigation. COVID-19 by November 3, 2021.5 Millions more have been seriously ill, and thousands are long-haulers who face long-term health problems.6 Communities of color and immigrants have been especially hard hit, both by the disease and its economic and social fallouts.7 Many factors impeded the nation’s response to COVID-19.8 For present purposes, three appear espe- cially relevant. First, is political polarization. Although there was bipartisan consensus for the initial round of emergency orders issued in March 2020, it quickly faded.9 By April 2020, the pandemic had taken on a distinctly political hue, with Republicans less con- cerned about the coronavirus and less supportive of state emergency orders than Democrats.10 That politi- cal divide continued during a presidential campaign in which one candidate (then President Trump) mini- mized the pandemic and the other (now President Congress did provide significant support through the CARES20 and the Families First Coronavirus Response Acts21 in the spring of 2020. journal of law, medicine & ethics journal of law, medicine & ethics The Journal of Law, Medicine & Ethics, 49 (2021): 564-579. © The Author(s), 2021. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. DOI: https://doi.org/10.1017/jme.2021.80 https://doi.org/10.1017/jme.2021.80 Published online by Cambridge University Press Parmet deference should not be absolute, states should not be precluded from protecting the public’s health. Biden) made it his number one priority.11 Given the pre-existing political alignment between religios- ity and party affiliation,12 not to mention President Trump’s emphasis on re-opening church services, par- tisan differences over the pandemic easily converted into a divide between religiosity and secularism.13 This paper develops these arguments. Part One briefly reviews the nation’s failed response to COVID and the state orders that have impacted worship. Part Two summarizes the application of the Free Exercise law to public health measures prior to and early in the pandemic. Part Three surveys the Supreme Court’s changing approach. Part Four interrogates the new approach, noting its most important features and highlighting areas of uncertainty. The Conclusion considers the potential impact of the COVID-cases on vaccine mandates and other public health laws post-pandemic. Second, was the lack of a coordinated, federal response. Under the Constitution, states have primary responsibility for public health protection.14 Never- theless, pandemics cross state lines and necessitate a level of national coordination that has been largely absent during the pandemic.15 As a result, states were largely left to go their own way as they tried contain the pandemic while mitigating its economic and social effects.16 This led to a confounding and often incoher- ent patchwork of orders.17 https://doi.org/10.1017/jme.2021.80 Published online by Cambridge University Press Part One: A Patchwork of Orders The December 2020 Coronavirus Response and Relief Supplemental Appropriations Act of 2021 offered additional aid,22 as did the American Rescue Plan Act that President Biden signed into law in March 2021.23 The support that these acts offered, however, did not reach every- one, and the delay in enacting further relief in the late summer and fall of 2020 added to the challenge that 565 first amendment values in health care • winter 2021 The Journal of Law, Medicine & Ethics, 49 (2021): 564-579. © 2021 The Author(s) first amendment values in health care • winter 2021 The Journal of Law, Medicine & Ethics, 49 (2021): 564-579. © 2021 The Author(s) https://doi.org/10.1017/jme.2021.80 Published online by Cambridge University Press https://doi.org/10.1017/jme.2021.80 Published online by Cambridge University Press SYMPOSIUM states faced as they tried to balance human and eco- nomic health.24 The results were not pretty. Initially, most states issued a series of emergency orders that shuttered some, but not all businesses, and limited many, but not all, social gatherings. Then, as pan- demic fatigue, economic stress, and partisan divisions grew, states began to “reopen.”25 Once cases re-surged in winter 2020-2021, some governors re-imposed some, but not all, of the restrictions.26 sachusetts law requiring all residents to be vaccinated against smallpox or pay a $5 fine. The defendant, Henning Jacobson, was a Lutheran pastor who had both religious and secular objections to vaccination.43 Yet, because the Supreme Court had yet to apply the Free Exercise Clause to the states,44 he based his chal- lenged on the due process clause, not the Free Exercise clause.45 In a complex and multi-faceted opinion by Justice Harlan, the Court rejected Jacobson’s contentions, emphasizing that a community has the “right to pro- tect itself against an epidemic of disease which threat- ens the safety of its members.”46 This did not mean that communicable disease laws were wholly beyond judicial review. Part One: A Patchwork of Orders Rather, the Court recognized that the police power extended only to “reasonable regulations, as the safety of the general public may demand,”47 and that courts should step in when public health laws have “no real or substantial relation” to their “objects,” or are “beyond all question, a plain, palpable invasion of rights secured by the fundamental law.”48 The Court also noted that some regulations might be “so arbitrary and oppressive in particular cases, as to justify the interference of the courts.”49 Still, Jacobson provided strong support for the principle that states can limit individual liberty to prevent the spread of communi- cable diseases, and that courts should provide con- siderable deference to the elected branches, and the health officials to whom they delegate power, to deter- mine what steps are needed to stop an epidemic.50 This less-than-coherent approach extended to reli- gious worship. Early on, it became clear that religious worship and gatherings could serve as super-spreader events.27 South Korea’s initial outbreak, for example, was tied to services in a charismatic religious com- munity.28 In March 2020, an Arkansas church service was associated with 61 cases and four deaths.29 As 2020 progressed, evidence accumulated that indoor activities where people are close to one another for an extended period, especially where there is singing or loud talking, are especially risky.30 Nevertheless, the CDC did not recommend restrictions on worship, not- ing that millions of Americans “embrace worship as an essential part of life.”31 In spring 2020, when COVID-restrictions were at their most stringent, most states exempted religious services from orders that shuttered mass gatherings.32 According to the Pew Research Center, only 10 states barred in-person religious services in April 2020.33 About one-third of states placed no caps at all on in- person religious gatherings.34 Three states deemed religious worship to be “essential services.”35 Still, religious services did not escape regulation. In April 2020, 22 states limited religious gatherings to 10 or fewer persons.36 Some states had even stricter and some had looser requirements.37 For more than 100 years, Jacobson remained the Court’s leading infectious disease case, and primary authority for the constitutionality of vaccine mandates (even in the absence of an epidemic).51 Moreover, although Jacobson was not a Free Exercise case, the Court cited it in several notable religious liberty cases. For example, the Court referenced it in Prince v. Part One: A Patchwork of Orders Mas- sachusetts while rejecting a religious liberty challenge to a child labor law.52 The Court also cited Jacobson in Sherbert v. Verner,53 which held that the denial of unemployment benefits to a Seventh-Day Adventist who refused to work on her Sabbath violated the Free Exercise Clause, for the proposition that the Consti- tution does not require accommodations to laws that regulate actions that “pose[] some substantial threat to public safety, peace or order.”54 In the summer and fall of 2020, even as infections surged, more states “opened up,” lifting restrictions on religious worship, as well as other activities.38 Other states, including New York and California, maintained significant restrictions.39 As the discussion below shows, challenges to these laws helped to reshape the Court’s understanding of how the Free Exercise Clause applies to public health laws. journal of law, medicine & ethics The Journal of Law, Medicine & Ethics, 49 (2021): 564-579. © 2021 The Author(s) https://doi.org/10.1017/jme.2021.80 Published online by Cambridge University Press Part Two: Doctrinal Roots and the Early COVID Cases In the spring and summer of 2020, most lower courts followed past practice and rejected Free Exer- cise challenges to public health orders regarding COVID-19.69 Although they used different approaches to reconcile Jacobson with contemporary Free Exer- cise cases, courts generally read Jacobson as requir- ing them to grant substantial deference to public health emergency orders.70 Most courts also relied on Smith to conclude that strict scrutiny was inapplicable because the state had restricted a range of comparable secular activities, and hence acted in a manner that was neutral toward religion.71 Still, the heated political debates over the treatment of religious services, combined with the fact that all states included multiple exemptions to their emer- gency orders, created anger and constitutional peril. On April 14, 2020, Attorney General William Barr warned that “government may not impose special restrictions on religious activity that do not also apply to similar nonreligious activity … Religious institu- tions must not be singled out for special burdens.”72 Gorsuch’s focus on the state’s perceived lack of neu- trality in Masterpiece Cake echoed Justice Alito’s 2016 dissent in Stormans, Inc. v. Wiseman 61 Stormans chal- lenged a Washington State law that required pharma- cists to sell contraceptives, including Plan B. Relying on Smith and Lakumi, the Ninth Circuit concluded that because the state’s rule was neutral and generally applicable, strict scrutiny was not required.62 Some courts agreed. For example, in Maryville Baptist Church v. Beshear, the Sixth Circuit held that orders by Kentucky Governor Beshear prohibiting drive-in services “by name” while allowing secular, “‘life-sustaining’ businesses [including] law firms, laundromats, liquor stores, and gun shops to continue to operate so long as they follow social-distancing and other health-related precautions” were likely uncon- stitutional.73 The court stated: In a dissent from the Court’s denial of certiorari, Justice Alito, joined by the Chief Justice and Justice Thomas, argued that because the Washington allowed pharmacies to refuse to fill prescriptions when they did not accept the customer’s insurance it was neither neutral nor generally applicable; hence strict scrutiny was required.63 This analysis suggested — or foretold — that the existence of any secular exemption from a regulation that also implicated a religious practice would trigger strict scrutiny. Assuming all of the same precautions are taken, why is it safe to wait in a car for a liquor store to open but dangerous to wait in a car to hear morning prayers? Part Two: Doctrinal Roots and the Early COVID Cases Smith overruled Sherbert, but in doing so, the Court did not reject the point that Sherbert drew from Jacob- son. Rather, Justice Scalia’s opinion in Smith ruled that all generally applicable regulations of conduct, and not simply those that seek to prevent a substantial threat to public safety, peace or order, were subject to rational basis review, even if they burdened someone’s exercise of religion.55 Prior to COVID-19, the application of the Free Exer- cise clause to communicable disease laws was rela- tively stable, if under-theorized. Three cases formed the foundation for the analysis: Jacobson v. Mas- sachusetts,40 Employment Division v. Smith,41 and Church of the Lukumi Babalu Aye v. Hialeah.42 Strictly speaking, Jacobson was not a Free Exercise case. The 1905 decision concerned a Cambridge, Mas- 566 Parmet Lukumi added an important limitation to Smith.56 In Lukumi, the Court clarified that laws that were facially neutral, but targeted religion, were subject to strict scrutiny, and were constitutional only if they were narrowly tailored to a compelling state interest.57 In Masterpiece Cakeshop v. Colorado Civil Rights Commission, the Court relied on Lukumi to hold that the Colorado Civil Rights Commission violated the Free Exercise Clause because it acted with hostil- ity toward the religious beliefs of a baker who refused to decorate a cake celebrating a same-sex marriage.58 Tellingly, Justice Gorsuch, in a concurring opinion, wrote “Smith remains controversial in many quar- ters.”59 However, he did not call for overruling Smith. Instead, he argued that the state had failed to act with neutrality in applying an intent requirement to the state’s civil rights laws to bakeshops that refused service.60 Exercise challenges.68 The existence of other exemp- tions — for example, for medical reasons — did not change the conclusion. https://doi.org/10.1017/jme.2021.80 Published online by Cambridge University Press first amendment values in health care • winter 2021 The Journal of Law, Medicine & Ethics, 49 (2021): 564-579. © 2021 The Author(s Part Three: The Supreme Court Steps In The Court’s Early COVID Cases: y Between May and November 2020, the composition of the Supreme Court changed. So, too, did its approach to Free Exercise challenges to COVID orders. As the views of the justices who were initially in the dissent became those of the majority, the Court established a The Court’s second COVID case, Calvary Chapel Dayton Valley v. Sisolak, concerned Nevada’s 50-per- Between May and November 2020, the composition of the Supreme Court changed. So, too, did its approach to Free Exercise challenges to COVID orders. As the views of the justices who were initially in the dissent became those of the majority, the Court established a new doctrinal framework that devalued public health evidence and could subject almost any public health law to strict scrutiny. son cap on religious services; certain other activities, including gaming, were allowed to admit 50% of their maximum occupancy.85 By another 5-4 vote, again from the shadow docket and without an opinion, the majority rejected an emergency petition to enjoin the occupancy limit. Justices Alito, Gorsuch, and Kavana- ugh published three separate dissents previewing the arguments that the majority would later adopt. new doctrinal framework that devalued public health evidence and could subject almost any public health law to strict scrutiny. y On May 22, 2020, the Supreme Court issued its first decision regarding a COVID-restriction in South Bay United Pentecostal Church v. Newsom (South Bay I).76 Like the other COVID-cases that the Court would hear, South Bay I was an emergency petition decided from the “shadow docket,”77 without the benefit of argument or full briefing. Part Two: Doctrinal Roots and the Early COVID Cases Why can someone safely walk down a grocery store aisle but not a pew? And why can someone safely interact with a brave deliverywoman but not with a stoic minister? The Commonwealth has no good answers. While the law may take periodic naps during a pandemic, we will not let it sleep through one.74 The interest among some justices in narrowing Smith was also evident by the Court’s February 2020 decision to grant certiorari in Fulton v. City of Phil- adelphia.64 In Fulton, a Catholic foster care agency challenged Philadelphia’s refusal to enter into new contracts with the agency due to its refusal to place children with same-sex couples. The Third Circuit had found that the city’s policy was a generally applicable law, subject under Smith, to rational basis review.65 The grant of certiorari included the question whether Smith should be overruled.66 A few days later, the same panel in Roberts v. Neace enjoined the Governor’s ban on in-door services.75 The Sixth Circuit’s decisions pointed to the dilemma that courts faced during the pandemic. In the absence of federal coordination, inadequate financial sup- port, and changing epidemiological and political conditions, state officials imposed orders that often appeared perplexing. Why exempt liquor stores but not churches? Laundromats but not worship? An Despite these forewarnings, until COVID-19, lower courts usually upheld communicable disease laws against Free Exercise claims. This was especially apparent with regard to state vaccine laws.67 For example, even after California and New York repealed religious exemptions for school-based mandates, courts relied on Smith and/or Jacobson to reject Free 567 SYMPOSIUM epidemiologist might answer that because worship brings many people together for an extended period, with singing and chanting, it creates a greater risk than retail stores or laundromats. The Sixth Circuit, however, did not consider public health evidence, rely- ing instead on its own assessment of risks. Soon the Supreme Court would do likewise. reluctant to second-guess officials when they “’under- take[] to act in areas fraught with medical and scien- tific uncertainties.’”82 This reluctance, he added, was particularly appropriate in deciding an emergency petition.83 In a strongly worded dissent, Justice Kavanaugh, joined by Justices Thomas and Gorsuch, argued that California had not imposed the identical occupancy limit on “comparable secular businesses.”84 Tellingly, he pointed to no evidence to support the conclusion that exempt businesses were “comparable” to religious services. journal of law, medicine & ethics The Journal of Law, Medicine & Ethics, 49 (2021): 564-579. © 2021 The Author(s) https://doi.org/10.1017/jme.2021.80 Published online by Cambridge University Press Part Two: Doctrinal Roots and the Early COVID Cases Nor did he explain how courts should deter- mine the relevant comparators. Part Three: The Supreme Court Steps In The Court’s Early COVID Cases: Between May and November 2020, the composition of the Supreme Court changed. So, too, did its approach to Free Exercise challenges to COVID orders. As the views of the justices who were initially in the dissent became those of the majority, the Court established a Part Three: The Supreme Court Steps In The Court’s Early COVID Cases: The issue before the Court was California Governor Gavin Newsom’s order limit- ing attendance at places of worship to 25% of capacity or a maximum of 100 attendees.78 Many other secu- lar activities, including lecture halls, concerts, movie theaters, and sports events faced similar limits, but others, including retail stores, restaurants, and hair salons faced less strict limits.79 In his dissent, Alito, joined by Thomas and Kavana- ugh, argued that the petitioner was likely to succeed on the merits of its Free Exercise claim because the state had “made no effort” to show that the religious services were riskier than activities that were permit- ted, such as “going to the gym” or “what goes on in casinos.”86 Thus like Kavanaugh in South Bay, Alito appeared to assume that the state bore the burden of establishing that the services were not comparable to the exempted activities.87 He added that because Jacobson was not a First Amendment case it was not relevant, and that “a public health emergency does not give Governors and other public officials carte blanche to disregard the Constitution for as long as the medi- cal problem exists.”88 By a 5-4 vote, the Court rejected the emergency petition without issuing an opinion. Concurring, Chief Justice Roberts explained that the order appeared to treat religious worship similarly to “comparable secu- lar gatherings … where large groups of people gather in close proximity for extended periods of time.”80 Citing Jacobson, he explained that the Constitution “principally entrusts” health and safety to”‘politically accountable officials,’”81 and that courts should be In his own dissent, Kavanaugh pinpointed the prob- lem presented by the juxtaposition of restrictions and exemptions: “when a law on its face favors or exempts journal of law, medicine & ethics The Journal of Law, Medicine & Ethics, 49 (2021): 564-579. © 2021 The Author(s) 568 Parmet The majority, however, did not rely on extra-textual evidence of animus. Rather, it found that discrimina- tion existed because certain secular activities, includ- ing “acupuncture facilities, camp grounds, garages, as well as many whose services are not limited to those that can be regarded as essential,” were subject to less onerous restrictions.100 From this, and the fact that the restrictions specified religious services by name, the majority concluded, without pointing to any pub- lic health evidence, that the contested orders were not of general applicability. A New Approach: h l Two months later, Justice Ruth Bader Ginsburg, who had voted with the majority in South Bay I and Cal- vary Chapel, died.92 On October, 26, 2020 President Trump’s nominee, Amy Coney Barrett, was confirmed to the Supreme Court.93 One month later, in Roman Catholic Diocese of Brooklyn (RCD), the approach of the dissenters in South Bay I and Calvary Christian became that of the majority. 94 j y RCD concerned New York Governor Cuomo’s order barring more than 10 persons from attend- ing religious services in “red-zones” (areas identified as COVID-19 “hotspots”) and more than 25 persons from attending services in “orange zones” (areas adja- cent to red zones).”95 By the time the case had reached the Supreme Court, the Governor had reclassified the areas where the plaintiffs were located, enabling them to hold services at 50% of capacity.96 Both Gorsuch and Kavanaugh added strongly worded concurring opinions. In his, Gorsuch derided governors who “[A]t the flick of a pen, … have asserted the right to privilege restaurants, marijuana dispen- saries and casinos over churches, mosques, and tem- ples.”103 He also criticized the Chief Justice’s concur- rence in South Bay I for relying on Jacobson, which he termed a “modest” decision that applied to a different set of facts and a different constitutional claim.104 He warned that while the impulse for courts to “stay out of the way in times of crisis … may be understandable or even admirable in other circumstances, we may not shelter in place when the Constitution is under attack. Things never go well when we do.”105 Despite the fact that the plaintiffs were no longer subject to the order at issue, the Court took up the emergency appeal and by a 5-4 vote, in a short per curiam opinion, concluded that the plaintiffs had “made a strong showing that the challenged restric- tions violate ‘the minimum requirement of neutrality’ to religion.”97 In support of its claim, the plaintiff Agu- dath Israel of America had referenced statements by Cuomo that could be construed as targeting Orthodox Jews.98 The Court could have rested on those facts.99 Such a decision would have signaled that the defer- ence that Roberts commended in South Bay I did not extend to orders when there was evidence of animus toward a religious group, perhaps especially a reli- gious minority. Part Three: The Supreme Court Steps In The Court’s Early COVID Cases: In effect, as in the South Bay I and Christian Calvary dissents, the majority relied on its own intuition to determine which activities were comparable to the religious services that were restricted. The majority also appeared, without stat- ing, to treat the state as having the burden of persua- sion on that threshold issue. some secular organizations as opposed to religious organizations, a court … must determine whether the State has sufficiently justified the basis for the dis- tinction.”89 Recognizing that states were “struggling” to balance economic and health risks, he stated, “The Constitution does not tolerate discrimination against religion merely because religious services do not yield a profit.”90 He added, This Court’s history is littered with unfortunate examples of overly broad judicial deference to the government when the government has invoked emergency powers … The court of his- tory has rejected those jurisprudential mistakes and cautions us against an unduly deferential judicial approach, especially when questions of racial discrimination, religious discrimination, or free speech are at stake.91 Applying strict scrutiny, the majority held that the regulations were not narrowly tailored to the com- pelling state interest of preventing the transmission of COVID-19. In so doing, the Court noted that many other “hard-hit” jurisdictions had less onerous restric- tions, showing how the variation among states that had come to characterize the pandemic response could be used to establish a lack of narrow tailoring.101 The Court also pointed out that there were no reported outbreaks of COVID-19 at plaintiffs’ services, suggest- ing that states could not act to prevent the transmis- sion of the virus until a super-spreader event at a par- ticular religious facility was documented.102 https://doi.org/10.1017/jme.2021.80 Published online by Cambridge University Press A New Approach: h l In his concurrence, Kavanaugh accepted that the Constitution “’principally entrusts the safety and health of the people to the politically accountable officials of the States,’” but explained that “judicial deference in an emergency or a crisis does not mean wholesale judi- cial abdication, especially when important questions of religious discrimination, racial discrimination, free speech, or the like are raised.”106 He added that “once a state creates a favored class of businesses … the State 569 first amendment values in health care • winter 2021 The Journal of Law, Medicine & Ethics, 49 (2021): 564-579. © 2021 The Author(s) first amendment values in health care • winter 2021 The Journal of Law, Medicine & Ethics, 49 (2021): 564-579. © 2021 The Author(s) https://doi.org/10.1017/jme.2021.80 Published online by Cambridge University Press SYMPOSIUM must justify why houses of worship are excluded from that favored class.”107 He did not explain, however, how the Court should determine which favored “classes of businesses” were comparable to worship. justice stated that the petitioners did not “carry their burden,” suggesting that she thought they had the burden of establishing that they were entitled to relief from that ban.118 In contrast, in her dissent, Justices Kagan, joined by Breyer and Sotomayor, lamented the majority’s failure to credit the state’s scientific evi- dence and hoped that the Court’s decision would not “worsen the Nation’s COVID crisis.”119 p p In dissent, Justice Sotomayor warned of the poten- tial danger of this approach: “Justices of this Court play a deadly game in second guessing the expert judgment of health officials about the environment in which a contagious virus, now infecting a mil- lion Americans each week, spreads most easily.”108 In the three months that followed the Court’s decision, approximately 250,000 more Americans died from COVID-19.109 Still, on its own, RCD might have been read as a limited decision, motivated by the draco- nian nature of Governor Cuomo’s order, and serving to remind officials to tread carefully when restricting worship. Despite the absence of a majority opinion in South Bay II, on February 26, 2021, by a six-three vote, the Court in Gateway City Church v. Newsom,120 granted emergency relief to a church contesting restrictions on indoor gatherings.121 Although the restrictions in Gateway City Church were quite unlike the ones in the earlier cases in that they applied to all indoor gather- ings and did not specify worship, the Court ruled that the outcome was “dictated by this Court’s decision” in South Bay II.122 That was not to be. In the weeks and months that fol- lowed, the Supreme Court issued a series of decisions relating to the Free Exercise clause.110 Among the more interesting was South Bay United Pentecostal Church v. first amendment values in health care • winter 2021 The Journal of Law, Medicine & Ethics, 49 (2021): 564-579. © 2021 The Author(s) Newsom (South Bay II).111 In a short, unsigned opin- ion, once again from the shadow docket, a six justice majority (including Roberts, Thomas, Alito, Gorsuch, Kavanaugh, and Barrett) blocked California’s ban on indoor services, but left in place a 25% capacity limit plus a ban on singing and chanting.112 Then on April 9, the Court, by a 5-4 vote — again from the shadow docket — issued its most far-reach- ing COVID decision in Tandon v. Newsom.123 Tandon challenged the application of California’s limits on the number of people from separate households who could gather in private homes.124 The plaintiffs claimed that the restrictions violated their rights under the Free Exercise Clause to conduct prayer meetings in homes because the state permitted more people to gather for secular purposes in certain public spaces, such as train stations and shopping malls.125 The Ninth Cir- cuit panel, by a vote of 2-1, disagreed, finding that such public settings were not comparable to in-home gath- erings “in terms of risk to public health or reasonable safety measures to address that risk.”126 The Appeals Court explained: p g g g Although the majority agreed to enjoin part of the state’s order, the separate opinions of the justices in the majority showed continuing disagreement. Now stating that deference had its “limits,” Roberts sup- ported enjoining the orders restricting worship, but would have kept in place the ban on singing, noting that he saw no basis for “overriding that aspect of the state public health framework.”113 In contrast, Gor- such, joined by Thomas and Alito, argued that the state had targeted religion, and that as a result, strict scrutiny was required.114 Regarding the ban on chant- ing, Gorsuch noted, “California’s powerful entertain- ment industry has won an exemption. https://doi.org/10.1017/jme.2021.80 Published online by Cambridge University Press journal of law, medicine & ethics The Journal of Law, Medicine & Ethics, 49 (2021): 564-579. © 2021 The Author(s) first amendment values in health care • winter 2021 The Journal of Law, Medicine & Ethics, 49 (2021): 564-579. © 2021 The Author(s) © 2021 The Author(s) https://doi.org/10.1017/jme.2021.80 Published online by Cambridge University Press https://doi.org/10.1017/jme.2021.80 Published online by Cambridge University Press Parmet phia’s policy was not neutral and generally applicable because the City’s contract with foster care agencies contained a provision granting it the sole discretion to create exceptions to its anti-discrimination require- ment.140 The Court also held that it need not decide if the City’s anti-discrimination law violated the Free Exercise clause because the agency plaintiff was not a public accommodation.141 gatherings were neither neutral nor generally applica- ble.129 In reaching its conclusion, the Court stated, “it is no answer that a State treats some comparable secu- lar businesses or other activities as poorly as or even less favorably than the religious exercise at issue.”130 The Court then explained that comparability “must be judged against the asserted government interest that justifies the regulation at issue,” and that comparabil- ity is concerned “with the risks various activities pose, not the reasons why people gather.”131 In concurring opinions, however, five justices expressed dissatisfaction with Smith. Barrett, who was joined by Kavanaugh, stated that the “textual and structural arguments against Smith are more com- pelling” than those supporting it.142 Nevertheless, she noted that overruling Smith would raise a host of dif- ficult questions that the Court need not answer for the reasons explained in the majority’s decision. Applying those principles, the Court determined that strict scrutiny was required, and that the restric- tions could not pass that high bar. In reaching that decision, the Court overlooked the testimony that was offered by the state’s experts, and pointed again to the exemptions the state offered for some secular activities, stating that the state “cannot ‘assume the worst when people go to worship but assume the best when people go to work.’”132 In effect, the very factors that led the Court to conclude that strict scrutiny was required led it to find that the order was not narrowly tailored, and hence failed strict scrutiny. The Court added that the fact that the state had changed its pol- icy after the petition for certiorari was filed made no difference, stating that “officials with a track record of ‘moving the goalposts’ retain authority to reinstate those heightened restrictions at any time.”133 Alito felt no such compunctions. first amendment values in health care • winter 2021 The Journal of Law, Medicine & Ethics, 49 (2021): 564-579. © 2021 The Author(s) So once more we appear to have a State playing favorites during a pandemic …”115 In a separate statement, Alito indi- cated that he would stay the injunction on capacity limits and singing and chanting for 30 days, to be lifted unless the state “demonstrates clearly that noth- ing short of those measures will reduce the commu- nity spread of COVID-19 at indoor religious gather- ings to the same extent as do the restrictions the State enforces with respect to other activities it classifies as essential.”116 [T]he district court found that the State reason- ably concluded that when people gather in social settings, their interactions are likely to be longer than they would be in a commercial setting; that participants in a social gathering are more likely to be involved in prolonged conversations; that private houses are typically smaller and less ventilated than commercial establishments; and that social distancing and mask-wearing are less likely in private settings and enforcement is more difficult.127 Having rejected the analogy to gatherings in public spaces, the Ninth Circuit concluded that the state’s restriction on private gatherings was a neutral law of general applicability, and not subject to strict scrutiny.128 In contrast, Barrett, joined by Kavanaugh, agreed that the capacity limits should also be blocked, but was content to accept the state’s limits on singing and chanting.117 In reaching that conclusion, the newest The Supreme Court disagreed. In a per curiam opin- ion, the Court held that the restrictions on in-home 570 journal of law, medicine & ethics The Journal of Law, Medicine & Ethics, 49 (2021): 564-579. Part Four: Themes and Questions The protection of the public’s health, especially but not solely from outbreaks of communicable disease, has long been considered a core component of the states’ police power.146 The Court’s most recent COVID-Free Exercise cases portend a fundamental change in the Court’s assessment of such laws, and raise many ques- tions about the state’s ability to protect public health in the years to come. first amendment values in health care • winter 2021 The Journal of Law, Medicine & Ethics, 49 (2021): 564-579. © 2021 The Author(s) In a lengthy con- curring opinion that Gorsuch and Thomas joined, he argued that an originalist interpretation of the First Amendment compelled the Court to overrule Smith and apply strict scrutiny to all laws that burden the exercise of religion.143 Although he did not rely on the COVID cases, he pointed to them to demonstrate that the Court’s current approach under Smith in deter- mining comparability was unworkable.144 This point was echoed in Gorsuch’s concurrence, which Alito and Thomas joined.145 In dissent, Kagan, who was joined by Breyer and Sotomayor, argued that because the state had adopted a “blanket restriction on at-home gatherings of all kind, religious and secular alike,” it had not treated religious activity less favorably than comparable secu- lar activities.134 The First Amendment, she claimed, does not demand “that the State equally treat apples and watermelons.”135 She added that the majority had ignored the lower courts’ factual findings that in- home gatherings posed a greater risk than the com- mercial activities that were less stringently regulated in other ways.136 She concluded by lamenting that the Court “once more commands California ’to ignore its experts’ scientific findings,’” thereby weakening its ability to address the health emergency.137 Less than three weeks later, the Court issued its third order in the South Bay litigation, this time vacating without an opinion the Ninth Circuit’s judgment.138 https://doi.org/10.1017/jme.2021.80 Published online by Cambridge University Press first amendment values in health care • winter 2021 The Journal of Law, Medicine & Ethics, 49 (2021): 564-579. © 2021 The Author(s) A. The Decline of Deference In its place, the Court seems to be relying on the justices’ own intu- ition as to what secular activities pose risks that are comparable to the activities that the petitioners seek to have exempt. Thus the Court assumes that retail establishments, casinos, and acupuncture are com- parable in terms of risk to in-person worship, but at least in South Bay II, some justices appeared to accept that in-person singing and chanting are more danger- ous.152 The justices offered no evidence in support of these distinctions. The Court, however, may not and should not read Fulton as holding that any broad grant of discretion to executive officials — including discretion over enforce- ment — compels strict scrutiny. Doing so would evis- cerate the ability of all administrative agencies to exer- cise discretion over their enforcement priorities. It would also make it difficult for officials to impose just the type of carefully tailored and measured responses that strict scrutiny theoretically favors. The Court, therefore, should limit Fulton’s reach to the type of contractual grant of discretion at issue in that case. Even so, the COVID cases show that the mere exis- tence of exemptions from public health laws can trig- ger strict scrutiny. The Court has also not addressed the critical ques- tion of which party has the burden of persuasion in establishing what secular activities present the appro- priate comparator for the religious exercise that has been burdened. Although the state clearly has the burden of proof once strict scrutiny is found to be applicable, the plaintiff should have had the burden of establishing comparability, as it is a necessary ele- ment for invoking strict scrutiny.153 In RCD, the Court hinted that the plaintiffs had that burden, pointing to their “strong showing” on the issue of comparabil- ity.154 In later cases, however, the Court failed to point to any evidence produced by the plaintiffs to establish comparability. In effect, the Court appeared to assume (without explicitly saying) that the state has the burden of showing that the secular activities it regulated more lightly were not comparable to the religious activities that were subject to stricter regulations. A. The Decline of Deference At the start of the pandemic, most courts, usually cit- ing Jacobson, granted substantial deference to state health officials in deciding whether restrictions on religious worship violated the Free Exercise Clause.147 In his concurring opinion in South Bay I, Roberts sig- naled that such deference was appropriate; the dis- senters disagreed.148 Out from the Shadows: On June 17, 2021, the Court emerged from its shadow docket and released its long-awaited decision in Ful- ton v. City of Philadelphia.139 By a unanimous vote, the Court held that Philadelphia had violated the Free Exercise clause. However, in his opinion for the Court, which never cited the COVID cases, Roberts declined to overrule Smith, finding instead that Philadel- Once the dissenters became the majority, deference diminished.149 Starting with RCD, the majority has not cited Jacobson; nor has it offered any deference to state health officials. Even the Chief Justice appears to have changed his tone, noting in South Bay II that, while courts “owe significant deference to politically accountable officials,” deference has its “limits.”150 Those limits, it now appears, extend not only to the 571 first amendment values in health care • winter 2021 The Journal of Law, Medicine & Ethics, 49 (2021): 564-579. © 2021 The Author(s) first amendment values in health care • winter 2021 The Journal of Law, Medicine & Ethics, 49 (2021): 564-579. © 2021 The Author(s) SYMPOSIUM deference granted to health officials. As Kagan sug- gested in Tandon, the Court now also seems unwill- ing to defer to the factual findings — based on public health evidence — of the lower courts.151 during the pandemic grant executive officials broad discretion to determine the type and level of restric- tions imposed on different activities.159 Other public health laws, such as quarantine laws, have typically been applied on an individualized basis; inevitably officials use their discretion in determining when to issue orders. Under Fulton, a religious litigant chal- lenging any of these laws could potentially argue that the mere existence of discretion and the possibility (in some cases) of an individualized analysis demands strict scrutiny. Critically, the Court has not replaced deference to public health officials or trial courts with a search- ing or even casual review of the scientific evidence. Instead, starting with RCD, the Court has ignored the public health evidence in the record. journal of law, medicine & ethics The Journal of Law, Medicine & Ethics, 49 (2021): 564-579. © 2021 The Author(s) https://doi.org/10.1017/jme.2021.80 Published online by Cambridge University Press A. The Decline of Deference the case, a court might conclude emergency rooms are not comparable to in-home prayer meetings because the former are more critical to society writ large dur- ing a pandemic than the latter. In Tandon, however, the Court insisted that comparability depends solely on the “risks various activities pose, not the reasons why people gather.”163 That approach allows the Court to avoid deriding the exercise of religion as “non- essential.” It also means that as long as hospitals pose as a great a risk of transmission as in-person worship (a likely assumption early in a pandemic), a court might treat the two activities as comparable, requiring the state to defend, subject to strict scrutiny, its deci- sion to allow the former but not the latter. Undoubtedly, the Court’s approach to comparability in the COVID cases responded at least in part to the messy and often quite questionable mix of laws and exemptions that characterized the state response to the pandemic.172 In the absence of a uniform national approach to pandemic mitigation, states adopted, rescinded, and re-imposed a dizzying array of restric- tions. Given the inconstancies between jurisdictions, and the ever-changing orders within jurisdictions (some due to new evidence and the virus’ shifting epi- demiology and some due to political and economic pressures), it is not surprising that the Court ques- tioned the application of strict measures to religious worship.173 Still, it is difficult to see how states can protect the public from disease threats without grant- ing officials substantial discretion, and implementing some distinctions between activities. Moreover, in the early days of a new pandemic, when the science is still evolving, the exercise of discretion will invariably be messy. Officials will make mistakes, and measures that appear to be necessary at one point of time may later be shown to be either unnecessary or ineffective. If we want officials to be able to save lives in the early stages of a pandemic, we need to give them some leeway. The Court, however, seems to be in an unforgiving mood. Importantly, the COVID cases show that states cannot escape the trap by treating religious activities more favorably than many other secular activities. Indeed, by singling out some types of religious activ- ity (e.g. worship), and treating it more favorably than some types of secular activity (e.g. entertainment ven- ues), the state may be found to have targeted religion. A. The Decline of Deference Interestingly, the state appears to have this burden even when plain- tiffs are seeking emergency petitions to stay refusals by the lower courts to enjoin state laws.155 In the COVID-cases, the key issue was comparabil- ity: whether the secular activities that were regulated less strictly were comparable to the religious practices that were regulated more strictly. As noted above, the Court appeared to rely on its own intuition, rather than deference or an evaluation of the public health evidence, in making the comparability determina- tion.160 The approach creates enormous uncertainty and risk for states that seek to implement non-phar- maceutical interventions during a public health emer- gency, forcing them to choose between implausibly restricting all activities or providing religious objec- tors “most-favored nation status.”161 Critically, states cannot avoid the problem by offer- ing no exemptions. Shuttering everything is simply not possible. People need health care, especially in a pandemic. They also need food and medicine, and the people who work in health care and food distribution need access to transportation and often childcare. Yet, by granting these necessary exemptions, states treat some secular activities more favorably than some religious activities (in-person worship). This sets a comparability trap, in which the state has to show — apparently without the benefit of deference — that none of the exempted activities is comparable to the religious activity asserted by the plaintiff. B. The Dangers of Exemptions Since RCD, the existence of exemptions, as in Justice Alito’s Stormans’ dissent, has proven critical to the Court’s Free Exercise analysis.156 In Fulton, the Court held that strict scrutiny was required because a pro- vision in the City’s contract with foster care agencies gave it discretion to offer individualized exemptions.157 The fact that the City had no intention of granting such exemptions was, according to the Court, irrelevant.158 p g The impact of that analysis to public health laws remains unclear. Few public health laws include the type of contractual provision at issue in Fulton. On the other hand, many of the emergency powers laws used Prior to Tandon, Caroline Corbin argued that com- parability should be based on two factors: the danger- ousness of the activity and its essentiality.162 If that were 572 https://doi.org/10.1017/jme.2021.80 Published online by Cambridge University Press Parmet preventing deaths from COVID-19 may not remain a compelling state interest.171 If so, no public health law that implicates religion could survive strict scrutiny. A. The Decline of Deference According to Sotomayor, this is precisely what hap- pened in RCD.164 The state regulated worship more strictly than some secular activities, but less strictly than others that the state deemed comparable. Still, the majority saw the state as impermissibly discrimi- nating against religious activities.165 g g g Theoretically, strict scrutiny need not doom a public health measure. Indeed, it may well be that although the Court will require strict scrutiny in most Free Exer- cise cases, that test will not always prove to be “fatal in fact.”166 In his concurrence in Fulton, Alito argued that certain peace and public safety laws, recognized at the time of the founding, should survive strict scrutiny.167 He did not include public health laws in that category, even though courts in the ante-bellum period accepted restraints on religion that related to health.168 He also pointed to some potential laws, including bans on cir- cumcision that could be defended on public health grounds, as examples of anti-religious measures that warranted strict scrutiny.169 It therefore seems pos- sible that Alito and the justices who joined his con- currence might not endorse a more relaxed approach to strict scrutiny for public health laws. Nor did the majority in the COVID cases seem willing to apply a less-than-fatal form of strict scrutiny. Indeed, Tandon suggests that the very fact that a comparable secular activity faces less stringent restrictions can serve to establish that the state has less restrictive means of protecting the public’s health.170 https://doi.org/10.1017/jme.2021.80 Published online by Cambridge University Press first amendment values in health care • winter 2021 The Journal of Law, Medicine & Ethics, 49 (2021): 564-579. © 2021 The Author(s) C. Beyond Worship f h l One of the unusual features of the Supreme Court’s initial COVID-Free Exercise cases is that in each instance, the challengers claimed that the state regula- tion burdened their ability to worship. As a result, the Court did not have to consider the impact of its less deferential and changing stance to public health laws that regulated other exercises of religion. Many other Free Exercise cases, however, focus on laws that burden religion without regulating worship. In Fulton, for example, the Court accepted that the city’s policy burdened the plaintiff’s religious exercise “by putting it to the choice of curtailing its mission or approving relationships inconsistent with its belief.”174 Although the religious activity infringed upon was not worship, the Court insisted that the plaintiff’s asser- tion that the law restricted its religious beliefs should be accepted.175 This is the typical approach.176 What happens when the Court’s well-established deferential stance to determining what constitutes a burden on religion meets its new less deferential approach to public health laws? Will the mere exis- tence of exemptions (or per Fulton, the mere possibil- ity of exemptions) mean that any religious litigant can demand an exemption to any public health law, even if More chilling, in a dissent to a later case in which the majority refused, without opinion, to block a COVID vaccine mandate for health care workers, Gorsuch, who was joined by Thomas and Alito, suggested that 573 first amendment values in health care • winter 2021 The Journal of Law, Medicine & Ethics, 49 (2021): 564-579. © 2021 The Author(s) SYMPOSIUM it restricts practices that most people would regard as purely secular. This is the issue that has arisen in liti- gation that has challenged COVID-vaccine mandates, but it is not limited to such cases. a religious activity, they are simply not comparable to hospital cafeterias and nursing home dining rooms? Or, would the Court follow the logic of the COVID cases and apply strict scrutiny? Unfortunately, the COVID-cases offer little basis for answering those questions. Tandon and Fulton raised the issue. The law in Tan- don, for example did not regulate worship qua worship, it simply impacted worship by regulating in-home gatherings. Other public health laws may implicate other activities that individuals may feel are related to their exercise of religion. Consider for example, an outbreak of a deadly gastrointestinal disease that seems to be spreading unchecked in restaurants. C. Beyond Worship f h l Early in the outbreak, health officials have little information about the specific practices that are spreading the dis- ease. They only know that several fatal outbreaks have been associated with restaurants; and that the death toll is climbing quickly. To slow the spread, they shut- ter restaurants, but allow food services to continue in hospitals and congregate care facilities. The possibility that courts would strike down public health orders that do not touch upon commonly rec- ognized forms of worship or religious activity is not far-fetched. Indeed, the uncertainty as to what Tandon and Fulton may require has already spawned a wave of litigation challenging COVID-vaccine mandates on Free Exercise grounds. Although many courts have rejected such challenges, ruling that the mandates are neutral laws of general applicability,176 others have held that by offering medical but not religious exemp- tions, the mandates violate the Free Exercise clause.177 To date, the Supreme Court has not ruled on this issue. On October 29, 2021, however, the court Bad facts make bad law. There is no doubt that the facts during the pandemic have been awful. The ever-changing and inconsistent patchwork of regulations and exemptions that tried to balance health and economic imperatives were often hard to fathom and difficult to explain. The sense of anger and grievance that much of the country felt regarding the COVID- restrictions, some of it justified and much of it stoked by President Trump and his allies, certainly added to the perception that state restrictions were motivated by animus and bigotry towards the faith-based community. Now imagine that a restaurant owner — Plaintiff X — claims that her religion compels her to cook and serve meals to strangers. She claims that the order shuttering restaurants burdens her ability to exercise her religion. She points to the fact that hospitals and nursing homes are permitted to remain open. They too could spread the disease. The state, she claims, has not treated comparable secular activities comparably to her religious practice of running her restaurant. rejected an emergency appeal in case denying a Free Exercise challenge to Maine’s requirement that health care workers be vaccinated against COVID-19.178 The majority did not write an opinion. journal of law, medicine & ethics The Journal of Law, Medicine & Ethics, 49 (2021): 564-579. © 2021 The Author(s) https://doi.org/10.1017/jme.2021.80 Published online by Cambridge University Press C. Beyond Worship f h l In a brief concur- ring opinion, Barrett, who was joined by Kavanaugh, stated that the Court should not use its discretion to take the case without benefit of “full briefing and oral argument.”179 In a heated dissent, Justice Gorsuch, who was joined by Thomas and Alito, argued that medical exemptions are comparable to religious exemptions and that strict scrutiny was required.180 How would the Court decide such a case? Would the fact that restaurants are not typically thought of as a religious activity result in the Court giving greater weight to the testimony of health officials than it did in the cases concerning the regulation of worship? In other words, would the Court, perhaps without saying so, be more willing to defer to health officials when reviewing claims that do not fall within the justices’ own pre-existing assumptions as to what constitutes a religious activity? Would the Court instead rely on its own intuition to decide that even if restaurants are To date, it is not clear whether the Court will take another vaccine case, or how it will resolve one should it do so. What is certain is that Fulton plus the COVID cases suggests that the Court does not mean to cabin its approach to laws that regulate worship qua wor- ship.181 Nor should the Court do so. Those whose prac- tice their faith by selling food or educating students should not be given less protection than those who practice their faith by attending church on Sunday. 574 Parmet The problem is that when the appropriately expan- sive notion of what constitutes a religious practice is combined with the less deferential approach to com- parability, all laws that seek to preserve the safety and well-being of society — during a pandemic and other- wise — are threatened. Any law can burden someone’s religious practice; and all laws have exemptions. Yet, freed from deference, and unconcerned with empirical facts, the Court is left with little but its own intuition to determine which secular activities pose health risks that are comparable to the regulated activities that the plaintiff sincerely views as religious. The result may be a Free Exercise jurisprudence that dramatically limits the states’ ability to protect public health, except when the Justices’ intuition tells them that the religious activity at issue is not comparable to the exempt secu- lar activities. Conclusion d f k Bad facts make bad law. There is no doubt that the facts during the pandemic have been awful. The ever- changing and inconsistent patchwork of regulations and exemptions that tried to balance health and eco- nomic imperatives were often hard to fathom and difficult to explain. The sense of anger and grievance that much of the country felt regarding the COVID- restrictions, some of it justified and much of it stoked by President Trump and his allies, certainly added to the perception that state restrictions were moti- vated by animus and bigotry towards the faith-based community. Also imperiled are day-to-day laws and regulations that protect population health. Fire safety laws, food inspection laws, and tobacco control laws, to name just a few examples, may face new challenges by individu- als who claim that compliance burdens their exercise of religion. Will all such laws be subject to strict scru- tiny as long as a litigant can show that officials have broad discretion, or that the laws are under-inclusive? Will we have anything more than judicial intuition to ensure that the mass of laws that keep us safe are not toppled? Still, by dispensing with deference, disregarding public health evidence, and limiting the determina- tion of comparability to the risks posed by activities without any consideration of their benefits, the Court opened a Pandora’s Box that threatens to undermine the public’s health. While punting on the question of Smith’s fate, Fulton did little to close that box. Rather it has invited more litigation on the impact of broad grants of discretion. Perhaps, after the pandemic is over, the Supreme Court’s eagerness to police public health orders through its shadow docket will diminish. Importantly, Justices Barrett and Kavanaugh have voiced their concerns about ruling on vaccine mandates without the benefit of full briefing and argument.186 Hopefully, when the Court next speaks, it will not be from the shadow docket, and the justices will provide us with an opinion that relies less on the rage and intuition that seemed to propel the Court’s COVID-cases and offer instead a more thoughtful and nuanced analysis of how to reconcile the Constitution’s protections for religious liberty with the protection of public health. Such an approach might accept a narrowed Smith, but might also make clear that public health evidence matters in the determination of comparability and the application of strict scrutiny. C. Beyond Worship f h l Judicial intuition, however, seems a thin reed upon which to rest the public’s health. all do,184 are subject to strict scrutiny.185 Further, a decision by a state to mandate vaccination in some employment settings — say nursing homes — but not others — say prisons — could also fall victim to the comparability trap. Of course, a court might find that nursing homes are not comparable to prisons, or that vaccine mandates for nursing home workers can sur- vive strict scrutiny. The problem is that the outcome of all of such questions seems now to depend on judicial intuition more than public health evidence. Future social distancing laws may also be at risk. COVID-19 will not be the last pandemic. When the next one strikes, the protection of the public may once again require the imposition of some forms of social distancing measures until a vaccine or treatment is developed. Ideally, those measures will be more care- fully crafted and more consistently applied than they have been during the COVID-19 pandemic. Neverthe- less, the Court’s new jurisprudence suggests that the existence of any exemptions may lead to strict scrutiny, and that the state’s careful reliance on public health evidence may prove to be of little help to the state. https://doi.org/10.1017/jme.2021.80 Published online by Cambridge University Press References 1. See F.M. Snowden, Epidemics and Society: From the Black Death to the Present (New Haven, CT: Yale Univ. Press, 2019): at 62-68; W.E. Parmet, “Health Care and the Constitution: Public Health and the Role of the State in the Framing Era,” Hastings Law Review 20, no. 2 (1993): 268-335, at 268-93 (discussing religious responses to epidemics in colonial New England); S. K. Cohn, Jr. “The Black Death and the Burning of Jews,” Past & Present 196, no. 1 (2007): 3-36. 18. H. J. Aaron, “The social safety net: The gaps that COVID-19 spotlights,” Brookings, Jun. 23, 2020, available at <https:// www.brookings.edu/blog/up-front/2020/06/23/the-social- safety-net-the-gaps-that-covid-19-spotlights/> (last visited October 1, 2021). 19. L.F. Wiley and S.R. Bagenstos, “The Personal Responsibility Pandemic: Centering Solidarity in Public Health and Employ- ment Law,” Arizona State Law Journal 52, no. 4 (2021): 1235- 1302; see also C.N. Couglin, “Public Health Policy: Revising the Need for A Compensation System for Quarantine to Maxi- mize Compliance,” Wake Forest Journal of Law and Policy 7, no. 3 (2017): 415-446. 2. Calvary Chapel Dayton Valley v. Sisolak, 591 U.S. __, 140 S. Ct. 2603, 2614 (2020); South Bay United Pentecostal Church v. Newsom (South Bay I), 590 U.S. ___, 140 S.C. 1613, 1613- 1614 (2020). ( ) 3. 141 S. Ct. 63 (2020). 3. 141 S. Ct. 63 (2020). 20. Coronavirus Aid, Relief, and Economic Security Act, Pub. L. No. 116-136, 134 Stat. 281 (2020). 4. W. Baude, “Foreword: The Supreme Court’s Shadow Docket,” New York University Journal of Law & Liberty 9, no. 1 (2015): 1-63; S. Vladeck Case Selection and Review at the Supreme Court, Hearing before the Presidential Commission on the Supreme Court, June 30, 2021, available at <https://www. justsecurity.org/wp-content/uploads/2021/06/Vladeck-SCO- TUS-Commission-Testimony-06-30-2021.pdf> (last visited October 1, 2021). 21. Families First Coronavirus Response Act, Pub. L. No. 116-127, 134 Stat. 178 (2021). 22. Consolidated Appropriations Act, 2021, Pub. L. 116-260, 134 Stat. 1182 (2021). 23. American Rescue Act Plan of 2021, Pub. L. 117-2, 135 Stat. 4 (2021). 24. M. Madowitz and J. Leibenluft, “A Coronavirus Recovery Demands Substantial, Durable Aid for State and Local Gov- ernments, Center for American Progress,” Apr. 17, 2020, avail- able at <https://www.americanprogress.org/issues/economy/ news/2020/04/17/483461/coronavirus-recovery-demands- substantial-durable-aid-state-local-governments/> (last vis- ited October 1, 2021). 5. M. Fisher, L. Rozsa, and K. Ruble, “750,000 Death: In Too Many Families, Unity in Pain But Division in Mourning,” Wash- ington Post, Nov. Conclusion d f k It might also accept that states should be able to consider not only the risk of an activity subject to regulation, but also its benefits. By offering such an approach, the Court could continue As a result, all public health laws now face uncer- tainty. This cloud extends to vaccine mandates, not only for COVID, but also for measles, mumps, rubella, and other long-required vaccinations. As noted above, for more than a century, courts looked to Jacobson to affirm the state’s right to mandate vaccination.182 Smith provided further support.183 Now, with the majority ignoring Jacobson, and five justices ques- tioning Smith, these laws face new dangers. Most omi- nously, the Court’s analysis of exemptions in both Ful- ton and the COVID cases raises the question whether vaccine mandates that include any exemptions, as 575 first amendment values in health care • winter 2021 The Journal of Law, Medicine & Ethics, 49 (2021): 564-579. © 2021 The Author(s) https://doi.org/10.1017/jme.2021.80 Published online by Cambridge University Press https://doi.org/10.1017/jme.2021.80 Published online by Cambridge University Press SYMPOSIUM able at <https://jamanetwork.com/journals/jama/fullarti- cle/2775687> (last visited October 1, 2021). the important task of policing anti-religious animus, especially aimed at religious minorities, without sub- jecting all public health laws to the comparability trap. / 77 7 ( , ) 8. For a discussion of the multitude of legal and policy failures, see Public Health Law Watch, COVID-19 Policy Playbook: Legal Recommendations for a Safer, More Equitable Future, (Burris et al., eds.) Mar. 2021, available at <https://static1. squarespace.com/static/5956e16e6b8f5b8c45f1c216/t/606 4ad386b6e756cabb56f96/1617210684660/COVIDPolicy- Playbook-March2021.pdf> (last visited October 1, 2021); S. Woolhander et al., “Public Policy and Health in the Trump Era,” The Lancet Commissions, Feb. 10, 2021, avail- able at <https://www.thelancet.com/action/showPdf?pi i=S0140-6736%2820%2932545-9> (last visited October 1, 2021). COVID-19 has stressed our society and our juris- prudence in a multitude of ways. Unfortunately, the next pandemic may be more lethal. It is also likely to have a different epidemiological profile, and require a very different mix of interventions than those that states used in 2020. To guide us through the inevita- ble clashes between religious liberty and public health that will then arise, we need a Free Exercise doctrine that takes both the science and the potentially adverse consequences of religious liberty more seriously than the opinions from the shadow docket. ) 9. See W.E. Note Prof. Parmet reports after completing the manuscript, but prior to its publication, she was awarded a subgrant to a grant from the Robert Wood Johnson Foundation to ChangeLabs to work on a project relating to public health authorities, which may turn out to relate to some of the issues discussed in this paper. In addition, also after the manuscript was submitted, Parmet signed onto an amicus brief in two cases before the Supreme Court on some of the issues discussed in the paper. 14. L.F. Wiley, “Federalism in Pandemic Prevention and Response,” in Public Health Law Watch, COVID-19 Policy Playbook supra note 8, at 84. 17. Id.; J. Blackman, “‘The Essential’ Free Exercise Clause,” Har- vard Journal of Law and Public Policy 44 (forthcoming 2021), available at <https://privpapers.ssrn.com/sol3/papers. cfm?abstract_id=3707739> (last visited October 1, 2021). Conclusion d f k Parmet, “The COVID-Cases: A Preliminary Assess- ment of Judicial Review of Public Health Powers During a Partisan and Polarized Pandemic,” San Diego Law Review 57, no. 4 (2020): 999-1048, at 1003-1010. 10. Id. at 1008-1010. 11. J. Oliphant, “U.S. Election Year Shaped by Pandemic and Trump’s Defiance,” Reuters, available at <https://www.reuters. com/article/global-poy-usa-election/u-s-election-year-shaped- by-pandemic-and-trumps-defiance-idUSKBN28K1FU> (last visited October 1, 2021). Acknowledgements Many thanks to Claudia Haupt. Jeremy Paul, and Dorit Rubinstein Reiss for their comments on an earlier draft of this paper and to Emily Kaiser and Riley Grinkis for their outstanding research assistance. 12. E.g., D.E. Campbell, “The Perils of Politicized Religion,” Dae- dalus 149, no. 3 (2020): 87-104. 13. T. B. Edsall, “In God We Divide,” New York Times, March 25, 2020, available at <https://www.nytimes.com/2020/03/25/ opinion/religion-democrats-republicans.html> (last visited October 1, 2021). References 3, 2021, available at <https://www.wash- ingtonpost.com/health/750000-covid-deaths/2021/11/03/ d637daaa-35c1-11ec-9bc4-86107e7b0ab1_story.html> (last visited Nov. 6, 2021).i 6. C. Barber, “The Problem of ‘Long Haul’ COVID,” Scientific American, Dec. 29, 2020, available at <https:www.scientifi- camerican.com/article/the-problem-of-long-haul-covid> (last visited October 1, 2021). 25. Parmet, supra note 9, at 1008-10. 26. F. Wiley, “Democratizing the Law of Social Distancing” Yale Journal of Health Policy, Law and Ethics, 19, no. 3 (2020): 50-121. 27. E.g. M. Koran, “California Megachurch Linked to Spread of More than 70 Coronavirus Cases,” The Guardian, April 7. L. Lopez III et al., “Racial and Ethnic Health Dispari- ties Related to COVID-19,” JAMA, Jan. 22, 2021, avail- 576 https://doi.org/10.1017/jme.2021.80 Published online by Cambridge University Press journal of law, medicine & ethics journal of law, medicine & ethics journal of law, medicine & ethics The Journal of Law, Medicine & Ethics, 49 (2021): 564-579. © 2021 The Author(s) The Journal of Law, Medicine & Ethics, 49 (2021): 564-579. © 2021 The Author(s) Parmet 3, 2020, available at <https://www.theguardian.com/ world/2020/apr/03/california-church-coronvirus-outbreak- sacramento> (last visited October 1, 2021); T. Salaum, “Special Report: Five Days of Worship that Set a Virus Time Bomb in France,” Reuters, March 30, 2020, available at <https://www. reuters.com/article/us-health-coronavirus-france-church- spec/special-report-five-days-of-worship-that-set-a-virus- time-bomb-in-france-idUSKBN21H0Q2> (last visited Octo- ber 1, 2021); N. Lanese, “Superspread’ in South Korea Infects Nearly 40 People with Coronavirus,” Live Science, Feb. 23, 2020, available at <https://www.livescience.com/coronavirus- superspreader-south-korea-church.html> (last visited October 1, 2021). 3, 2020, available at <https://www.theguardian.com/ world/2020/apr/03/california-church-coronvirus-outbreak- sacramento> (last visited October 1, 2021); T. Salaum, “Special Report: Five Days of Worship that Set a Virus Time Bomb in France,” Reuters, March 30, 2020, available at <https://www. reuters.com/article/us-health-coronavirus-france-church- spec/special-report-five-days-of-worship-that-set-a-virus- time-bomb-in-france-idUSKBN21H0Q2> (last visited Octo- ber 1, 2021); N. Lanese, “Superspread’ in South Korea Infects Nearly 40 People with Coronavirus,” Live Science, Feb. 23, 2020, available at <https://www.livescience.com/coronavirus- superspreader-south-korea-church.html> (last visited October 1, 2021). 48. Id. at 31. 49. Id. at 38. 50. Id. at 27. 51. See Zucht v. King, 257 U.S. 650 (1921); Phillips v. New York, 775 F.3d 538 2d Cir. (2015). 52. 321 U.S. 158 (1944). 53. 374 U.S. 398, 403 (1963). For a discussion of the Court’s cita- tion of Jacobson in Sherbert, see J. Blackman, supra note 17, at 46-47. 54. 374 U.S. at 403. 55. Employment Div., Dep’t of Human Resources of Oregon v. Smith, 494 U.S. at 872, 878-885 (1990). , , 56. Church of the Lukumi Babalu Aye, Inc. v. City of Hialeah, 508 U.S. 520 (1993). 28. A. Kuhn, “Secretive Church Sect at the Center of South Korea’s Coronavirus Outbreak,” NPR, Feb. 24, 2020, available at <https://www.npr.org/sections/goatsand- soda/2020/02/24/808914718/secretive-church-sect-at-the- center-of-south-koreas-coronavirus-outbreak> (last visited October 1, 2021); Lanese, supra note 27. 57. Id. at 533. 57. Id. at 533. 58. 138 S. Ct. 1719, 1729-1732 (2018). 59. Id. at, 1734, 1734 (2018)(Gorsuch, J., concurring). 60. Id. at 1736. 60. Id. at 1736. 61. 138 S. Ct. 2433 (2016)(mem.) 29. A. James et al., “High COVID-19 Attack Rate Among Attend- ees at Events at a Church — Arkansas,” Morbidity and Mortal- ity Weekly Report 69, no. 20 (2020): 632-635. 62. Stormans, Inc. v. Wiseman, 794 F.3d 1064, 1074-1086 (9th Cir. 2015). 63. Id. at 2439. journal of law, medicine & ethics y y p , ( ) 30. T T.A. Henry, “5 Reasons Why Religious Services Pose High Risk of COVID-19 Spread,” AMA News, Dec. 7, 2020, avail- able at <https://www.ama-assn.org/delivering-care/public- health/5-reasons-why-religious-services-pose-high-risk- covid-19-spread> (last visited October 1, 2021). Religious services continued into 2021 to be associated with outbreaks. See A. Salcedo, “An Oregon Church Sued Over COVID-19 Restrictions. Now an Outbreak There has Sickened 74,” Washington Post, May 7, 2021, available at <https://www. washingtonpost.com/nation/2021/05/07/oregon-peoples- church-covid-outbreak/?utm_campaign=wp_main&utm_ source=twitter&utm_medium=social> (last visited October 1, 2021). 64. 140 S. Ct. 1104 (2020)(mem.). 65. Fulton v. City of Philadelphia, 922 F.3d 140, 147 (3d Cir. 2019), rev’d 141 S .Ct.1868 (2021). 65. Fulton v. City of Philadelphia, 9 2019), rev’d 141 S .Ct.1868 (2021). 66. Petition for Writ of Certiorari, Fulton v. City of Philadelphia, 2019 WL 3380520 (No. 19-123) (July 22, 2019). 67. See E. Tomrick, Note “The Public Health Demand for Revok- ing Non-Medical Exemptions to Compulsory Vaccination Statutes,” Journal of Law and Health 34, no.1 (2020): 131-156. 68. See, e.g., Brown v. Smith, 235 Cal. Rptr. 3d 218 (Cal. Ct. App. 2018); Whitlow v. Cal. Dep’t of Educ., 203 F. Supp. 3d. 1079 (S.D. Cal. 2016); F.F. v. State of New York, 114 N.Y.S.3d 852 (N.Y. Sup. Ct. 2019). 69. Parmet, supra note 9, at 1026. 69. Parmet, supra note 9, at 1026. 31. Centers for Disease Control and Prevention, “Consider- ations for Communities of Faith,” Dec. 30, 2020, available at <https://www.cdc.gov/coronavirus/2019-ncov/community/ faith-based.html> (last visited October 1, 2021). 70. Id. at 1002. 71. E.g., Elim Romanian Pentecostal Church v. Pritzker, No. 201811, 2020 WL 2517093 (7th Cir. May 16, 2020); South Bay United Pentecostal Church v. Newsom, 959 F.3d 938 (9th Cir. 2020), aff’g 140 S. Ct. 1613 (2020); Antietam Battlefield KOA v. Hogan, No. CCB-20-1130, 2020 WL 2556496 (D. Md. May 20, 2020). fif 32. Pew Research Center, “Most States have Religious exemptions to COVID-19 Social Distancing Rules,” April 27, 2020, avail- able at <https://www.pewresearch.org/fact-tank/2020/04/27/ most-states-have-religious-exemptions-to-covid-19-social- distancing-rules> (last visited October 1, 2021). 72. U.S. Department of Justice, “Office of Public Affairs, Attorney General William P. Barr Issues Statement on Religious Prac- tice and Social Distancing; Department Files State of Inter- est in Mississippi Church Case,” April 14, 2020, available at <https://www.justice.gov/opa/pr/attorney-general-william-p- barr-issues-statement-religious-practice-and-social-distanc- ing-0> (last visited March 2, 2021). 33. Id. See D.R. Reiss and M. Thomas, “More than a Mask: Stay- at-Home Orders and Religious Freedom,” San Diego Law Review 57, no. 3, 2020, available at <https://www.theguardian.com/ world/2020/apr/03/california-church-coronvirus-outbreak- sacramento> (last visited October 1, 2021); T. Salaum, “Special Report: Five Days of Worship that Set a Virus Time Bomb in France,” Reuters, March 30, 2020, available at <https://www. reuters.com/article/us-health-coronavirus-france-church- spec/special-report-five-days-of-worship-that-set-a-virus- time-bomb-in-france-idUSKBN21H0Q2> (last visited Octo- ber 1, 2021); N. Lanese, “Superspread’ in South Korea Infects Nearly 40 People with Coronavirus,” Live Science, Feb. 23, 2020, available at <https://www.livescience.com/coronavirus- superspreader-south-korea-church.html> (last visited October 1, 2021). https://doi.org/10.1017/jme.2021.80 Published online by Cambridge University Press journal of law, medicine & ethics 889 (2020)(per curiam), he also warned that if that commu- nity did not comply with his orders “we’ll close the institution down.” Agudth Israel of America, 979 F.3d 177, 183 (2d Cir. 2020)(Park J., dissenting), rev’d 141 S, Ct, 889 (2020) (per curiam). 132. Id. at 1297 (quoting Roberts v. Neace, 958 F.3d 409, 414 (6 th Cir. 2020). 133. Id. at 1297 (citing South Bay II, 141 S. Ct. at 720 (statement of Gorsuch, J.)). 134. 141 S. Ct. at 1298 (Kagan, J., dissenting). 135. Id. 136. Id. at 1298. 137. Id. at 1299 (quoting South Bay Pentecostal Church v. Newsom, 141 S.Ct. 717, 722 (Kagan J., dissenting)) 141 S.Ct. 717, 722 (Kagan J., dissenting)) 99. Masterpiece Cakeshop, Ltd. v. Colo. Civil Rights Comm’n, 138 S. Ct. 1719, 1719 (2018). 138. South Bay Pentecostal v. Newsom, 2021 WL 1602607 (U.S. 2021)(citing Tandon, 141 S. Ct . at 1294). 100. Roman Catholic Diocese of Brooklyn, 141 S. Ct. at 66. 139. Fulton v. City of Philadelphia, 141 S. Ct. 1868 (2021). 140. Id. at 1878-1879. 141. Id. at 1880. 141. Id. at 1880. 142. Id. at 1880 (2021)(Barrett J., concurring). 103. Id. at 69 (Gorsuch, J., concurring). 104. Id. at 70-71. 143. Id. at 1883 (2021)(Alito, J., concurring). 144. Id. at 1921. 144. Id. at 1921. 106. Id. at 73 (Kavanaugh, J., concurring (quoting South Bay United Pentecostal Church v. Newsom, 590 U.S. __, 140 S. Ct. 1613 (Roberts, C.J. concurring)). 145. Id. at 1926 (2021)(Gorsuch, concurring). 146. See W.E. Parmet, Populations, Public Health and the Law (Georgetown University Press, 2009): at 41-47. 147. See Parmet, supra note 9, at 1010-1012. 108. Id. at 79 (Sotomayor, J., dissenting). In his dissent, Chief Jus- tice Roberts argued that although Gov. Cuomo’s orders were troubling, the Court had no need to act because the orders were no longer affecting the petitioners. 148. See supra text accompanying notes 80-84. 149. See C. Sunstein, “Our The Anti-Korematsu,” December 29, 2020, available at <https://papers.ssrn.com/sol3/papers. cfm?abstract_id=3756853> at 5 (last visited October 1, 2021). 109. By Thanksgiving, the U.S. had recorded 269,000 deaths from COVID-19. S. Kim, “1,311 People Die of COVID on Thanksgiving Day in the U.S.,” Newsweek, Nov. 27, 2000, available at <https://www.newsweek.com/coronavirus-us- death-toll-thanksgiving-travel-infections-cases-hospitaliza- tions-1550760> (last visited June 1, 2021). On February 22, 2021, the nation recorded its 500,000 death. P. Huang, “‘A Loss to the Whole Society’: U.S. journal of law, medicine & ethics Justice Alito also found that the petitioner was likely to succeed on the merits of a free speech claim. d 118. Id. 118. Id. 119. Id. at 723 (Kagan, J., dissenting). 120. Gateway City Church v. Newsom, 141 S. Ct. 1460, 1460 (2020). 87. Id. 121. A. Howe, “Court Clears Way for Indoor Worship Services in Northern California,” SCOTUSBlog, Feb. 26, 2020, available at <https://www.scotusblog.com/2021/02/court-clears-way- for-indoor-worship-services-in-northern-california/> (last October 1, 2021). 88. Id. at 2605 -2608. 88. Id. at 2605 -2608. 89. Id. at 2612 (Kavanaugh J., dissenting). 90. Id. at 2614. 91. Id. at 2615. 122. Gateway City Church. v. Newsom, 141 S. Ct. at 1460 (2021). 92. L. Greenhouse, “Ruth Bader Ginsburg, Supreme Court’s Fem- inist Icon, is Dead at 87,” New York Times, Sept. 24, 2020, available at <https://www.nytimes.com/2020/09/18/us/ruth- bader-ginsburg-dead.html> (last visited October 1, 2021).i 123. Tandon v. Newsome, 141 S. Ct. 1294, 1294-99 (2021)(per curiam). 124. Tandon v. Newsom, 992 F.3d 916 (9th Cir. 2021), vacated by 141 S. Ct. 1294 (2021). 93. B. Sprunt, “Amy Coney Barrett Confirmed to Supreme Court, Takes Constitutional Oath,” National Public radio, Oct. 26, 2020, available at <https://www.npr.org/2020/10/26/927640619/ senate-confirms-amy-coney-barrett-to-the-supreme-court> (last visited October 1, 2021). 125. Id. at 920. 126. Id. 126. Id. 127. Id. at 925 (citing Tandon v. Newsom, 2021 WL 411375, No. 20-CV-07108-LHK (N.D. Cal. Feb. 5, 2021), at *30). 94. Roman Catholic Diocese of Brooklyn v. Cuomo, 141 S. Ct. 63 (2020)(per curiam). 128. Id. at 920. 128. Id. at 920. 129. Tandon, 141 S. Ct. at 1296 (2021). d h l 95. Id. at 65. 95. Id. at 65. 130. Id. citing Roman Catholic Diocese of Brooklyn v. Cuomo, 592 U.S. ___, 141 S. Ct. 63, 66-67 (2020)(Kavanaugh, J., concurring). 96. Id. at 68. 97. Id. at 66 (citing Church of Lukumi Babalu Aye, Inc. v. Hia- leah, 508 U.S. 520, 533 (1993)). g 131. Id. citing 141 S. Ct. at 67 (per curiam); 141 S. Ct. at 66 (Gor- such, J., concurring). 98. Id. at 67. For example, although the Governor stated his “love for the Orthodox community,” Roman Catholic Diocese of Brooklyn v. Cuomo, 495 F. Supp. 3d 118, 122 (E.D.N.Y. 2020), aff’d Agudath Israel of Americ v. Cuomo, 979 F.3d 177 (2d Cir. 2020), rev’d Roman Catholic Diocese v. Brooklyn, 1414 S.Ct. journal of law, medicine & ethics 4 (2020): 947-972. 34. Pew Research Center, supra note 32. 34. Pew Research Center, supra note 32. 35. Id. , p 35. Id. 35. Id. 73. 957 F.3d 610, 614 (6 th Cir. 2020). 36. Id. 37. Id. 37. Id. 37. Id. 74. Id. at 615. 74. Id. at 615. 75. 958 F.3d 409, 416 (6 th Cir. 2020). 38. Parmet, supra note 9; B.J. Buchanan, “Covid-19 and the First Amendment: A Running Report, Free Speech Center,” Feb. 5, 2021, available at <https://www.mtsu.edu/first-amendment/ post/613/covid-19-and-the-first-amendment-a-running- report-may-21> (last visited October 1, 2021). 76. 140 S. Ct. 1613 (2020). 77. M. Walsh, “The Supreme Court’s ‘Shadow Docket’ is Draw- ing Increasing Scrutiny,” ABA Journal, Aug. 20, 2020, avail- able at <https://www.abajournal.com/web/article/scotus- shadow-docket-draws-increasing-scrutiny#:~:text=The%20 Supreme%20Court’s%20’shadow%20docket’%20is%20 drawing%20increasing%20scrutiny,-By%20Mark%20 Walsh&text=Image%20from%20Shutterstock.com.,of%20 argued%20cases%20and%20decisions> (last visited October 1, 2021). 39. See infra text accompanying notes 91-118. 40. 197 U.S. 11 (1905). 41. 494 U.S. 872 (1990). 42. 508 U.S. 520 (1993). 43. K. L. Wallach, The Antivaccine Heresy: Jacobson v. Massa- chusetts and the Troubled History of Compulsory Vaccination in the United States (Rochester, NY, Univ. Rochester Press, 2015): at 182-184. 78. 140 S. Ct. 1613 (Roberts, C.J., concurring). 79. Id. (Kavanaugh, J., dissenting). 44. W.E. Parmet, “Rediscovering Jacobson in the Era of COVID- 19,” Boston University Law Review Online 100 (2020): 117-33; D. Farber, “The Long Shadow of Jacobson v. Massachusetts: Public Health: Fundamental Rights, and the Courts,” San Diego Law Review 57 no. 4 (2020): 833-63. 80. Id. (Roberts, CJ, concurring). 81. Id. (citing and quoting 197 U.S. at 38). 82. Id. (quoting Marshall v. United States, 414 U.S. 417 (1974)). 83 Id 82. Id. (quoting Marshall v. United States, 414 U.S. 417 (1974)). 83. Id. 84. Id. at 1614 (Kavanaugh J., dissenting). 45. 197 U.S. at 22. 45. 197 U.S. at 22. 85. Calvary Chapel Dayton Valley v. Sisolak, 140 S. Ct. 2603, 2603-09 (2020) (Alito, J., dissenting) (finding that petitioner was likely to succeed on the merits of a free speech claim 46. Id. at 27. 46. Id. at 27. 47. Id. at 29. 577 first amendment values in health care • winter 2021 The Journal of Law, Medicine & Ethics, 49 (2021): 564-579. © 2021 The Author(s) The Journal of Law, Medicine & Ethics, 49 (2021): 564-579. © 2021 The Author(s) https://doi.org/10.1017/jme.2021.80 Published online by Cambridge University Press SYMPOSIUM 86. Id. at 2604 (Alito J., dissenting). 117. South Bay United Pentecostal Church v. Newsom, 141 S. Ct. 717-18 (2021)(Barrett, J. concurring). The Journal of Law, Medicine & Ethics, 49 (2021): 564-579. © 2021 The Author(s) https://doi.org/10.1017/jme.2021.80 Published online by Cambridge University Press journal of law, medicine & ethics Kroto- szynski, Jr., “If Judges Were Angels: Religious Equality, Free Exercise, the (Underappreciated) Merits of Smith,” Northwest- ern Law Review 102 (2008): 1189-1274. 174. 141 S. Ct. 1868, 1876 (2021). 175. Id. 154. Roman Catholic Diocese of Brooklyn v. Cuomo, 141 S. Ct. 63, 67 (2020)(per curiam). 176. For a discussion of how courts assess claims of substantial bur- den, and the problems with deferring to the plaintiff’s asser- tion, see F. M. Gedicks “‘Substantial’ Burdens: How Courts May (and Why they Must) Judge Burdens on Religion Under RFRA,” George Washington Law Review 85 (2017): 94-151. 155. Vlacdeck, supra note 4, at 16. 156. See supra text accompanying notes 61-63. 157. Fulton v. City of Philadelphia, 141 S. Ct. 1868, 1878 (2021). 158. Id. 177. E.g., Dahl v. Bd. of Trustees, Western Michigan University, 14 F. 4th 728 (2021): Dr. A. v. Hochul, 1:21-CV-1009, 2001 WL 4734404 (N. D. N.Y. Oct. 12, 2021), vacated, We the Patriots USA v. Hochul, No. 21-2566, 2021 WL 5121983 (2d Cir. Nov. 4, 2021). 159. Wiley, supra note 26. 160. See supra text accompanying notes 151-152. 161. D. Laylock, “The Remnants of Free Exercise,” Supreme Court Review (1990): 1-69, at 49. 178. 595 U.S. ___, 2001 WL 5027177 (Oct. 29, 2021). 162. C. Corbin, “Religious Liberty in a Pandemic,” Duke Law Jour- nal Online 70 (2020): 1-28, at 15-26, available at <https://dlj. law.duke.edu/2020/09/religiouspandemic/> (last visited June 1, 2021). 179. Id. (Barrett, J., concurring). 180. Id. (Gorsuch, J., concurring). 181. See also Danville Christian Acad., Inc. v. Beshear, 141 S. Ct. 527, 527-28 (2020)(Gorusch, J., dissenting)(questioning con- stitutionality on Free Exercise grounds of health order closing all schools). 163. Tandon v. Newsom, 141 S. Ct. 1294, 1296 (citing Roman Cath- olic Diocese of Brooklyn v. Cuomo, 141 S. Ct. 63, 66 (Gorsuch, J., concurring)). 164. Roman Catholic Diocese of Brooklyn v. Cuomo, 141 S. Ct. 63, 79 (2020)(Sotomayor, dissenting). 182. See supra text accompany notes 45-68. 183. See supra text accompany notes 67-68. 165. Id. at 65-67. 184. National Conference of State Legislatures, “States with Reli- gious and Philosophical Exemptions from School Immuni- zation Requirements,” available at <https://www.ncsl.org/ research/health/school-immunization-exemption-state-laws. aspx> (last visited October 1, 2021). 166. See A. Winkler, “Fatal in Theory and Strict in Fact: An Empiri- cal Analysis of Strict Scrutiny,” Vanderbilt Law Review 59 (2006): 793-871 (discussing study showing that strict scrutiny was often not fatal). 167. Fulton v. journal of law, medicine & ethics City of Philadelphia, 141 S. CT. 1868, 1883 (2021) (Alito, J., concurring). 185. See supra text accompanying notes 181-184. For a discussion of the potential impact of the COVID-cases and Fulton on vaccine mandates, see D. R. Reiss, “Vaccines Mandates and Religion: Where are We Headed with the Current Supreme Court?” Journal of Law, Medicine & Ethics 49, no. 4 (2021): 552-563. g 168. J.F. Witt, American Contagion: Epidemics and the Law From Smallpox to COVID-19 (New Haven, CT, Yale Univ. Press, 2020): at 24. 169. 141 S. CT. 1884 (Alito, J. concurring). 169. 141 S. CT. 1884 (Alito, J. concurring). 7 S i 186. 595 U.S. ___, 2001 WL 5027177 (Oct. 29, 2021)(Barrett, J., concurring). 170. See supra text accompanying notes 132-134. 171. Does 1-3 v. Mills, 2021 WL 5027177 (Oct. 29, 2021)(Gorsuch, J., dissenting). 172. See supra notes 8-39. 172. See supra notes 8-39. 579 journal of law, medicine & ethics COVID-19 Death Toll Reaches 500,000,” NPR, Feb. 22, 2021, available at <https://www.npr. org/sections/health-shots/2021/02/22/969494791/a-loss-to- the-whole-society-u-s-covid-19-death-toll-reaches-500-000> (last visited October 1, 2021). 150. S. Bay United Pentecostal Church v. Newsom, 141 S .Ct. 716, 716 (2021)(Roberts, CJ, concurring). Less than a month later, the Chief Justice, however, took a different position in Food and Drug Administration v. American College of Obstetricians and Gynecologists, 141 S.Ct. 578 (2021)(Roberts, C.J. concur- ring). In that brief opinion concurring with the Court’s deci- sion to stay a lower court order that would have required the FDA to allow pharmacists to dispense mifespristone (which is used in medical abortions) without an in-person visit, Roberts restated his comments about deference from South Bay I. 1. See Tandon, 141 S. Ct. at 1298-99 (Kagan, J., dissentin 152. See South Bay United Pentecostal Church v. Newsom (South Bay II), 141 S. Ct. 716, 716 (2021)(mem). 110. See infra text accompanying notes 111-138; High Plains Har- vest Church v. Polis, 141 S. Ct. 527, 527 (2020)(mem.). 153 See Winter v. Natural Resources Defense Council, Inc., 555 U.S. 7, 20 (2008)(explaining that a party seeking a prelimi- nary injunction “must establish that he is likely to succeed on the merits, that he is likely to suffer irreparable harm in the absence of preliminary relief, that the balance of equities tips in his favor, and that an injunction is in the public inter- est); Schaffer ex re. Schaffer v. Weast, 546 U.S. 49 (2005)(“we have usually assumed without comment that plaintiffs bear the burden of persuasion regarding the essential aspects of their claim.”). For a further discussion of burdens of proof and 111. South Bay United Pentecostal Church v. Newsom (South Bay II), 141 S. Ct. 716, 716 (2021). ), , ( ) 112. Id. 113. Id. at 717 (Roberts, C.J. concurring). 114. Id. at 719 (statement of Gorsuch, J). 114. Id. at 719 (statement of Gorsuch, J). 115. Id. 7 9 ( , ) 115. Id. 116. Id. at 716 (statement of Alito, J. ). 117. South Bay United Pentecostal Church v. Newsom, 141 S. Ct. 717-18 (2021)(Barrett, J. concurring). 578 The Journal of Law, Medicine & Ethics, 49 (2021): 564-579. © 2021 The Author(s) Parmet 173. Roman Catholic Diocese of Brooklyn v. Cuomo, 141 S. Ct. 63 at 66-67 (2020). standards applicable to Free Exercise claims, see R.J. 186. 595 U.S. ___, 2001 WL 5027177 (Oct. 29, 2021)(Barrett, J., concurring). first amendment values in health care • winter 2021 The Journal of Law, Medicine & Ethics, 49 (2021): 564-579. © 2021 The Author(s) https://doi.org/10.1017/jme.2021.80 Published online by Cambridge University Press
https://openalex.org/W2903971524	https://europepmc.org/articles/pmc6420443?pdf=render	English	null	Pupil size and search performance in low and high perceptual load	Cognitive, affective & behavioral neuroscience	2,018	cc-by	8,273	Abstract The ability to focus on a task while disregarding irrelevant information is an example of selective attention. The perceptual- load hypothesis argues that the occurrence of early or late selection mechanisms is determined by task-relevant perceptual load. Additionally, evidence shows that pupil size serves as proxy of locus coeruleus-norepinephrine (LC-NE) activity, a system associated with cognitive and attentional mediation. Here, we assessed pupil baseline (and pupil dilation) as predictors of load-related early and late selection performance. Participants were asked to search for a target in conditions of high and low perceptual load, while ignoring irrelevant stimuli. The results showed that pupil baseline size, measured prior trial onset, significantly predicted the upcoming search efficiency only in low perceptual load, when—according to the perceptual-load hypothesis—all perceptual information receives attentional resources. In addition, pupil dilation was linked to the time course of perceptual processing and predicted response times in both perceptual load conditions, an association that was enhanced in high load. Thus, this study relates attentional selection mechanisms, as defined by the perceptual-load theory, with pupil-related LC-NE activity. Because pupil baseline predicted attentional performance in low load but not in high load, this suggests that different attentional mechanisms are involved, one in which the LC-NE system plays a key role (low load) and one in which it is less relevant (high load). This suggests that the degree with which LC-NE influences behavioral performance is related to the perceptual load of the task at hand. Keywords Perceptual load · Pupillometry · Locus coeruleus · Visual search · Attention Pupil size and search performance in low and high perceptual load Manuel Oliva1,2 © The Author(s) 2018 Published online: 14 December 2018 1 Cognitive Science, Lund University, Lund, Sweden 2 MAPP, Aarhus University, Aarhus, Denmark https://doi.org/10.3758/s13415-018-00677-w Cognitive, Aﬀective & Behavioral Neuroscience (2019) 19:366–376 https://doi.org/10.3758/s13415-018-00677-w Cognitive, Aﬀective & Behavioral Neuroscience (2019) 19:366–376 Manuel Oliva manueloliva@gmail.com Introduction On the contrary, high tonic activity correlated with poorer performance (higher rate of false-positive responses to non- target stimuli) and diminished LC-NE phasic responses. et al., 1994; Privitera et al., 2010). The LC-NE system can have periods of higher or lower tonic (basal) activity, which have been associated with shifts in attentional performance (Usher, 1999; Gilzenrat et al., 2010). Most of this evidence comes from electrophysiological studies in monkeys performing go/no-go tasks, in which epochs of low LC tonic activity correlated with better attentional performance (reflected by lower rates of false-positive responses to non-target stimuli) and pronounced phasic spike bursts after the perceptual detection of the targets. On the contrary, high tonic activity correlated with poorer performance (higher rate of false-positive responses to non- target stimuli) and diminished LC-NE phasic responses. Deutsch and Deutsch (1963) proposed a model capable of explaining selection in tasks with low perceptual load, such as the flanker task. In contrast to early selection, this model posits that perception proceeds in parallel across all stimuli. According to this account, selection of the target stimulus occurs “late” in processing, as a result of the need to provide a pertinent behavioral response. Late selection models explain flanker interference effects by predicting that because of the absence of early perceptual filtering, irrelevant stimuli would compete with the target stimulus and influence response times. These seemingly contradictory differences between models led (Kahneman & Treisman, 1984) to suggest the existence of two different attentional mechanisms acting in different circumstances, a hypothesis that was further developed in the perceptual load hypothesis (Lavie, 1995; Lavie & Tsal, 1994). The perceptual load model integrates early and late selection accounts by proposing that the perceptual load of the task at hand is the main factor determining whether early or late mechanisms will occur. As in the late account, it proposes that perception is an automatic process, in the sense that it proceeds in parallel across all stimuli without voluntary control. The perceptual load hypothesis adds that perception proceeds automatically only until the perceptual system runs out of capacity, in which case not all perceptual information receives further processing. By manipulating the degree of perceptual load of a flanker task, Lavie and Cox (1997) showed that high perceptual load can prevent the interference produced by a competing flanker. Introduction In addition, (De Fockert et al., 2001) showed that cortical functions are important for selective attention in conditions of low perceptual load. In such cases, all information is fully perceived and working memory seems to play a key role in maintaining the prioritization of relevant information. In a series of experiments, it was shown that by taxing the participants’ working memory system, selective attention was impaired in low load but not in high load (De Fockert, 2013; Lavie et al., 2004). Although accumulating evidence links noradrenergic activity with attention and cognitive processes, the role of LC-NE activity within attentional selection mechanisms is not yet understood. In the present study, we used pupil size measures of tonic and phasic LC-NE activity to predict performance in an attentional task. Perceptual load and the locus of selection Researchers have long been interested in detecting how behaviorally relevant information is selected within the course of attentional processing. The first influential theory that accounted for selective attention was proposed by Broadbent (1958) and later updated by Treisman (1969). In this theory, they proposed a two- stage perceptual mechanism where first, physical features of the stimuli are extracted in parallel and filtered, so that only the stimulus of interest will receive further processing. According to this theory, selection occurs in an early processing stage after which irrelevant stimuli receive no further analysis. Early selection models are well suited to explain selection in perceptually difficult tasks, such as in “shadowing” experiments (Cherry, 1953), which involve high perceptual load (i.e., complex target stimulus, large set size). In these experiments, participants had to hear two auditory messages played each on different ears (usually by means of headphones), and repeat out loud (or to “shadow”) only one of the messages. Along with early selection models, these experiments showed that individuals are good at efficiently selecting one channel while at the same time disregarding the irrelevant one (Treisman, 1969). However, this model failed to explain selection under low perceptual load (i.e., simple target stimulus, small set size). A clear example of the latter are flanker tasks (Eriksen & Eriksen, 1974) where participants are asked to report the presence of one out of two possible targets while at the same time ignoring a peripheral distractor. This paradigm shows that under low load, individuals are unable to ignore irrelevant stimuli, which translates into slower responses compared to when no distractor is present. Introduction Fluctuations in pupil size have been associated with the time course of perceptual processing (de Gee et al., 2014) and decision-making (Einh¨auser et al., 2010; Oliva & Anikin, 2018). This relationship arises because under isoluminance conditions, pupil dilation is largely caused by norepinephrine (NE) release from the locus coeruleus (LC) (Joshi et al., 2016). The LC sends inputs to different prefrontal brain areas involved in control functions and attentional processing (Foote et al., 1991; Joshi et al., 2016). Norepinephrine release on these target areas is believed to act by increasing neural gain (Aston-Jones & Cohen, 2005), which enhances the signal-to-noise ratio in the processing of sensory input (Sara & Bouret, 2012b; Mather et al., 2016a; Arnsten & Rubia, 2012). In fact, a recent neuroimaging study showed that LC-NE activity improves the precision of cortical representations of perceptual signals (Warren et al., 2016). Among other functions, the LC-NE system seems to be highly involved in the detection of behaviorally relevant stimuli. When a target is detected, the LC produces a phasic activation that is subsequently accompanied by a task-evoked pupil response (Usher, 1999; Aston-Jones When a person is engaged in studying, playing sports, or focused on reading this article, that individual is likely to become simultaneously unaware of events happening in the surroundings. These examples of selective attention occur as the result of processing limitations, where either due to bottom-up or top-down mechanisms, only a limited amount of the information received from the environment is fully processed for meaning. An increasing body of evidence points to the importance of noradrenergic activity during perceptual processing. Therefore, this study investigates the link between visual perceptual load processing with locus coeruleus-norepinephrine function as measured through tonic and phasic changes in pupil size. Manuel Oliva manueloliva@gmail.com Manuel Oliva manueloliva@gmail.com 1 Cognitive Science, Lund University, Lund, Sweden 2 MAPP, Aarhus University, Aarhus, Denmark Cogn Aﬀect Behav Neurosci (2019) 19:366–376 367 et al., 1994; Privitera et al., 2010). The LC-NE system can have periods of higher or lower tonic (basal) activity, which have been associated with shifts in attentional performance (Usher, 1999; Gilzenrat et al., 2010). Most of this evidence comes from electrophysiological studies in monkeys performing go/no-go tasks, in which epochs of low LC tonic activity correlated with better attentional performance (reflected by lower rates of false-positive responses to non-target stimuli) and pronounced phasic spike bursts after the perceptual detection of the targets. Participants Nineteen participants (mean age = 26, age range = 21– 41) with normal or corrected-to-normal vision voluntarily participated in the experiment and received a cinema ticket in return. Data from two participants were discarded due to poor eye-tracking data quality (more than 50% of data loss, see Data Analysis). As described above, this paradigm predicts that under low load, all stimuli from the search array will receive perceptual resources and analyzed in parallel. This leads to a condition where all perceptual information access awareness and selection is then resolved after stimuli identification—in which case, cognitive control and work- ing memory are critical for successfully selecting and prior- itizing relevant information (Lavie et al., 2004; De Fockert, 2013). In this context, we expect that visual search perfor- mance should be modulated by the LC-NE tonic activity reflected by baseline pupil size—measured just before trial onset—particularly in conditions of low perceptual load. If LC-NE modulates cognitive processing, this modulation should be reflected in low load, when all stimuli receive full perception. In high perceptual load, in contrast, not all perceptual information is perceptually processed at once. This is because perceptual information is filtered out due to capacity limits. In such a case, we expect pupil baseline size not to predict search performance. Ethical statement In accordance with the Swedish law (SFS 2003: 460, 16 §) all participants gave written consent for participating in the experiment. The present study was exempt from the requirement for ethical approval. The present study In the present study, we examined the relationship between LC-NE activity—as measured through pupil size—and the efficiency of visual search for a target in conditions of high and low perceptual load. For such a purpose, we adapted a task previously used for the study of perceptual load and selective attention (Lavie, 1995; Lavie & Cox, 1997; Theeuwes et al., 2004) so that it could be used under isoluminant conditions. In this task, participants are instructed to report the appearance of a target letter (X or N) within a central search array that contains the target together with other five non-target letters (Fig. 1). Simultaneously, participants have to ignore a peripheral distractor. The Cogn Aﬀect Behav Neurosci (2019) 19:366–376 368 Fig. 1 a Participants started fixating at a central cross and after a non-aging foreperiod a search array was presented and participants had to report the target stimulus. b Examples of the search arrays from each condition a b a Participants started ing at a central cross and a non-aging foreperiod a h array was presented and cipants had to report the t stimulus. b Examples of earch arrays from each ition a a b Fig. 1 a Participants started fixating at a central cross and after a non-aging foreperiod a search array was presented and participants had to report the target stimulus. b Examples of the search arrays from each condition b a a is an indirect measure of perceptual load effect, which reflects different attentional selection strategies (Lavie, 2010). is an indirect measure of perceptual load effect, which reflects different attentional selection strategies (Lavie, 2010). distractor letter can be compatible (i.e., same as target letter) or incompatible (i.e., alternative target letter). The perceptual load of the task is manipulated by varying the similarity between the target with the non-target letters (Fig. 1). In high load, the non-target letters in the array are more similar to the target than in conditions of low load. In this way, perceptual load is manipulated while keeping similar set sizes between the low and high load search conditions. Stimuli The baseline pupil diameter for each trial was calculated as the average diameter over a period of 1 s before trial onset (during the inter-trial foreperiod). For the analysis of baseline, trial baseline values from each participant were normalized by the respective average resting state pupil size measured during 40 s of passive fixation (see Procedure), so as to compare the behavioral state of the participant prior trial onset against their resting state. Task- evoked pupil responses were computed as the relative difference in pupil size between the trial baseline and the peak pupil dilation measured until 3 s after the participant’s key press. Pupil dilation peak timing was determined as the difference in time between the peak pupil dilation and trial onset. Pupil data were processed in Python (2.7.11) to detect blinks and gaze displacement. Blinks and other artifacts where removed from the resting state baseline average calculation. Trials containing blinks between trial onset and the peak dilation and/or when gaze was displaced from the central fixation cross were excluded from all analyses. Trials in which periods of blinks, missing data and gaze displacement represented more than 20% of the total trial samples were also excluded. Under these criteria, two participants were excluded from the analyses for excessive data loss (less than 50% usable trials). All the included participants had above 74% of usable data. We used R (RStudio, v1.0.153) to perform the analyses of the relationship between response time and pupil size. Response times were positively skewed. A common approach to correct for deviations of normality is to inverse transform response times (1/RT), however, applying nonlinear transformations can affect the interpretation of interactions. Thus, we used generalized linear mixed-effect Bayesian models assuming an inverse Gaussian distribution with inverse (-1/RT) link, which provide a solution to this problem by satisfying normality assumptions without the need for transformation (Lo & Andrews, 2015). Because pupil dilation and pupil baseline were partially correlated (r = .39), their effects were assessed in two separate models. Errors rates were analyzed through logistic regressions. Statistical significance of predictors was tested with likelihood ratio tests using lme4 (Bates et al., 2015). To extract confidence intervals, we fit analogous Bayesian models, which arguably offer more robust estimates in the context of multilevel regression. Bayesian models were created in Stan (http://mc-stan.org/) and brms package (B¨urkner, 2017). Apparatus The presentation of the stimuli was controlled using Psy- chopy (Peirce, 2007) (v2.85). The stimuli were presented on a 1280 × 1080 monitor screen (Samsung 931C) with a refresh rate of 75 Hz. Pupil size and gaze position were recorded with a tower-mounted eyetracker (SMI, Teltow, Germany) at 500 Hz. Participants used a chinrest and main- tained a viewing distance of 65 cm. Isoluminant colors for the letters and the background were approximated using the YUV color encoding system and later adjusted to be perceptually isoluminant with the flicker-fusion procedure The perceptual load of the main task will also influence distractor processing. Low perceptual load arrays may allow the perception of the distractor, which may interfere with response selection in the case of incompatible trials. High perceptual load displays, in contrast, will deplete resources and distractor compatibility should have little influence on response times. As such, the degree of distractor processing Cogn Aﬀect Behav Neurosci (2019) 19:366–376 369 (Lambert et al., 2003). The resulting colors had the RGB values of 69, 149, 24 for the background and 223, 61, 61, for the letters. Under these conditions, the luminance was kept constant throughout the experiment at 56 cd/m2. distractor. Feedback about their performance (response times and error rates) was displayed on the computer screen after the completion of each block. Stimuli To improve convergence and guard against overfitting (McElreath & Smaldino, 2015), we specified The target letters that participants were instructed to report were X and N. In the low load condition, the non-target letters were all “O”. In the high load condition, the non- target letters were the letters “W”, “Z”, “F”, “H”, “T”. In this way, there were always five non-target letters in both the high and low conditions, although, the processing demands were higher for the high perceptual load condition (see Fig. 1). Each letter subtended 1.1◦in height and 0.8◦ in width. The letters were presented randomly at 45◦, 90◦, 135◦of arc on an imaginary hexagon at an eccentricity of 3.5◦. The distractor letter was presented randomly to the left or right sides of the letters array with a random position varying between +/- 10◦of arc. The distractor was displayed at an eccentricity of 4.5◦from the fixation point. Perceptual load and classic interference The attentional task utilized in this study was adapted from a commonly used visual search task for the study of perceptual load effects (Lavie 1995, 2005). In this task, stimuli are usually displayed without controlling for luminance. However, because pupil size reflects both LC- NE activity and the light reflex, we adapted this task so that the stimuli were isoluminant with the background. Isoluminance may reduce contrast between the stimuli and the background and therefore we first assessed whether the classic effects of perceptual load were maintained under our manipulation. Fig. 2 Pupil size fluctuations during passive fixation and isoluminance conditions. Spontaneous changes in pupil size correlate with to LC- NE activity (Joshi et al., 2016). An average resting state baseline was extracted from each participant in order to normalize pupil size We hypothesized that perceptual load should modulate interference caused by a peripheral distractor (Lavie & Cox, 1997). In this, we expected an interaction between perceptual load and compatibility of distractor, where incompatible distractors should delay response times when they are processed (i.e., under low load where individuals still have available perceptual resources). In contrast, little or no distractor interference is expected in high perceptual load, where there is little spare capacity to process the peripheral distractor. of compatibility of the distractor had no significant main effect (853 vs. 840 ms; χ2 = 2.28, df = 1, p = 0.131). However, in line with the perceptual load theory, there was a significant interaction between load (high vs. low) and type of distractor (compatible vs. incompatible) (Table 1). In high load the compatibility effect was only - 5 ms, while in Fig. 3 The pupil baseline prior the onset of each trial was normalized by a resting state baseline recorded during passive fixation. Trial baselines preceding each trial were on average smaller compared to a resting state baseline. However, there were no differences between conditions. Such decrease in pupil size seems to reveal attentional predispositions towards the upcoming attentional task. Error bars represent SEM In order to analyze the independent effects of load and compatibility on response time, we conducted a linear regression analysis of response time. The model included load (high/low) and compatibility (compati- ble/incompatible) as main effects, random intercepts for participants, and random slopes for load. The results showed that, as expected, low-load displays yielded significantly faster RTs than did the high load (1005 vs. Design and procedure Participants received 192 experimental trials separated in four blocks of 48 trials each with optional breaks in between blocks. Blocks of high and low load were presented in counterbalanced order. There was an equal number of compatible and incompatible trials on each block and the position of the distractor was randomized in every trial. Participants completed at least 48 practice trials. If necessary, the practice session was extended until participants reached at least 70% of correct trials on each load condition. After calibration of the eyetracker, the resting state baseline size was measured. For this purpose, participants were asked to passively fixate for 40 s on a central fixation circle. This prolonged window allowed to average out local fluctuations in pupil size, so as to calculate the mean pupil size for each participant when they are not engage in any specific cognitive task. The experimental trials began with the presentation of a fixation cross at the center of the screen, which was presented following a non-ageing foreperiod of 5-11 s. A non-ageing foreperiod reduces the effect of target onset expectations (Oswal et al., 2007). The relatively long foreperiod allowed the pupil to subside back to baseline levels. Subsequently, the central search array and distractor were displayed for 183 ms (see Fig. 1). The short presentation time was intended to avoid the use of eye movements for the visual search. If an X was presented, the participants had to press the “2” key; if an N was presented, they had to press the “0” key. Participants were instructed to report the target present in the central search array and to ignore the peripheral Cogn Aﬀect Behav Neurosci (2019) 19:366–376 370 Fig. 2 Pupil size fluctuations during passive fixation and isoluminance conditions. Spontaneous changes in pupil size correlate with to LC- NE activity (Joshi et al., 2016). An average resting state baseline was extracted from each participant in order to normalize pupil size mildly informative conservative priors. Python and R scripts for analyses are available in supplementary materials (osf.io/4r7wf). Perceptual load and classic interference 715 ms; χ2 = 22.52, df = 1, p<0.001). The effect Table 1 Response times (SE in parenthesis) for the different load and compatibility conditions Table 1 Response times (SE in parenthesis) for the different load and compatibility conditions Compatible Incompatible I - C Low Load 701 (35) 729 (34) 27∗ Errors (%) 1.5 2.9 − High Load 1008 (40) 1003 (46) -5 Errors (%) 14.3 14.8 − There was a significant interaction between load and distractor com- patibility, indicating that our manipulation successfully reproduced the effect of perceptual load on irrelevant distractor processing. Error rates (in percentage) are presented for each condition. * p < 0.01 Compatible Incompatible I - C Low Load 701 (35) 729 (34) 27∗ Errors (%) 1.5 2.9 − High Load 1008 (40) 1003 (46) -5 Errors (%) 14.3 14.8 − Fig. 3 The pupil baseline prior the onset of each trial was normalized by a resting state baseline recorded during passive fixation. Trial baselines preceding each trial were on average smaller compared to a resting state baseline. However, there were no differences between conditions. Such decrease in pupil size seems to reveal attentional predispositions towards the upcoming attentional task. Error bars represent SEM There was a significant interaction between load and distractor com- patibility, indicating that our manipulation successfully reproduced the effect of perceptual load on irrelevant distractor processing. Error rates (in percentage) are presented for each condition. * p < 0.01 There was a significant interaction between load and distractor com- patibility, indicating that our manipulation successfully reproduced the effect of perceptual load on irrelevant distractor processing. Error rates (in percentage) are presented for each condition. * p < 0.01 Cogn Aﬀect Behav Neurosci (2019) 19:366–376 371 low load it was 27 ms, indicating an interaction estimate of 33 ms (χ2 = 7.45, df = 1, p = 0.006). Total error rates were relatively low (9 %). There were less errors in the low load than in the high condition (χ2 = 28.20, df = 1, p<0.001). Compatibility did not affect error rates (χ2 = 1.38, df = 1, p = 0.236) and there was no interaction between load and compatibility (χ2 = 3.19, df = 1, p = 0.068). Overall, these results indicate that we were successful in designing isoluminant high- and low-load conditions that resulted in compatibility effects in the low-load condition and a no compatibility effect in the high-load condition. Perceptual load and classic interference attentional performance (Aston-Jones, 2005). In particular, low tonic LC activity was linked with better performance in attentional tasks (Aston-Jones & Cohen, 2005; Gilzenrat et al., 2010). Therefore, if the pupil baseline preceding each trial reflects activity in the LC, we hypothesized that it should have a significant effect on search efficiency. g y In order to compare the effect of baseline, the baselines preceding each trial were normalized by a resting state pupil size recorded after the calibration procedure of the eyetracker (see Fig. 2). As shown in Fig. 3, the baseline preceding trial onsets were smaller in both the low (0.885, 95% CI [0.945; 0.824]) and high (0.862, 95% CI [0.916; 0.807]) perceptual load conditions compared to a resting state baseline. However, a comparison between load conditions showed that their baselines did not differ significantly between each other (χ2 = 0.703, df = 1, p = 0.402). A transition towards a smaller pupil size suggests a decrease in LC tonic activity, usually associated Pupil baseline vs. perceptual load Pupil baseline fluctuations (see Figures 2 and 6) have been shown to be an indicator of LC-NE tonic activity (Joshi et al., 2016). LC-NE can have periods of higher or lower basal activity, which have been associated with shifts in 600 900 1200 0.4 0.8 1.2 Normalized Pupil Baseline Response Time (ms) Load H L Fig. 4 The effect of baseline pupil size on response time for high (H) and low (L) perceptual load. There was a significant relationship between pupil baseline and response time only in low perceptual load. This difference suggest the involvement of distinct attentional mech- anisms, where pupil baseline seems to only reflect the influence one of such mechanisms. The plot displays fitted values and 95% credible intervals obtained from a Bayesian mixed model. Each data point rep- resents aggregated reaction time data. For display purposes together with model data, RTs were centered around each participant’s average response time and around the respective perceptual load group average. Smoothed RT histograms were restricted to fit plot limits 600 900 1200 0.4 0.8 1.2 Normalized Pupil Baseline Response Time (ms) Load H L Response Time (ms) Normalized Pupil Baseline intervals obtained from a Bayesian mixed model. Each data point rep- resents aggregated reaction time data. For display purposes together with model data, RTs were centered around each participant’s average response time and around the respective perceptual load group average. Smoothed RT histograms were restricted to fit plot limits Fig. 4 The effect of baseline pupil size on response time for high (H) and low (L) perceptual load. There was a significant relationship between pupil baseline and response time only in low perceptual load. This difference suggest the involvement of distinct attentional mech- anisms, where pupil baseline seems to only reflect the influence one of such mechanisms. The plot displays fitted values and 95% credible Cogn Aﬀect Behav Neurosci (2019) 19:366–376 372 low perceptual load. In low load, the effect of baseline going from a minimum to a maximum size is to slow down RTs by 121 ms (95% CI [62; 190] ms), where the smaller the baseline, the faster the participant’s RTs. In contrast, the baseline did not predict search efficiency in high load (χ2 = 0.164, df = 1, p = 0.685). The interaction between baseline and compatibility was not reliable (χ2 = 2.31, df = 1, p = 0.128). with improved attentional performance. Task-evoked pupil dilation The results indicated that the effect of the interaction between perceptual load and distractor compatibility, now controlling for pupil baseline, was found to remain significant compared to when no pupil size information was included (χ2 = 9.29, df = 1, p = 0.002). In fact, a comparison between models showed that adding pupil baseline information significantly improved the model’s fit (χ2 = 18.91, df = 2, p<0.001). Task-evoked pupil responses were analyzed with a regres- sion model that shared the same structure as that for pupil baseline. As depicted in Figs. 5 and 6, high perceptual load caused significantly larger peak pupil dilations than low load (16 vs. 21%, χ2 = 102.58, df = 1 p<0.001). The analyses showed that there was a significant effect of pupil dilation on response time, where larger pupil dilations correlated with slower response times (χ2 = 78.61, df = 1, p<0.001), which can be observed in Fig. 7. As for the baseline, there was a significant interaction between pupil dilation and perceptual load, where the role of pupil dilation was enhanced in predicting response times in high perceptual load (χ2 = 9.39, df = 1, p = 0.002). The effect of pupil dilation going from a minimum to a maximum is to slow down RTs by 110 ms (95% CI [47; 187]) in low load, and 558 ms (95% CI [377; 807] in high load. This indicates that the amplitude of pupil dilation was positively related to the latency of the response, where slower RTs led to larger pupil dilation. Lastly, there were no differences in the amplitude of pupil dilation as function of distractor compatibility (χ2 = 1.069, df = 1, p = 0.301). As shown in Fig. 4, there was a significant interaction between load and pupil baseline in explaining search performance (χ2 = 9.29, df = 1, p = 0.002). This interaction revealed an enhanced effect of baseline in conditions of Fig. 5 The figure shows pupil dilation relative to baseline for low and high perceptual load. The amplitude of pupil dilation was larger in high load (p<0.001). Error bars denote SEM Larger pupil dilations were associated with an increase of 3.97 in the log odds of making an error (χ2 = 18.35, df = 1, p<0.001). Pupil baseline vs. perceptual load In the case of our study, this shift suggest some attentional predisposition of the participants towards the upcoming attentional task. Baseline as predictor of response time We then investigated whether fluctuations in pupil baseline would predict search efficiency (as reflected by response times). To tease apart the independent effect of baseline, we conducted a linear regression analysis that included normalized trial baseline, perceptual load, and distractor compatibility as fixed effects. The model also controlled for trial order effects and included random intercepts for participants and random slopes for load. In addition to slowing down responses, pupil baseline did not significantly increase the log odds of committing an error (χ2 = 2.17, df = 1, p = 0.141). This indicates that the shift in response times cannot be attributed to a speed/accuracy trade-off. Pupil peak dilation timing The timing to peak dilation followed a dynamic similar to that observed for pupil dilation amplitudes reported in the previous section. There was a main effect of peak dilation time in predicting response times (χ2 = 188.54, df = 1, p<0.001), indicating that the timing of pupil dilation followed the timing of decoding and responding to the target letter. There was also a significant interaction with load (χ2 = 72.26, df = 1, p<0.001), revealing that the correlation Fig. 5 The figure shows pupil dilation relative to baseline for low and high perceptual load. The amplitude of pupil dilation was larger in high load (p<0.001). Error bars denote SEM 373 Cogn Aﬀect Behav Neurosci (2019) 19:366–376 between peak pupil dilation timing and the behavioral response was enhanced in high load. during passive fixation (Fig. 2). This finding is in line with previous evidence showing that low tonic LC-NE activity (also revealed by relatively smaller pupil size) correlates with periods of good attentional performance in go/no-go tasks (Gilzenrat et al., 2010; Usher, 1999) as well as with improved cortical representations of sensory inputs (Warren et al., 2016). Therefore, we interpret these results as suggesting that shifts in pupil baseline size reveal attentional preparatory mechanisms in anticipation for perceptual processing. Discussion This study is the first to relate LC-NE function, as measured through pupil size, with search performance in conditions of high and low perceptual load. According to recent evidence, fluctuations in pupil size serve as a proxy of LC-NE activity (Joshi et al., 2016), which, in turn, is associated with cognitive and attentional mediation (Aston-Jones & Cohen, 2005). The analyses presented here show that the extent at which pupil size fluctuations predicted task performance was modulated by task load. Specifically, pupil baseline predicted response times only in low load, whereas task- evoked pupil dilation predicted response times both in low and high load, although this relationship was enhanced in high load. In addition, the timing of the task-evoked pupil response also predicted search performance in both conditions. The LC presents spontaneous fluctuations in tonic level that translate into changes in pupil size, as observed during passive fixation (see Figs. 2 and 6). Here, we show that this variability in baseline size across trials predicted search performance, specifically in conditions of low perceptual load. In the context of a visual search task, low perceptual load allows participants to perform an efficient search for the target (Lavie & Cox, 1997), where all stimuli receive perceptual resources and information can be extracted in parallel across stimuli. Contrary to conditions of high perceptual load, selection in low perceptual load has been shown to rely more on control functions. For instance, decreases in attentional performance were reported, specifically in low perceptual load, when working memory capacity is taxed (De Fockert, 2013; Lavie, 2010). This interaction between control functions and the degree of perceptual load may explain the results presented here between pupil baseline and perceptual load. If selection in low load is more dependent on cognitive control, and cortical functions are modulated by the LC-NE system, then a modulation of performance in low load is to be expected. LC activity is believed to modulate arousal and cortical functions, with extensive influence over behavior and cognitive states (Aston-Jones & Cohen, 2005). Indeed, recent reviews suggest an important role of transient fluctuations in arousal on the modulation of cognitive and learning processes (Sara & Bouret, 2012a; Eldar et al., 2013; Mather et al., 2016b). The present analysis of pupil baseline shows that there was a significant decrease in pupil size as participants engaged in the experimental tasks (Fig. 3) compared to when pupil size was measured ig. Discussion 6 Pupil responses for each ondition. For visualization urposes, pupil responses are hown relative to resting aseline size. Pupil responses re time-locked to the moment f stimulus onset (0 s). As shown n the figure, pupil baselines rior trial onset were on average maller than during passive xation. Shaded areas represent EM at each time point Fig. 6 Pupil responses for each condition. For visualization purposes, pupil responses are shown relative to resting baseline size. Pupil responses are time-locked to the moment of stimulus onset (0 s). As shown in the figure, pupil baselines prior trial onset were on average smaller than during passive fixation. Shaded areas represent SEM at each time point Cogn Aﬀect Behav Neurosci (2019) 19:366–376 374 Recently, the perceptual load model was rivaled by an alternative account suggesting that the reduction in distractor interference under high perceptual load is due to ‘dilution’ of the distractor within the irrelevant letters in the search array (Tsal & Benoni, 2010; Benoni & Tsal, 2013; Wilson et al., 2011; Cave & Chen, 2016). Although both models differ on the selection mechanisms involved in high load, both agree on the fact that distractors are more likely to be processed under low perceptual conditions, as all information is perceived at once. Regardless of the differences between these views, the results presented here fit with predictions derived from both the perceptual load and dilution accounts, in the sense that they denote the occurrence of different attentional effects. with increased false positives responses (Aston-Jones et al., 1994; Usher, 1999; Gilzenrat et al., 2010). This difference may rely in the fact that “go” responses in such tasks had to be performed within strict time constraints, forcing speeded responses. In addition, the task used in the present study differs from go/no-go paradigms in the sense that it assesses attention allocated across stimuli within a search display, rather than the monitoring of a rapid visual stimuli presentation. Pupil dilation amplitude and timing The processing of the search arrays was followed by a phasic increase in pupil size. In line with previous reports (Lisi et al., 2015; Porter et al., 2006; Wahn et al., 2016), higher perceptual load elicited larger pupil dilation amplitudes (Fig. 7). In addition, the present results revealed an interaction between load and pupil responses in predicting search performance. While the timing and amplitude of pupil dilation were significant predictors of response time in both low and high, the link between Pupil baseline predicted search efficiency—as reflected by faster response times—and this was not connected to changes in response error rates. First, this is indicative that the improvement in response times cannot be attributed to speed/accuracy trade-off mechanisms. Secondly, this contrasts with experiments in monkeys using a go/no-go task which report that high tonic LC activity were associated 600 900 1200 0.0 0.2 0.4 0.6 Pupil Dilation Response Time (ms) Load H L 600 900 1200 0.0 0.2 0.4 0.6 Pupil Dilation Response Time (ms) Load H L Fig. 7 Pupil dilation and response times for high (H) and low (L) load. There was a positive relationship between pupil dilation and response times in both conditions, a relationship that was enhanced in conditions of high perceptual load. The plot displays fitted values and 95% cred- ible intervals obtained from a Bayesian mixed model. Each data point represent aggregated reaction time data. RTs were centered around each participant’s average response time and around the respective per- ceptual load group average. Because of uneven pupil dilation tails, 1.5% of data was excluded only for aggregation and display purposes. Smoothed RT histograms were restricted to fit plot limits Response Time (ms) Fig. 7 Pupil dilation and response times for high (H) and low (L) load. There was a positive relationship between pupil dilation and response times in both conditions, a relationship that was enhanced in conditions of high perceptual load. The plot displays fitted values and 95% cred- ible intervals obtained from a Bayesian mixed model. Each data point represent aggregated reaction time data. RTs were centered around each participant’s average response time and around the respective per- ceptual load group average. Because of uneven pupil dilation tails, 1.5% of data was excluded only for aggregation and display purposes. Pupil dilation amplitude and timing Smoothed RT histograms were restricted to fit plot limits Cogn Aﬀect Behav Neurosci (2019) 19:366–376 375 pupil dilation and response time was significantly more pronounced in conditions of high load, when the system was perceptually overloaded (see Fig. 7). and low visual perceptual load. The results indicate that pupil baseline only predicts selection performance in condi- tions of low perceptual load, where all perceptual informa- tion presented in the display can be processed in parallel. The fact that baseline predicts visual search performance only in low load, reflects the involvement of different atten- tional processes, one that seems to be mediated by the LC-NE system and one that is not. In line with previous studies, the time course of pupil responses did also reflect the tim- ing of perceptual processing in both high and low perceptual load, but this relationship was enhanced in high load. Pupil dilation has generally received more attention than pupil baseline in psychological research. For instance, pupil dilation has been long associated with memory load and mental effort (Kahneman & Beatty, 1966). In particular, several articles reported that pupil dilation reflects the time course of decision-making during perceptually challenging tasks involving both visual (de Gee et al., 2014; Einh¨auser et al., 2008) and affective processing (Oliva & Anikin, 2018). In line with these previous reports, the results presented here show that both the time course and amplitude of pupil dilation predicted the timing of the participants’ responses. In addition, we show that this link between pupil dilation and response time was enhanced in high load. However, task-evoked responses present some limitations compared to pupil baseline measures. Whereas task-evoked pupil dilation is measured during task processing and even after participants have responded (because of its slow latency), pupil baseline is measured right before task onset, therefore providing actual anticipatory information. Acknowledgements I gratefully acknowledge Lund Humanities Lab for their help during data collection and Andrey Anikin for his insights in statistical analysis. I would also like to thank Sebastiaan Mathˆot. and two other anonymous reviewers for their helpful comments on the manuscript. 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. References Arnsten, A. F., & Rubia, K. (2012). Neurobiological circuits regulating attention, cognitive control, motivation, and emotion: disruptions in neurodevelopmental psychiatric disorders. Journal of the American Academy of Child & Adolescent Psychiatry, 51(4), 356–367. Aston-Jones, G., & Cohen, J. D. (2005). An integrative theory of locus coeruleus-norepinephrine function: adaptive gain and optimal performance. Annual Review of Neuroscience, 28(1), 403–50. Aston-Jones, G., Rajkowski, J., Kubiak, P., & Alexinsky, T. (1994). Locus coeruleus neurons in monkey are selectively activated by attended cues in a vigilance task. The Journal of Neuroscience, 14(July), 4467–4480. All in all, the results of the present study reveal an interaction between pupil baseline and attentional performance as a function of perceptual load that is in line with the perceptual-load hypothesis and with current views of LC-NE function (Aston-Jones & Cohen, 2005). The results show that the degree with which LC-NE influences behavioral performance is related to the perceptual load of the task at hand. Thus, this study links early and late selection mechanisms, as defined by the perceptual-load hypothesis, with LC-NE function, as measured by pupil size. y Bates, D., M¨achler, M., Bolker, B., & Walker, S. (2015). Fitting linear mixed-effects models using lme4. Journal of Statistical Software, 67(1), 1–48. https://doi.org/10.18637/jss.v067.i01 Benoni, H., & Tsal, Y. (2013). Conceptual and methodological concerns in the theory of perceptual load. Frontiers in Psychology, 4(AUG), 1–7. ( ), Broadbent, D. E. (1958). The selective nature of learning. B¨urkner, P.-C. (2017). Bayesian distributional non-linear multilevel modeling with the R package brms. arXiv: 1705.11123. Cave, K. R., & Chen, Z. (2016). Identifying visual targets amongst interfering distractors: Sorting out the roles of perceptual load, dilution, and attentional zoom. 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https://openalex.org/W4239768707	https://www.researchsquare.com/article/rs-141870/v1.pdf?c=1610516761000	English	null	Physical Simulation for Water Invasion and Water Control Optimization in Water Drive Gas Reservoirs	Research Square (Research Square)	2,021	cc-by	31,175	Physical Simulation for Water Invasion and Water Control Optimization in Water Drive Gas Reservoirs xuan xu PetroChina Research Institute of Petroleum Exploration and Development Xizhe li (  xuxuan69@petrochina.com.cn ) PetroChina Research Institute of Petroleum Exploration and Development yong hu PetroChina Research Institute of Petroleum Exploration and Development yu shi Xi'an Shiyou University qingyan mei Research Institute of Southwest Oil and Gas Field Company, PetroChina chunyan jiao PetroChina Research Institute of Petroleum Exploration and Development Research Article Keywords: WDGRs, water, gas, energy Posted Date: January 13th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-141870/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License  Corresponding authors. Xizhe Li and Yong Hu. Research Article License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at Scienti¦c Reports on March 18th, 2021. See the published version at https://doi.org/10.1038/s41598-021-85548-0. Physical Simulation for Water Invasion and 1 Water Control Optimization in Water Drive 2 Gas Reservoirs 3 Xuan Xu and Xizhe Li and Yong Hu, PetroChina Research Institute of 4 Petroleum Exploration and Development; and Yu Shi, Xi'an Shiyou University, 5 Xi'an, Shanxi; and Qingyan Mei, Research Institute of Southwest Oil and Gas 6 Field Company, PetroChina, Chengdu, Sichuan; and Chunyan Jiao, PetroChina 7 Research Institute of Petroleum Exploration and Development 8  Corresponding authors. Xizhe Li and Yong Hu. Abstract 9 The development of water drive gas reservoirs (WDGRs) with fractures or strong heterogeneity is 10 severely influenced by water invasion. Accurately simulating the rules of water invasion and 11 drainage gas recovery countermeasures in fractured WDGRs, thereby revealing the mechanism of 12 water invasion and an appropriate development strategy, is important for formulating water 13 management measures and enhancing the recovery of gas reservoirs. In this work, physical 14 simulation methods were proposed to gain a better understanding of water invasion and to optimize 15 the water control of fractured WDGRs. Five groups of experiments were designed and conducted to 16 probe the impacts of the distance between the fractures and the gas well, the drainage position, the 17 drainage timing and the aquifer size on the water invasion and production performance of a gas 18 reservoir. The gas and water production and the internal pressure drop were monitored in real time 19 during the experiments. Based on the above experimental works, a theoretical analysis was 20 conducted to quantitatively evaluate the performance of the gas reservoir recovery via the gas well 21 production performance, water invasion, dynamic pressure drop and residual gas and water 22 distribution analysis. The results show that when the fracture scale was appropriate, a gas well 23 drilled close to a fracture (Experiment 1-3) or a high-permeability formation could also produce gas 24 and achieve drainage efficiently. The recovery factor of Experiment 1-3 reached 62.5%, which was 25 24.6% and 21.1% higher than those of Experiments 1-1 and 1-2, respectively, which had wells 26 drilled in low-permeability areas. Draining water near an aquifer can effectively inhibit water 27 invasion during the early stage of gas recovery. The setup in Experiment 2-1 effectively inhibited 28 1 water invasion and avoided the formation of water-sealed volumes of gas to recover 30% more gas 29 than recovered with that of Experiment 1-1 without drainage wells. A shorter distance between the 30 drainage well and the aquifer increased the drainage capacity and decreased the gas production 31 capacity, respectively (Well 2 at Point A vs Point B). A larger aquifer had a lower gas recovery, 32 which reduced the economic benefit. For example, due to an infinitely large aquifer, the reserves in 33 Experiment 4-1 were developed by a single well, the gas recovery was only 33.4%. Abstract 9 These research 34 results are expected to be beneficial for the preparation of development plans and the optimization 35 of water control measures for WDGRs. 36 Introduction 37 Most of the gas reservoirs discovered and developed in China are affected by water invasion, albeit 38 to different degrees. Such effects have been observed to be more serious for WDGRs with fractured 39 or highly heterogeneous formations. The edge water or bottom water tends to intrude easily through 40 fractures or high-permeability zones during gas production. This splits the gas reservoir, resulting 41 in water production in the gas wells, which considerably decreases gas production (Beattie and 42 Roberts 1996; Liu et al. 1999; Hernandez et al. 2006; Dotoku et al. 2019). 43 Most of the gas reservoirs discovered and developed in China are affected by water invasion, albeit 38 to different degrees. Such effects have been observed to be more serious for WDGRs with fractured 39 or highly heterogeneous formations. The edge water or bottom water tends to intrude easily through 40 fractures or high-permeability zones during gas production. This splits the gas reservoir, resulting 41 in water production in the gas wells, which considerably decreases gas production (Beattie and 42 Roberts 1996; Liu et al. 1999; Hernandez et al. 2006; Dotoku et al. 2019). 43 WDGRs have the advantage of supplemented energy. For a relatively homogeneous reservoir, 44 the water intrudes evenly, forming the so-called tongue, which acts to replenish energy (Agarwal et 45 al. 1965, Farah et al. 2017). However, water intrusion poses a larger risk to gas reservoir development 46 than to oil reservoir development. The edge and bottom water will "channel in" through the high- 47 permeability zones in a fractured or heterogeneous reservoir, decreasing the efficiency of gas 48 reservoir development. After the water in the gas reservoir is discharged, the formation water 49 intrudes through the gas reservoir and will be split due to the formation water invasion via the 50 fractures (or high-permeability zones). Then, some gas zones will be sealed and eventually form a 51 dead zone, resulting in a significant decrease in the ultimate recovery of the gas reservoir. 52 Additionally, after the gas wells are discharged, gas and water two-phase flow occurs in the 53 formation, which will lead to an increased gas reservoir abandonment pressure and decreased gas 54 production. In the later stage, due to the need for drainage to stabilize the production and tap 55 potential, the difficulty and the costs of development increase (Li and Pan 2011). Introduction 37 Among the three types of water control countermeasures, water drainage uses water 65 wells to actively produce water to consume water energy and reduce the pressure difference between 66 the gas zones and water zones to control water invasion. Water drainage, especially a multiwell 67 combined drainage scheme implemented across a gas reservoir, is an active water control strategy 68 that is widely used in practice. However, due to the complex reservoir conditions and water invasion 69 mechanisms, as well as the constraints imposed by the technical, economic, and environmental 70 conditions, it is difficult to determine the best water control measures and timing, posing great 71 challenges to the formulation and implementation of water control development in gas reservoirs 72 (Feng et al. 2015). 73 Therefore, for formulating water management measures and enhancing the recovery ratio of 74 gas reservoirs, it is important to accurately simulate the water invasion and drainage gas recovery 75 countermeasures in fractured WDGRs to reveal the water invasion mechanism and plan the reservoir 76 development strategy (Warren and Root 1963; Xia 2002; Hearn 2010; Jin and Wojtanowicz 2010; Kabir 77 et al. 2015; Glumov et al. 2017). However, due to the complexity of the water invasion mechanism 78 and the limitations of experimental techniques, there have been few targeted theoretical studies 79 performed on water control in such a WDGR. Lakatos et al. (2009) illustrated the water-induced 80 formation damage via a flow experiment and high-pressure Hg porosimetry of tight sandstone. Li 81 & Zhang (2011) conducted an experimental study of water shut-off by gas wettability alteration and 82 investigated the feasibility of reducing water production in gas wells by changing the wettability of 83 the gas zone. Xu et al. (2019) investigated the water drainage effect of reducing water invasion via 84 physical simulation experiments. Fang et al. (2019) investigated the influence of reservoir 85 parameters on water invasion in fractured carbonate gas reservoirs. A few indoor studies were 86 conducted on the entire development cycle of gas reservoirs, including water invasion in the early stages 87 and water control in the later stages (Rezaee et al. 2013; Liu et al. 2015). Introduction 37 56 WDGRs have the advantage of supplemented energy. For a relatively homogeneous reservoir, 44 the water intrudes evenly, forming the so-called tongue, which acts to replenish energy (Agarwal et 45 al. 1965, Farah et al. 2017). However, water intrusion poses a larger risk to gas reservoir development 46 than to oil reservoir development. The edge and bottom water will "channel in" through the high- 47 permeability zones in a fractured or heterogeneous reservoir, decreasing the efficiency of gas 48 reservoir development. After the water in the gas reservoir is discharged, the formation water 49 intrudes through the gas reservoir and will be split due to the formation water invasion via the 50 fractures (or high-permeability zones). Then, some gas zones will be sealed and eventually form a 51 dead zone, resulting in a significant decrease in the ultimate recovery of the gas reservoir. 52 Additionally, after the gas wells are discharged, gas and water two-phase flow occurs in the 53 formation, which will lead to an increased gas reservoir abandonment pressure and decreased gas 54 production. In the later stage, due to the need for drainage to stabilize the production and tap 55 potential, the difficulty and the costs of development increase (Li and Pan 2011). 56 Numerous efforts have been made regarding gas reservoir water invasion control by researchers 57 in the oil and gas industry. There are currently three main types of water control countermeasures, 58 2 Numerous efforts have been made regarding gas reservoir water invasion control by researchers 57 in the oil and gas industry. There are currently three main types of water control countermeasures, 58 2 i.e., water drainage, gas well deliquification, and water shut-off (Hearn 2010; Yang et al. 2013; Mattey 59 et al. 2017). Water shut-off is performed to reduce the permeability of fractures by injecting chemical 60 agents, such as gelant, into them, thus preventing water invasion. Ghosh et al. (2020) presented 61 experimental results of water shut-off and noted that the reservoir matrix must be well protected 62 while plugging fractures. To achieve this, different agents may need to be injected into the reservoir 63 at different times. This study suggested that it is not easy to solve the problem of water invasion via 64 water shut-off. Introduction 37 Such a model is aimed at simulating the 90 relationship between the fractures and gas well locations, the water invasion in fractured WDGRs 91 with different drainage locations, the drainage timing and the water body sizes. The pressure profiles 92 of different gas reservoirs are monitored and recorded during the experiments to reveal the water 93 invasion dynamics, i.e., the water sealing mechanism. Accordingly, reserve development rules and 94 the distribution of the residual gas are analyzed. On these bases, reasonable countermeasures are 95 proposed, providing technical support for the preparation of development plans and water control 96 development measures for this type of gas reservoir. 97 89 Geological Model 99 The water control practices in the Longdiao gas reservoir, a Carboniferous gas field in eastern 100 Sichuan, China, reflect the complexity and challenges of on-site water control (Chen et al. 1999; 101 Yang et al. 2003; Gou et al. 2002). Well Chi 39 in the Longdiao gas reservoir is located at the northern 102 end of the Diaozhongba high spot of the gas field, with single-well-controlled reserves of 26.5×108 103 m3. This well was put into production in March 1992. The initial daily gas production rate (Qg) was 104 35.0×104 m3/d. In March 1994, formation water was suddenly produced at a daily water production 105 rate (Qw) of 3 m3/d. The daily Qg was then reduced to 7.3×104 m3/d. Studies showed that Well Chi 106 39 underwent water invasion typical of reservoirs with large fractures: two large water-conducting 107 faults bring water to the Well Chi 39 area, as shown in Fig. 1. 108 The water control practices in the Longdiao gas reservoir, a Carboniferous gas field in eastern 100 Sichuan, China, reflect the complexity and challenges of on-site water control (Chen et al. 1999; 101 Yang et al. 2003; Gou et al. 2002). Well Chi 39 in the Longdiao gas reservoir is located at the northern 102 end of the Diaozhongba high spot of the gas field, with single-well-controlled reserves of 26.5×108 103 m3. This well was put into production in March 1992. The initial daily gas production rate (Qg) was 104 35.0×104 m3/d. In March 1994, formation water was suddenly produced at a daily water production 105 rate (Qw) of 3 m3/d. The daily Qg was then reduced to 7.3×104 m3/d. Studies showed that Well Chi 106 39 underwent water invasion typical of reservoirs with large fractures: two large water-conducting 107 faults bring water to the Well Chi 39 area, as shown in Fig. 1. 108 In this case, the water invasion control measures for the gas reservoir were as follows: 109 In this case, the water invasion control measures for the gas reservoir were as follows: 109 4 g In May 1998, a large-scale water drainage for pressure relief was performed for Well Chi 27; 110 at the same time, gas lift drainage was conducted for Well Chi 39 using two additional wells for 111 water control. Introduction 37 88 Therefore, for formulating water management measures and enhancing the recovery ratio of 74 gas reservoirs, it is important to accurately simulate the water invasion and drainage gas recovery 75 countermeasures in fractured WDGRs to reveal the water invasion mechanism and plan the reservoir 76 development strategy (Warren and Root 1963; Xia 2002; Hearn 2010; Jin and Wojtanowicz 2010; Kabir 77 et al. 2015; Glumov et al. 2017). However, due to the complexity of the water invasion mechanism 78 and the limitations of experimental techniques, there have been few targeted theoretical studies 79 performed on water control in such a WDGR. Lakatos et al. (2009) illustrated the water-induced 80 formation damage via a flow experiment and high-pressure Hg porosimetry of tight sandstone. Li 81 & Zhang (2011) conducted an experimental study of water shut-off by gas wettability alteration and 82 investigated the feasibility of reducing water production in gas wells by changing the wettability of 83 the gas zone. Xu et al. (2019) investigated the water drainage effect of reducing water invasion via 84 physical simulation experiments. Fang et al. (2019) investigated the influence of reservoir 85 parameters on water invasion in fractured carbonate gas reservoirs. A few indoor studies were 86 conducted on the entire development cycle of gas reservoirs, including water invasion in the early stages 87 and water control in the later stages (Rezaee et al. 2013; Liu et al. 2015). 88 3 3 In this work, a typical gas reservoir is adapted as a prototype to establish a geological model 89 considering different fracture and matrix parameters. Such a model is aimed at simulating the 90 relationship between the fractures and gas well locations, the water invasion in fractured WDGRs 91 with different drainage locations, the drainage timing and the water body sizes. The pressure profiles 92 of different gas reservoirs are monitored and recorded during the experiments to reveal the water 93 invasion dynamics, i.e., the water sealing mechanism. Accordingly, reserve development rules and 94 the distribution of the residual gas are analyzed. On these bases, reasonable countermeasures are 95 proposed, providing technical support for the preparation of development plans and water control 96 development measures for this type of gas reservoir. 97 In this work, a typical gas reservoir is adapted as a prototype to establish a geological model 89 considering different fracture and matrix parameters. Geological Model 99 The dynamic monitoring curves of the gas production depicted that the pressure in 112 the water zones of Well Chi 27 was obviously decreased due to the pumping and drainage, and the 113 daily Qw of Well Chi 39 was effectively stabilized. After pumping was stopped in October 1999, 114 the pressure in the water zones around Well Chi 27 rapidly recovered. The formation filtration 115 conditions at Well Chi 39 deteriorated rapidly, with the rapid decrease in casing pressure. The Qw 116 increased from 18 m3/d at pumping stoppage to approximately 40 m3/d. The positive and negative 117 production changes indicated that the pumping drainage pressure relief had a significant effect on 118 4 In May 1998, a large-scale water drainage for pressure relief was performed for Well Chi 27; 110 at the same time, gas lift drainage was conducted for Well Chi 39 using two additional wells for 111 water control. The dynamic monitoring curves of the gas production depicted that the pressure in 112 the water zones of Well Chi 27 was obviously decreased due to the pumping and drainage, and the 113 daily Qw of Well Chi 39 was effectively stabilized. After pumping was stopped in October 1999, 114 the pressure in the water zones around Well Chi 27 rapidly recovered. The formation filtration 115 conditions at Well Chi 39 deteriorated rapidly, with the rapid decrease in casing pressure. The Qw 116 increased from 18 m3/d at pumping stoppage to approximately 40 m3/d. The positive and negative 117 production changes indicated that the pumping drainage pressure relief had a significant effect on 118 inhibiting the further deterioration of water invasion and reducing the water invasion damage. 119 Although there was a favorable trend in the initial stage of drainage, after nearly four years of 120 drainage, the overall water control effect in the area of Well Chi 27 has not achieved its intended 121 purpose. The main reason is that the water displacement from Well Chi 27 did not meet the designed 122 requirement, and the energy of the movable water was greater than predicted. The water control 123 practice applied in the Well Chi 27-39 area shows that the uncertainties of the water-energy 124 prediction and the actual drainage conditions on site are important factors affecting the water control 125 effectiveness in the well area. Geological Model 99 141 Experimental Method 142 The Chi 27-39 well area of the Longdiao gas reservoir mentioned above has the typical 127 characteristics of a WDGR. As shown in Fig. 2, three geological models of typical multiscale 128 fractured WDGR are developed, taking the geological and development examples of the gas zone 129 as a prototype. Water invasion and water control experiments are designed and performed. The left 130 end of the model is connected to the aquifer, and the right side is a heterogeneous reservoir, which 131 is composed of fractured zones (FZs) and matrix zones (MZs) with different physical properties. 132 The FZs are located above and below the MZs. The MZs are divided into MZ 1 and MZ 2 according 133 to different physical properties (indicated by the different filling colors in Fig. 2). The main 134 differences among the three geological models are the range of the right MZ 2 (see the boundaries 135 of the three models shown in Fig. 2) and the location of Well 1. The gas wells in Geological Models 136 1 and 2 were deployed at MZ 2, representing a gas reservoir with low-permeability matrix barriers; 137 in these cases, the gas wells and aquifer are not completely connected via fractures. In Model 3, 138 Well 1 is directly connected to the FZ, which represents a gas reservoir in which the gas wells are 139 directly connected with the aquifer through fractures. Compared to the other models, in Model 1, 140 Well 1 is the farthest from the FZ; in Model 3, Well 1 is directly connected to the FZ. 141 Experimental Method 142 Corresponding to the geological model, a novel experimental device and method for water invasion 143 and water control in a complex and fractured WDGR were proposed and established (Fig. 3 and Fig. 144 4). 145 As shown in Fig. 3, Fig. 4 and Table 1, the experimental reservoir was mainly composed of 146 four groups of cores with different physical properties. The core holders were equipped with 147 multiple pressure probes to monitor the gas reservoir pressure profile in real time. 148 As shown in Fig. 3, Fig. 4 and Table 1, the experimental reservoir was mainly composed of 146 four groups of cores with different physical properties. The core holders were equipped with 147 multiple pressure probes to monitor the gas reservoir pressure profile in real time. Geological Model 99 126 inhibiting the further deterioration of water invasion and reducing the water invasion damage. 119 Although there was a favorable trend in the initial stage of drainage, after nearly four years of 120 drainage, the overall water control effect in the area of Well Chi 27 has not achieved its intended 121 purpose. The main reason is that the water displacement from Well Chi 27 did not meet the designed 122 requirement, and the energy of the movable water was greater than predicted. The water control 123 practice applied in the Well Chi 27-39 area shows that the uncertainties of the water-energy 124 prediction and the actual drainage conditions on site are important factors affecting the water control 125 effectiveness in the well area. 126 The Chi 27-39 well area of the Longdiao gas reservoir mentioned above has the typical 127 characteristics of a WDGR. As shown in Fig. 2, three geological models of typical multiscale 128 fractured WDGR are developed, taking the geological and development examples of the gas zone 129 as a prototype. Water invasion and water control experiments are designed and performed. The left 130 end of the model is connected to the aquifer, and the right side is a heterogeneous reservoir, which 131 is composed of fractured zones (FZs) and matrix zones (MZs) with different physical properties. 132 The FZs are located above and below the MZs. The MZs are divided into MZ 1 and MZ 2 according 133 to different physical properties (indicated by the different filling colors in Fig. 2). The main 134 differences among the three geological models are the range of the right MZ 2 (see the boundaries 135 of the three models shown in Fig. 2) and the location of Well 1. The gas wells in Geological Models 136 1 and 2 were deployed at MZ 2, representing a gas reservoir with low-permeability matrix barriers; 137 in these cases, the gas wells and aquifer are not completely connected via fractures. In Model 3, 138 Well 1 is directly connected to the FZ, which represents a gas reservoir in which the gas wells are 139 directly connected with the aquifer through fractures. Compared to the other models, in Model 1, 140 Well 1 is the farthest from the FZ; in Model 3, Well 1 is directly connected to the FZ. Geological Model 99 148 5 Different lengths were selected for Core 4 according to the simulated geological models: 50 152 cm for Model 1; 25 cm for Model 2; and 0 cm for Model 3. For Well 1 in the three models, the 153 fracture penetration rates were 50%, 67%, and 100%, respectively (Table 1). 154 Five groups of different types of experiments were designed to investigate the main geological 155 and production conditions affecting the water invasion and water control in the gas reservoirs. The 156 purpose, characteristics, models, and gas well parameters of the experiments are shown in Table 2 157 and are described in detail below: 158 Five groups of different types of experiments were designed to investigate the main geological 155 and production conditions affecting the water invasion and water control in the gas reservoirs. The 156 purpose, characteristics, models, and gas well parameters of the experiments are shown in Table 2 157 and are described in detail below: 158 Five groups of different types of experiments were designed to investigate the main geological 155 and production conditions affecting the water invasion and water control in the gas reservoirs. The 156 purpose, characteristics, models, and gas well parameters of the experiments are shown in Table 2 157 and are described in detail below: 158 The first group of experiments (Group I) was designed to unveil the impact of the positional 159 relationship between the gas wells and FZs on the water invasion. During the single-well production 160 of Well 1, three different geological models were formed by adjusting the length of Core 4, as 161 described above. 162 The second group of experiments (Group II) was designed to reveal the impact of the drainage 163 positions on the water control effectiveness. Wells 1 and 2 were simultaneously initiated for 164 production, simulating multiple-well drainage and gas production. Note that Well 2 was placed at 165 four different positions (A, B, C, and D) in the FZs in the four different experiments of Group II. A 166 and C were set in the middle of Core 1 and Core 3, respectively. Meanwhile, B and D were 12.5 cm 167 to the left of A and C, respectively. 168 The third group of experiments (Group III) was designed to conduct research related to the 169 timing of drainage on water control effectiveness. Geological Model 99 All the 191 experimental gas wells were produced at a production rate of 400 mL/min with an abandonment 192 production constraint of 10 mL/min. 193 Considering the similar requirements and gas reservoir production matching experiences, the 186 experimental production rate was 15%-30% of the model open flow (Geertsma et al. 1956; Jiao et 187 al. 2019). In this study, the measured open flows of the different models were in the range of 1000- 188 3000 mL/min, so the experimental Qg met the open flow requirements of 150-900 mL/min. The 189 abandoned production range of the gas reservoir and the detection accuracy of the flow meter was 190 considered in the experimental study. The abandoned production was set to 2.5% of the Qg. All the 191 experimental gas wells were produced at a production rate of 400 mL/min with an abandonment 192 production constraint of 10 mL/min. 193 Geological Model 99 All the 191 experimental gas wells were produced at a production rate of 400 mL/min with an abandonment 192 production constraint of 10 mL/min. 193 Experimental Procedures 194 Step 1: Prepare the core models according to the experimental scheme, load all the cores into the 195 core holder, and then add a confining pressure of 35 MPa. 196 Step 2: Slowly saturate the core model from both ends with nitrogen until the pressure reaches 197 30 MPa. 198 St 3 L d th i l t d f ti t i t hi h i t t t i d l 199 The purpose of the fifth group of basic experiments (Group V) was a comparative analysis. 179 Experiments 5-1 and 5-2 were conducted with Model 1 and Model 3, respectively, to simulate 180 volumetric gas reservoirs. 181 The purpose of the fifth group of basic experiments (Group V) was a comparative analysis. 179 Experiments 5-1 and 5-2 were conducted with Model 1 and Model 3, respectively, to simulate 180 volumetric gas reservoirs. 181 In the first to third groups of experiments, the gas reservoirs were connected to the finite aquifer 182 in a total of 10 experiments. Considering that the elastic energy was released from the gas reservoir 183 rocks and the aquifer, an aquifer volume equivalent to 15 times the gas reservoir volume was 184 consistently set in the experiments. 185 In the first to third groups of experiments, the gas reservoirs were connected to the finite aquifer 182 in a total of 10 experiments. Considering that the elastic energy was released from the gas reservoir 183 rocks and the aquifer, an aquifer volume equivalent to 15 times the gas reservoir volume was 184 consistently set in the experiments. 185 Considering the similar requirements and gas reservoir production matching experiences, the 186 experimental production rate was 15%-30% of the model open flow (Geertsma et al. 1956; Jiao et 187 al. 2019). In this study, the measured open flows of the different models were in the range of 1000- 188 3000 mL/min, so the experimental Qg met the open flow requirements of 150-900 mL/min. The 189 abandoned production range of the gas reservoir and the detection accuracy of the flow meter was 190 considered in the experimental study. The abandoned production was set to 2.5% of the Qg. Experimental Procedures 194 Step 1: Prepare the core models according to the experimental scheme, load all the cores into the 195 core holder, and then add a confining pressure of 35 MPa. 196 Step 1: Prepare the core models according to the experimental scheme, load all the cores into the 195 core holder, and then add a confining pressure of 35 MPa. 196 Step 1: Prepare the core models according to the experimental scheme, load all the cores into the 195 core holder, and then add a confining pressure of 35 MPa. 196 Step 1: Prepare the core models according to the experimental scheme, load all the cores into the 195 core holder, and then add a confining pressure of 35 MPa. 196 Step 2: Slowly saturate the core model from both ends with nitrogen until the pressure reaches 197 30 MPa. 198 Step 2: Slowly saturate the core model from both ends with nitrogen until the pressure reaches 197 30 MPa. 198 Step 3: Load the simulated formation water into a high-pressure resistant container and apply 199 a pressure of 30 MPa. Change the aquifer setup as needed; if it is an infinite aquifer, then connect a 200 constant pressure gas source to the container to provide continuous pressure. 201 Step 4: Connect the container storing the brine to the core model at 100% nitrogen saturation. 202 According to the experimental scheme, produce the gas well at a production rate of 400 mL/min to 203 simulate gas reservoir exploitation. 204 Step 4: Connect the container storing the brine to the core model at 100% nitrogen saturation. 202 According to the experimental scheme, produce the gas well at a production rate of 400 mL/min to 203 simulate gas reservoir exploitation. 204 7 Step 5: During gas recovery, the pressure probes set on the core holder record the pressure 205 profile of the core in real time. The instantaneous gas, water production, accumulated gas, water 206 breakthrough time, and other parameters are recorded by the outlet flow meter and the gas-water 207 separator. Stop the experiment when Well 1 or Well 2 reaches the predetermined production target. 208 7 Step 5: During gas recovery, the pressure probes set on the core holder record the pressure 205 profile of the core in real time. Geological Model 99 Experiments 3-1 and 3-2 were completed with 170 Geological Model 2, which was compared with Experiment 1-2 (considering the same geological 171 model), and Experiment 3-3 was completed with Geological Model 3, which was compared with 172 Experiment 1-3 (considering the same model). 173 The fourth group of experiments (Group IV) was designed to investigate the impact of aquifer 174 size on water control effectiveness. Experiment 4-1 simulated gas reservoirs with an infinite aquifer, 175 while Experiment 4-2 simulated gas reservoirs without an aquifer. Experiments 4-1 and 4-2 were 176 compared with Experiment 3-1 because they used the same geological model. The volume of the 177 aquifer in Experiment 3-1 was 15 times the gas reservoir volume. 178 The fourth group of experiments (Group IV) was designed to investigate the impact of aquifer 174 size on water control effectiveness. Experiment 4-1 simulated gas reservoirs with an infinite aquifer, 175 while Experiment 4-2 simulated gas reservoirs without an aquifer. Experiments 4-1 and 4-2 were 176 compared with Experiment 3-1 because they used the same geological model. The volume of the 177 aquifer in Experiment 3-1 was 15 times the gas reservoir volume. 178 6 The purpose of the fifth group of basic experiments (Group V) was a comparative analysis. 179 Experiments 5-1 and 5-2 were conducted with Model 1 and Model 3, respectively, to simulate 180 volumetric gas reservoirs. 181 In the first to third groups of experiments, the gas reservoirs were connected to the finite aquifer 182 in a total of 10 experiments. Considering that the elastic energy was released from the gas reservoir 183 rocks and the aquifer, an aquifer volume equivalent to 15 times the gas reservoir volume was 184 consistently set in the experiments. 185 Considering the similar requirements and gas reservoir production matching experiences, the 186 experimental production rate was 15%-30% of the model open flow (Geertsma et al. 1956; Jiao et 187 al. 2019). In this study, the measured open flows of the different models were in the range of 1000- 188 3000 mL/min, so the experimental Qg met the open flow requirements of 150-900 mL/min. The 189 abandoned production range of the gas reservoir and the detection accuracy of the flow meter was 190 considered in the experimental study. The abandoned production was set to 2.5% of the Qg. Impact of the Distance Between the Wells and Fractures 212 The impacts of the water 236 The production curves of Group I are plotted in Fig. 5. For comparison, the production curves of 217 the volumetric gas reservoir in Experiment 4-2 are also included in Fig. 5. The main production 218 parameters are given in Table 3. 219 The experimental results show that the distance between the gas wells and the FZ can greatly 220 influence the production performance and recovery factor (R). 221 The experimental results show that the distance between the gas wells and the FZ can greatly 220 influence the production performance and recovery factor (R). 221 The experimental results show that the distance between the gas wells and the FZ can greatly 220 influence the production performance and recovery factor (R). 221 The gas wells in Gas Reservoirs 1-1 and 1-2 drilled in the low-permeability MZ 2, and there 222 are barriers in the FZ. Once water breakthrough occurred in the gas wells in both gas reservoirs, the 223 production rate dropped rapidly to the abandonment production rate. Specifically, water 224 breakthrough was observed at 74 mins in Experiment 1-1; then, at 76 mins, the Qg decreased to the 225 abandonment Qg due to the water breakthrough. Additionally, water breakthrough was observed at 226 47 mins in Experiment 1-2; then, at 49 mins, the gas production was stopped. Therefore, only the 227 water production curves of Experiment 1-3 are included in Fig. 5(b). For Experiment 1-3, with the 228 gas well directly connected to the FZ, after water breakthrough, the gas well still produced gas for 229 a long time. This is because the water in Well 1 could be quickly and fully drained during the 230 experiment, although a water breakthrough was observed 22 mins after Well 1 production was 231 initiated, when the production capacity stabilized. Stable production lasted for 32 mins, and both 232 gas and water were produced for 115 min. The R during the stage of producing both gas and water 233 reached 25.6%. 234 Considering the timing of the water breakthrough, a shorter distance between a gas well and 235 the FZ connected to an aquifer will result in a faster water breakthrough. Experimental Procedures 194 The instantaneous gas, water production, accumulated gas, water 206 breakthrough time, and other parameters are recorded by the outlet flow meter and the gas-water 207 separator. Stop the experiment when Well 1 or Well 2 reaches the predetermined production target. 208 7 Step 6: After the end of the experiment, weigh the cores to obtain the water saturations at 209 different positions. 210 Step 6: After the end of the experiment, weigh the cores to obtain the water saturation 9 Impact of the Distance Between the Wells and Fractures 212 In Group I, three different gas reservoir geological models were formed by varying the length of 213 Core 4 to reveal the impact of the distance from the gas well to the fractures on the water invasion 214 and gas production. 215 In Group I, three different gas reservoir geological models were formed by varying the length of 213 Core 4 to reveal the impact of the distance from the gas well to the fractures on the water invasion 214 and gas production. 215 Production Performance 216 The production curves of Group I are plotted in Fig. 5. For comparison, the production curves of 217 the volumetric gas reservoir in Experiment 4-2 are also included in Fig. 5. The main production 218 parameters are given in Table 3. 219 The experimental results show that the distance between the gas wells and the FZ can greatly 220 influence the production performance and recovery factor (R). 221 The gas wells in Gas Reservoirs 1-1 and 1-2 drilled in the low-permeability MZ 2, and there 222 are barriers in the FZ. Once water breakthrough occurred in the gas wells in both gas reservoirs, the 223 production rate dropped rapidly to the abandonment production rate. Specifically, water 224 breakthrough was observed at 74 mins in Experiment 1-1; then, at 76 mins, the Qg decreased to the 225 abandonment Qg due to the water breakthrough. Additionally, water breakthrough was observed at 226 47 mins in Experiment 1-2; then, at 49 mins, the gas production was stopped. Therefore, only the 227 water production curves of Experiment 1-3 are included in Fig. 5(b). For Experiment 1-3, with the 228 gas well directly connected to the FZ, after water breakthrough, the gas well still produced gas for 229 a long time. This is because the water in Well 1 could be quickly and fully drained during the 230 experiment, although a water breakthrough was observed 22 mins after Well 1 production was 231 initiated, when the production capacity stabilized. Stable production lasted for 32 mins, and both 232 gas and water were produced for 115 min. The R during the stage of producing both gas and water 233 reached 25.6%. 234 Considering the timing of the water breakthrough, a shorter distance between a gas well and 235 the FZ connected to an aquifer will result in a faster water breakthrough. Impact of the Distance Between the Wells and Fractures 212 The impacts of the water 236 invasion and the water production capacities of the gas wells are varied, along with the different 237 Considering the timing of the water breakthrough, a shorter distance between a gas well and 235 the FZ connected to an aquifer will result in a faster water breakthrough. The impacts of the water 236 invasion and the water production capacities of the gas wells are varied, along with the different 237 Considering the timing of the water breakthrough, a shorter distance between a gas well and 235 the FZ connected to an aquifer will result in a faster water breakthrough. The impacts of the water 236 invasion and the water production capacities of the gas wells are varied, along with the different 237 8 distances between the gas wells and the FZs. Although the Qg of the three types of gas reservoirs 238 were similar, due to the large differences in the gas reservoir reserves, the R of Experiment 1-1 239 was only 37.9%, and the R of Experiment 1-2 was 41.4%, while the gas R in Experiment 1-3 in 240 the zone directly connected to the FZ reached 62.5%. 241 distances between the gas wells and the FZs. Although the Qg of the three types of gas reservoirs 238 were similar, due to the large differences in the gas reservoir reserves, the R of Experiment 1-1 239 was only 37.9%, and the R of Experiment 1-2 was 41.4%, while the gas R in Experiment 1-3 in 240 the zone directly connected to the FZ reached 62.5%. 241 distances between the gas wells and the FZs. Although the Qg of the three types of gas reservoirs 238 were similar, due to the large differences in the gas reservoir reserves, the R of Experiment 1-1 239 was only 37.9%, and the R of Experiment 1-2 was 41.4%, while the gas R in Experiment 1-3 in 240 the zone directly connected to the FZ reached 62.5%. 241 The experimental results show that because the FZ is connected to the aquifer, the closer a gas 242 well is to the FZ, the higher the R is. First, the fractures have a high gas supply capacity because 243 they are high-speed gas flow channels, which results in a stable production capacity of the gas 244 reservoir; consequently, the reserves are developed fast. Impact of the Distance Between the Wells and Fractures 212 2010; 258 Xu et al. 2020). 259 9 Water Invasion Analysis 255 Currently, the main methods of water invasion degree identification can be divided into three main 256 methods: the pressure drop curve method, the apparent geological reserves method and the water 257 invasion volume coefficient method (Chen 1978; Abdul-Majeed and Al-Assal 1998; Siddiqui et al. 2010; 258 Xu et al. 2020). 259 Using the water invasion volume coefficient method, the θ~R curves of Group I were drawn; 260 details are presented in Appendix A. For comparison, the curve of the basic Experiment 5-2 (curve 261 of the volumetric gas reservoir Model 3 without an aquifer) was also drawn in Fig. 6. The 262 relationships between the Wp and the R for Experiments 1, 2 and 3 are also plotted in Fig. 6 for 263 ease of analysis. 264 Fig. 6 shows a consistency among the theoretical results. For the volumetric gas reservoir 265 depletion exploitation without an aquifer (Experiment 5-2), the θ~R curve basically conforms to 266 the 45° line. In the early stage of production in Group I, the θ~R curve curves upward, which 267 Water Invasion Analysis 255 Currently, the main methods of water invasion degree identification can be divided into three main 256 methods: the pressure drop curve method, the apparent geological reserves method and the water 257 invasion volume coefficient method (Chen 1978; Abdul-Majeed and Al-Assal 1998; Siddiqui et al. 2010; 258 Xu et al. 2020). 259 Using the water invasion volume coefficient method, the θ~R curves of Group I were drawn; 260 details are presented in Appendix A. For comparison, the curve of the basic Experiment 5-2 (curve 261 of the volumetric gas reservoir Model 3 without an aquifer) was also drawn in Fig. 6. The 262 relationships between the Wp and the R for Experiments 1, 2 and 3 are also plotted in Fig. 6 for 263 ease of analysis. 264 9 Fig. 6 shows a consistency among the theoretical results. For the volumetric gas reservoir 265 depletion exploitation without an aquifer (Experiment 5-2), the θ~R curve basically conforms to 266 the 45° line. In the early stage of production in Group I, the θ~R curve curves upward, which 267 9 Fig. 6 shows a consistency among the theoretical results. For the volumetric gas reservoir 265 depletion exploitation without an aquifer (Experiment 5-2), the θ~R curve basically conforms to 266 the 45° line. Impact of the Distance Between the Wells and Fractures 212 Second, the gas wells in the FZ can produce 245 gas with water for a long time because continuous drainage consumes the water energy and inhibits 246 the water invasion. Third, due to the high conductivity of the large fractures, even if the water 247 saturation is increased, they still have a high seepage capacity, resulting in stabilized gas production 248 at the gas wells after water breakthrough and maintaining long-term gas and water production. 249 Notably, the conclusions have preconditions. The results on the effect of a fracture on the gas 250 recovery are obtained from these experimental conditions (a specific fracture scale and matrix 251 permeability). Fang et al. (2019) noted that although fractures at certain scales can enhance gas 252 recovery, excessively large fractures would allow water to flow along the fractures, which could 253 rapidly decrease the gas recovery. 254 The experimental results show that because the FZ is connected to the aquifer, the closer a gas 242 well is to the FZ, the higher the R is. First, the fractures have a high gas supply capacity because 243 they are high-speed gas flow channels, which results in a stable production capacity of the gas 244 reservoir; consequently, the reserves are developed fast. Second, the gas wells in the FZ can produce 245 gas with water for a long time because continuous drainage consumes the water energy and inhibits 246 the water invasion. Third, due to the high conductivity of the large fractures, even if the water 247 saturation is increased, they still have a high seepage capacity, resulting in stabilized gas production 248 at the gas wells after water breakthrough and maintaining long-term gas and water production. 249 Notably, the conclusions have preconditions. The results on the effect of a fracture on the gas 250 recovery are obtained from these experimental conditions (a specific fracture scale and matrix 251 permeability). Fang et al. (2019) noted that although fractures at certain scales can enhance gas 252 recovery, excessively large fractures would allow water to flow along the fractures, which could 253 rapidly decrease the gas recovery. 254 Water Invasion Analysis 255 Currently, the main methods of water invasion degree identification can be divided into three main 256 methods: the pressure drop curve method, the apparent geological reserves method and the water 257 invasion volume coefficient method (Chen 1978; Abdul-Majeed and Al-Assal 1998; Siddiqui et al. Dynamic Pressure Drop 284 The gas reservoir material balance method can be adapted to estimate the overall water invasion 285 degree and the R of the gas reservoir. However, to calculate the R of the reservoir and the residual 286 gas distribution in different zones of the gas reservoir, a deep understanding of the residual pressure 287 of different regions of the gas reservoir is needed, and it is necessary to apply the gas reservoir 288 pressure contour method and other methods to study the water sealing condition in the gas reservoir 289 (Xu et al. 2012; Feng et al. 2013). During the experiments, the dynamic pressure of the gas reservoir 290 was monitored in real time to intuitively reflect the distribution of the residual sealed gas in the gas 291 reservoir and the R of the reserves, providing important analytical method and basis for 292 understanding the water invasion rules, the water sealing gas mechanism and the mechanism of the 293 remaining reserve development. 294 The gas reservoir material balance method can be adapted to estimate the overall water invasion 285 degree and the R of the gas reservoir. However, to calculate the R of the reservoir and the residual 286 gas distribution in different zones of the gas reservoir, a deep understanding of the residual pressure 287 of different regions of the gas reservoir is needed, and it is necessary to apply the gas reservoir 288 pressure contour method and other methods to study the water sealing condition in the gas reservoir 289 (Xu et al. 2012; Feng et al. 2013). During the experiments, the dynamic pressure of the gas reservoir 290 was monitored in real time to intuitively reflect the distribution of the residual sealed gas in the gas 291 reservoir and the R of the reserves, providing important analytical method and basis for 292 understanding the water invasion rules, the water sealing gas mechanism and the mechanism of the 293 remaining reserve development. 294 10 Fig. 7 shows the dynamic pressure drop profile of the volumetric gas reservoir in Experiment 295 4-2. Figs. 8-10 show the pressure drop profiles of the water drive in Experiments 1-1, 1-2, and 1-3. 296 For the convenience of analysis, the average pressure gradients in the near-well zones during the 297 10 Fig. 7 shows the dynamic pressure drop profile of the volumetric gas reservoir in Experiment 295 4-2. Figs. Impact of the Distance Between the Wells and Fractures 212 In the early stage of production in Group I, the θ~R curve curves upward, which 267 9 reflects the water invasion into the gas reservoirs. The θ~R curves of the later stage of production 268 for the three experiments show a difference in the water invasion as follows. 269 1. For Experiments 1-1 and 1-2, the slopes of the θ~R curves do not change considerably, and 270 they always plot above the 45° line, indicating that as the formation water continued to intrude, and 271 effective drainage was not fulfilled. The gas well was directly connected to the FZ in Experiment 1- 272 3. When R=0.37 or so, the relative pressure of the drawdown curve begins to shift downward, 273 showing strong drainage characteristics that correspond to the point where the gas well began to 274 produce water. This indicates that the θ~R curve can, in time, accurately reflect the change in the 275 water invasion degree on the basis of an accurate calculation of the average formation pressure and 276 geological reserves. As the Wp of the gas well increases, the relative pressure drawdown curve 277 crosses the 45° line when the R of the reserves R is approximately 0.4 and continues to tilt down, 278 indicating that the net intrusion of the aquifer continued to decrease at this stage and gradually 279 transformed into net production. 280 1. For Experiments 1-1 and 1-2, the slopes of the θ~R curves do not change considerably, and 270 they always plot above the 45° line, indicating that as the formation water continued to intrude, and 271 effective drainage was not fulfilled. The gas well was directly connected to the FZ in Experiment 1- 272 3. When R=0.37 or so, the relative pressure of the drawdown curve begins to shift downward, 273 showing strong drainage characteristics that correspond to the point where the gas well began to 274 produce water. This indicates that the θ~R curve can, in time, accurately reflect the change in the 275 water invasion degree on the basis of an accurate calculation of the average formation pressure and 276 geological reserves. Impact of the Distance Between the Wells and Fractures 212 As the Wp of the gas well increases, the relative pressure drawdown curve 277 crosses the 45° line when the R of the reserves R is approximately 0.4 and continues to tilt down, 278 indicating that the net intrusion of the aquifer continued to decrease at this stage and gradually 279 transformed into net production. 280 2. For Experiment 1-3, the θ~R curve decreases and finally approaches θ=0.25, which is in 281 accordance with the fact that the R of Experiment 1-3 is more than 20% higher than those of 282 Experiments 1-1 and 1-2. 283 2. For Experiment 1-3, the θ~R curve decreases and finally approaches θ=0.25, which is in 281 accordance with the fact that the R of Experiment 1-3 is more than 20% higher than those of 282 Experiments 1-1 and 1-2. 283 Dynamic Pressure Drop 284 8-10 show the pressure drop profiles of the water drive in Experiments 1-1, 1-2, and 1-3. 296 For the convenience of analysis, the average pressure gradients in the near-well zones during the 297 10 above four experiments are calculated and plotted according to the pressure parameters 298 (Experiments 1-1, 1-2 and 4-2 at both ends of Core 4, and Gas Reservoir 1-3 at both ends of the gas 299 reservoir), as shown in Fig. 11. 300 The experimental results show that the dynamic pressure drop profiles of the volumetric gas 301 reservoir and the WDGRs are significantly different, providing rich information about the gas 302 reservoir dynamics. 303 The pressures at various positions evenly decreased during the production of the volumetric 304 gas reservoir in Experiment 4-2. When the period of stabilized production ended, the R in the 305 reserves was nearly uniform. When the production was stopped at 88 mins, the residual pressure at 306 the various positions was nearly completely dissipated, which corresponded to a gas R greater than 307 98%. 308 Matrix 2 of Gas Reservoir 1-1 had the largest range, and Well 1 was 50 cm from the FZ. The 309 dynamic pressure drop process of the gas reservoir shows that the pressure drop in the entire gas 310 reservoir was relatively synchronous and occurred within the first 20 mins of gas production, 311 indicating that the initial reserve development was balanced and that the reservoir could provide a 312 stable gas supply to the gas well. In this stage, the maximum pressure gradient of the gas supply 313 path of the gas reservoir, which occurs in the near-well area of the low-pressure MZ 2 (Core 4), was 314 only 0.007 MPa/cm (Fig. 11). Thereafter, the pressure gradient in the immediate vicinity of the well 315 began to increase rapidly, reaching 0.18 MPa/cm at 25 mins and a peak value of 0.34 MPa/cm at 30 316 mins before stable production ended. The significant pressure drop funnel that formed around the 317 gas well indicated that a large amount of formation energy was lost in the immediate vicinity of the 318 well, i.e., the MZ. Dynamic Pressure Drop 284 When Well 1 was abandoned and production was stopped, the pressure gradient 319 near the well finally stabilized at 0.38 MPa/cm, and the residual pressure in the peripheral FZs and 320 MZ 1 (Cores 1-3) was as high as 19.6 MPa to 21.6 MPa. A large amount of residual reserves was 321 not developed due to water sealing. The R of the corresponding gas reservoir was only 37.9%. 322 Well 1 in Experiment 1-2 was 25 cm from the FZs, 50% of the distance in Experiment 1-1. 323 Similar to Experiment 1-1, the initial pressure drop in Experiment 1-2 was synchronous in the entire 324 gas reservoir. In the later stage, due to the intrusion of formation water in the near-well zone (MZ 325 2), the pressure gradient of the near-well zone increased rapidly. In the process of production decline, 326 the average pressure gradient of the near-well zone was stabilized at approximately 0.74 MPa/cm, 327 above four experiments are calculated and plotted according to the pressure parameters 298 (Experiments 1-1, 1-2 and 4-2 at both ends of Core 4, and Gas Reservoir 1-3 at both ends of the gas 299 reservoir), as shown in Fig. 11. 300 above four experiments are calculated and plotted according to the pressure parameters 298 (Experiments 1-1, 1-2 and 4-2 at both ends of Core 4, and Gas Reservoir 1-3 at both ends of the gas 299 reservoir), as shown in Fig. 11. 300 above four experiments are calculated and plotted according to the pressure parameters 298 (Experiments 1-1, 1-2 and 4-2 at both ends of Core 4, and Gas Reservoir 1-3 at both ends of the gas 299 reservoir), as shown in Fig. 11. 300 The experimental results show that the dynamic pressure drop profiles of the volumetric gas 301 reservoir and the WDGRs are significantly different, providing rich information about the gas 302 reservoir dynamics. 303 The pressures at various positions evenly decreased during the production of the volumetric 304 gas reservoir in Experiment 4-2. When the period of stabilized production ended, the R in the 305 reserves was nearly uniform. When the production was stopped at 88 mins, the residual pressure at 306 the various positions was nearly completely dissipated, which corresponded to a gas R greater than 307 98%. 308 Matrix 2 of Gas Reservoir 1-1 had the largest range, and Well 1 was 50 cm from the FZ. Dynamic Pressure Drop 284 Whether in the early or late stages of 330 development, the R in all parts of the gas reservoir Experiment 1-3 was relatively balanced, and the 331 near-well and peripheral pressures gently and synchronously reduced. Due to the high conductivity 332 of the fractures, gas and water were produced together from the gas well, and the energy of the 333 aquifer was simultaneously reduced. This reduces the physical damage due to water invasion, 334 allowing the gas to flow to the gas well through the fractures. 335 which was the highest value among the three WDGRs, approximately twice that in Experiment 1-1. 328 Well 1 in Experiment 1-3 was directly connected to the FZs. Its pressure drop profile was 329 significantly different from that of Experiments 1-1 and 1-2. Whether in the early or late stages of 330 development, the R in all parts of the gas reservoir Experiment 1-3 was relatively balanced, and the 331 near-well and peripheral pressures gently and synchronously reduced. Due to the high conductivity 332 of the fractures, gas and water were produced together from the gas well, and the energy of the 333 aquifer was simultaneously reduced. This reduces the physical damage due to water invasion, 334 allowing the gas to flow to the gas well through the fractures. 335 which was the highest value among the three WDGRs, approximately twice that in Experiment 1-1. 328 Well 1 in Experiment 1-3 was directly connected to the FZs. Its pressure drop profile was 329 significantly different from that of Experiments 1-1 and 1-2. Whether in the early or late stages of 330 development, the R in all parts of the gas reservoir Experiment 1-3 was relatively balanced, and the 331 near-well and peripheral pressures gently and synchronously reduced. Due to the high conductivity 332 of the fractures, gas and water were produced together from the gas well, and the energy of the 333 aquifer was simultaneously reduced. This reduces the physical damage due to water invasion, 334 allowing the gas to flow to the gas well through the fractures. 335 The gas reservoir pressure drop profiles in Figs. 8-10 also show that, with only Well 1 in 336 production, the R of the low-permeability MZ 1 (Core 2) surrounded by the FZs at the distal end 337 of the gas well depended on the peripheral FZs (Cores 1 and 3). Dynamic Pressure Drop 284 If the R of the reservoir in the FZ 338 was low (Experiments 1-1 and 1-2), the reserves in MZ 1 were sealed off and could not be developed; 339 if the R of the reservoir in the FZs was high (Experiment 1-3), the MZ 1 R would supply gas to 340 the gas well through the FZs and achieve a high gas reservoir R. 341 Distribution of Residual Water and Reserves 342 12 The cores representing the three types of WDGRs were removed immediately after the gas 343 production experiments ended. Then, the average water saturation of the cores in the different parts 344 of the gas reservoirs was obtained using the weighing method, as shown in Table 4. 345 The experiments show that in Experiments 1-1 and 1-2, the overall average water saturations 346 after production were not very different, both exceeding than 40%. Experiment 1-3 with the gas 347 well connected to the FZs produced gas with water for a relatively long time after water 348 breakthrough was observed in the gas well, and the drainage effect was good. The water saturation 349 was only 32.33%, which was approximately 10% lower than that in Experiments 1-1 and 1-2. 350 All the experimental results obtained for the three types of WDGRs show that the water 351 invasion in the FZs directly connected to the aquifer was the most serious, and correspondingly, the 352 water saturation was the highest, reaching 40%-55%. Although near-well MZ 2 was the farthest 353 from the aquifer, its water saturation was also approximately 45%. The high water saturation of MZ 354 2 indicated that the fracture was the main water intrusion channel and that the water spread into the 355 The cores representing the three types of WDGRs were removed immediately after the gas 343 production experiments ended. Then, the average water saturation of the cores in the different parts 344 of the gas reservoirs was obtained using the weighing method, as shown in Table 4. 345 The cores representing the three types of WDGRs were removed immediately after the gas 343 production experiments ended. Then, the average water saturation of the cores in the different parts 344 of the gas reservoirs was obtained using the weighing method, as shown in Table 4. 345 The experiments show that in Experiments 1-1 and 1-2, the overall average water saturations 346 after production were not very different, both exceeding than 40%. Experiment 1-3 with the gas 347 well connected to the FZs produced gas with water for a relatively long time after water 348 breakthrough was observed in the gas well, and the drainage effect was good. The water saturation 349 was only 32.33%, which was approximately 10% lower than that in Experiments 1-1 and 1-2. Dynamic Pressure Drop 284 The 309 dynamic pressure drop process of the gas reservoir shows that the pressure drop in the entire gas 310 reservoir was relatively synchronous and occurred within the first 20 mins of gas production, 311 indicating that the initial reserve development was balanced and that the reservoir could provide a 312 stable gas supply to the gas well. In this stage, the maximum pressure gradient of the gas supply 313 path of the gas reservoir, which occurs in the near-well area of the low-pressure MZ 2 (Core 4), was 314 only 0.007 MPa/cm (Fig. 11). Thereafter, the pressure gradient in the immediate vicinity of the well 315 began to increase rapidly, reaching 0.18 MPa/cm at 25 mins and a peak value of 0.34 MPa/cm at 30 316 mins before stable production ended. The significant pressure drop funnel that formed around the 317 gas well indicated that a large amount of formation energy was lost in the immediate vicinity of the 318 well, i.e., the MZ. When Well 1 was abandoned and production was stopped, the pressure gradient 319 near the well finally stabilized at 0.38 MPa/cm, and the residual pressure in the peripheral FZs and 320 MZ 1 (Cores 1-3) was as high as 19.6 MPa to 21.6 MPa. A large amount of residual reserves was 321 not developed due to water sealing. The R of the corresponding gas reservoir was only 37.9%. 322 W ll 1 i E i 1 2 25 f h FZ 50% f h di i E i 1 1 323 Well 1 in Experiment 1-2 was 25 cm from the FZs, 50% of the distance in Experiment 1-1. 323 Similar to Experiment 1-1, the initial pressure drop in Experiment 1-2 was synchronous in the entire 324 gas reservoir. In the later stage, due to the intrusion of formation water in the near-well zone (MZ 325 2), the pressure gradient of the near-well zone increased rapidly. In the process of production decline, 326 the average pressure gradient of the near-well zone was stabilized at approximately 0.74 MPa/cm, 327 11 which was the highest value among the three WDGRs, approximately twice that in Experiment 1-1. 328 Well 1 in Experiment 1-3 was directly connected to the FZs. Its pressure drop profile was 329 significantly different from that of Experiments 1-1 and 1-2. Distribution of Residual Water and Reserves 342 350 12 All the experimental results obtained for the three types of WDGRs show that the water 351 invasion in the FZs directly connected to the aquifer was the most serious, and correspondingly, the 352 water saturation was the highest, reaching 40%-55%. Although near-well MZ 2 was the farthest 353 from the aquifer, its water saturation was also approximately 45%. The high water saturation of MZ 354 2 indicated that the fracture was the main water intrusion channel and that the water spread into the 355 12 MZ far from the aquifer via the fracture. This is consistent with the conclusion of Sait (2019) from 356 a study of the water invasion mechanism of a fractured carbonate gas reservoir. 357 MZ 1, surrounded by the FZs, had the lowest average water saturation for each experimental 358 scheme, i.e., 27.60%, 21.45%, and 9.56% in Experiments 1-1, 1-2 and 1-3, respectively, which were 359 considerably lower than those in other zones. The reason for this is that the main water invasion 360 mechanism of MZ 1, the imbibition effect, was different from those of the other zones. Specifically, 361 the permeability of MZ 1 was 0.67 mD, which was considerably lower than that of the reservoirs in 362 the peripheral FZs, where the gas supply rate was slow and the development of the remaining 363 reserves was delayed. The pressure was always slightly higher than that of the connected FZs (this 364 can be verified with the measured pressure data). Physically, gas and water in porous media cannot 365 flow from a low-pressure FZs to a high-pressure MZ. Therefore, the mechanism of the water 366 invasion in MZ 1 can mainly be the imbibition caused by the capillary force. 367 Gas reservoirs with different geological conditions and even different zones of the same gas 368 reservoirs may have completely different water invasion mechanisms. Their water saturation and 369 residual gas distributions also may be significantly different. 370 The relative residual reserves of different zones (the ratio of the residual reserves to the total 371 gas reserves) can be calculated after converting the water saturation to the residual gas saturation 372 while considering the residual pressure and porosity of various zones in the gas reservoir. The 373 specific calculation method is as follows: 374 f= PrkSgk∅k ∑ PrkSgk∅k 4 k=1 . ………………………………………………………………………….………. Distribution of Residual Water and Reserves 342 385 saturation of residual gas into account. The amount of residual reserves in the near-well MZ 2 in the 381 near-well area of Experiments 1-1 and 1-2 was the lowest. The reserves were concentrated in the 382 peripheral FZs and MZ 1, and the gas reservoir reserves were not recovered uniformly; the 383 difference among the proportions of the residual reserves in the different zones of Experiment 1-3 384 is no more than 10%, and the reserve development was the most balanced. 385 saturation of residual gas into account. The amount of residual reserves in the near-well MZ 2 in the 381 near-well area of Experiments 1-1 and 1-2 was the lowest. The reserves were concentrated in the 382 peripheral FZs and MZ 1, and the gas reservoir reserves were not recovered uniformly; the 383 difference among the proportions of the residual reserves in the different zones of Experiment 1-3 384 is no more than 10%, and the reserve development was the most balanced. 385 Distribution of Residual Water and Reserves 342 (1) 375 The proportions of residual reserves in different zones after the production of the three types 376 of WDGRs for Group I were calculated and are shown in Table 5. 377 Table 5 shows that the distribution of the residual reserves in different zones of the same gas 378 reservoir was not uniform. Although the porosity of MZ 1 was not high in the three experiments, 379 the residual reserves of this zone were apparently higher than those of the other zones, taking a high 380 MZ far from the aquifer via the fracture. This is consistent with the conclusion of Sait (2019) from 356 a study of the water invasion mechanism of a fractured carbonate gas reservoir. 357 a study of the water invasion mechanism of a fractured carbonate gas reservoir. 357 MZ 1, surrounded by the FZs, had the lowest average water saturation for each experimental 358 scheme, i.e., 27.60%, 21.45%, and 9.56% in Experiments 1-1, 1-2 and 1-3, respectively, which were 359 considerably lower than those in other zones. The reason for this is that the main water invasion 360 mechanism of MZ 1, the imbibition effect, was different from those of the other zones. Specifically, 361 the permeability of MZ 1 was 0.67 mD, which was considerably lower than that of the reservoirs in 362 the peripheral FZs, where the gas supply rate was slow and the development of the remaining 363 reserves was delayed. The pressure was always slightly higher than that of the connected FZs (this 364 can be verified with the measured pressure data). Physically, gas and water in porous media cannot 365 flow from a low-pressure FZs to a high-pressure MZ. Therefore, the mechanism of the water 366 invasion in MZ 1 can mainly be the imbibition caused by the capillary force. 367 Gas reservoirs with different geological conditions and even different zones of the same gas 368 (1) 13 saturation of residual gas into account. The amount of residual reserves in the near-well MZ 2 in the 381 near-well area of Experiments 1-1 and 1-2 was the lowest. The reserves were concentrated in the 382 peripheral FZs and MZ 1, and the gas reservoir reserves were not recovered uniformly; the 383 difference among the proportions of the residual reserves in the different zones of Experiment 1-3 384 is no more than 10%, and the reserve development was the most balanced. Impact of Drainage Positions 386 The multiwell drainage gas production process is often adopted in the early stage of development 387 in fractured WDGRs. For fractured reservoirs or high-permeability zones connecting to the aquifer, 388 water drainage wells (such as Well Chi 27 in Fig. 1 and Well 2 in Fig. 2) are deployed to block 389 aquifer water intrusion. In Group II, the impact of the drainage position on the water control effect 390 was simulated by adjusting the position of the gas production well. As shown in Table 2 and Fig. 3, 391 Well 2 in Experiments 2-1 and 2-2 was set at Point A in the middle of the LFZ and Point B near the 392 water edge, respectively (the distance between Points A and B was 12.5 cm). Well 2 in Experiments 393 2-3 and 2-4 was located in the zone with small fractures, at Point C and Point D. In the experiments, 394 the operation of the Well 1 and Well 2 was simultaneously initiated. For joint gas production and 395 water draining, the well that reached an abandoned production rate first, had to keep open until the 396 other well reached the abandonment production rate. 397 The multiwell drainage gas production process is often adopted in the early stage of development 387 in fractured WDGRs. For fractured reservoirs or high-permeability zones connecting to the aquifer, 388 water drainage wells (such as Well Chi 27 in Fig. 1 and Well 2 in Fig. 2) are deployed to block 389 aquifer water intrusion. In Group II, the impact of the drainage position on the water control effect 390 was simulated by adjusting the position of the gas production well. As shown in Table 2 and Fig. 3, 391 Well 2 in Experiments 2-1 and 2-2 was set at Point A in the middle of the LFZ and Point B near the 392 water edge, respectively (the distance between Points A and B was 12.5 cm). Well 2 in Experiments 393 2-3 and 2-4 was located in the zone with small fractures, at Point C and Point D. In the experiments, 394 the operation of the Well 1 and Well 2 was simultaneously initiated. For joint gas production and 395 water draining, the well that reached an abandoned production rate first, had to keep open until the 396 other well reached the abandonment production rate. Impact of Drainage Positions 386 Experiment 2-2 had a better effect on the overall water 413 control of the gas reservoir. The stable production period of Well 1 at the distal matrix position 414 reached 40 min, and the R was as high as 50.29%, which were 1.48 times and 1.34 times those of 415 the corresponding Well 1 in Experiment 2-1, respectively. The R of the entire gas reservoir reached 416 78.87%, which was 6.08% higher than that in Experiment 2-1. 417 the deep part of the gas reservoir. In Experiment 2-2, Well 2 was closer to the aquifer and had a 411 higher drainage capacity and a lower gas production capacity. The R of Well 2 was 28.58%, which 412 was only 80.6% of that in Experiment 2-1. Experiment 2-2 had a better effect on the overall water 413 control of the gas reservoir. The stable production period of Well 1 at the distal matrix position 414 reached 40 min, and the R was as high as 50.29%, which were 1.48 times and 1.34 times those of 415 the corresponding Well 1 in Experiment 2-1, respectively. The R of the entire gas reservoir reached 416 78.87%, which was 6.08% higher than that in Experiment 2-1. 417 the deep part of the gas reservoir. In Experiment 2-2, Well 2 was closer to the aquifer and had a 411 higher drainage capacity and a lower gas production capacity. The R of Well 2 was 28.58%, which 412 was only 80.6% of that in Experiment 2-1. Experiment 2-2 had a better effect on the overall water 413 control of the gas reservoir. The stable production period of Well 1 at the distal matrix position 414 reached 40 min, and the R was as high as 50.29%, which were 1.48 times and 1.34 times those of 415 the corresponding Well 1 in Experiment 2-1, respectively. The R of the entire gas reservoir reached 416 78.87%, which was 6.08% higher than that in Experiment 2-1. 417 Clearly, the drainage gas recovery in Experiments 2-1 and 2-2 was better than Experiment 1-1, 418 not considering the technical limitations and economic costs. 419 Clearly, the drainage gas recovery in Experiments 2-1 and 2-2 was better than Experiment 1-1, 418 not considering the technical limitations and economic costs. 419 Both production parameters of Experiments 2-3 and 2-4 are tabulated in Table 6. Impact of Drainage Positions 386 397 Production Performance 398 Experiments 2-1 and 2-2 in which the drainage well was set in a LFZ were selected for a comparative 399 analysis. The gas production and water production results of both experiments are shown in Figs. 400 12 and 13. The production parameters are shown in Table 6. 401 Experiments 2-1 and 2-2 in which the drainage well was set in a LFZ were selected for a comparative 399 analysis. The gas production and water production results of both experiments are shown in Figs. 400 12 and 13. The production parameters are shown in Table 6. 401 Since multiwell water control was conducted in the early stage, the gas R of Experiments 2-1 402 and 2-2 was significantly improved compared with that of Experiment 1-1 with a single well, Well 403 1 for production, reaching 72.79% and 78.87%, respectively (the gas reservoir conditions were 404 exactly the same, and the R of Experiment 1-1 was only 37.9%). This shows that in the early stage 405 of development, the gas wells in reasonable positions perform joint drainage, greatly reduce the 406 impact of the water invasion and significantly improve the gas recovery. 407 14 The production curve shows that no water breakthrough occurred during the production of Well 408 1 in the distal MZ in Experiments 2-1 and 2-2 due to the high-efficiency drainage of Well 2. This 409 indicates that joint drainage and gas production effectively prevented the aquifer from invading into 410 14 The production curve shows that no water breakthrough occurred during the production of Well 408 1 in the distal MZ in Experiments 2-1 and 2-2 due to the high-efficiency drainage of Well 2. This 409 indicates that joint drainage and gas production effectively prevented the aquifer from invading into 410 14 The production curve shows that no water breakthrough occurred during the production of Well 408 1 in the distal MZ in Experiments 2-1 and 2-2 due to the high-efficiency drainage of Well 2. This 409 indicates that joint drainage and gas production effectively prevented the aquifer from invading into 410 14 the deep part of the gas reservoir. In Experiment 2-2, Well 2 was closer to the aquifer and had a 411 higher drainage capacity and a lower gas production capacity. The R of Well 2 was 28.58%, which 412 was only 80.6% of that in Experiment 2-1. Impact of Drainage Positions 386 The R of the 420 gas reservoirs reached 78.08% and 80.33%, respectively, which were also greatly improved 421 compared with the single-well production in Experiment 1-1: the differences in the production 422 performance metrics of Experiment 1-1 and Experiments 2-1 and 2-2 were noticeable. The impact 423 of the drainage position in the SFZ was small, and the difference in the R did not exceed 2.5%. 424 Water-control Experiments 2-3 and 2-4 had similar drainage effectiveness based on the drainage and 425 gas production capacities of the main drainage well, Well 2. Thus, their drainage functions and the 426 protective effects on the gas reservoirs were not considerably different. Compared with drainage 427 Well 2, which was set in the LFZ (Experiments 2-1 and 2-2), Well 2 in the SFZ (Experiments 2-2 428 and 2-3) had a delayed breakthrough time, and its drainage speed was slower, but the corresponding 429 gas production capacity increased, and the R reached 44%, which was more than 10%-15% higher 430 than that of Experiments 2-1 and 2-2. 431 Group II showed that the gas drainage effectiveness and the gas production capacity were 432 significantly affected by the location of the gas well upstream, performing the main drainage 433 function. The distance from the gas well to the aquifer and the seepage capacity of the reservoir in 434 the area were important factors. At the same time, these results also illustrated the complexity of the 435 development of a water control plan. The drainage position should be determined by 436 comprehensively considering the recovery of the entire gas reservoir, the technical conditions on 437 site and the economic benefits. 438 Group II showed that the gas drainage effectiveness and the gas production capacity were 432 significantly affected by the location of the gas well upstream, performing the main drainage 433 function. The distance from the gas well to the aquifer and the seepage capacity of the reservoir in 434 the area were important factors. At the same time, these results also illustrated the complexity of the 435 development of a water control plan. The drainage position should be determined by 436 comprehensively considering the recovery of the entire gas reservoir, the technical conditions on 437 site and the economic benefits. 438 15 volumetric gas reservoirs using the same model (basic Experiment 5-1) are also shown in Fig. 14. Impact of Drainage Positions 386 441 For the convenience of analysis, the relationships between the Wp and the R of the reserves for the 442 corresponding experiments are also plotted in Fig. 14. 443 The θ~R curves of Group II showed the characteristics of water drainage. As Well 2 in 444 Experiments 2-1 and 2-2 produced water earlier and faster, the relative pressure curve started to 445 decrease earlier, and the rate of decrease was greater, implying a stronger drainage effect. As the 446 production progressed, the curves decreased to greater extents, indicating that as the drainage 447 proceeded, the net water influx was reduced and converted to net water production in the later stage. 448 In Fig. 14, the upward Wp~R curve indicates that the unit gas production corresponded to an 449 increase in both the water production and the water-gas ratio. The water production of the four 450 experiments significantly increased once the R was greater than 50%-60%. Thus, the water-gas 451 ratio of the gas reservoir in the later stage of production increased significantly, which also indicates 452 that only the large-scale drainage in the later stage could maintain the gas production of the gas well. 453 Meanwhile, the corresponding relationship between the Wp and the relative formation pressure 454 decline curve was obvious, indicating that the θ~R relation curve could accurately reflect the water 455 invasion degree of the gas reservoir in a timely manner. 456 The θ~R curves of Group II showed the characteristics of water drainage. As Well 2 in 444 Experiments 2-1 and 2-2 produced water earlier and faster, the relative pressure curve started to 445 decrease earlier, and the rate of decrease was greater, implying a stronger drainage effect. As the 446 production progressed, the curves decreased to greater extents, indicating that as the drainage 447 proceeded, the net water influx was reduced and converted to net water production in the later stage. 448 In Fig. 14, the upward Wp~R curve indicates that the unit gas production corresponded to an 449 increase in both the water production and the water-gas ratio. The water production of the four 450 experiments significantly increased once the R was greater than 50%-60%. Thus, the water-gas 451 ratio of the gas reservoir in the later stage of production increased significantly, which also indicates 452 that only the large-scale drainage in the later stage could maintain the gas production of the gas well. Impact of Drainage Positions 386 453 Meanwhile, the corresponding relationship between the Wp and the relative formation pressure 454 decline curve was obvious, indicating that the θ~R relation curve could accurately reflect the water 455 invasion degree of the gas reservoir in a timely manner. 456 Dynamic Pressure Drop 457 The dynamic pressure drop profiles of Experiments 2-1 and 2-2 are included in Fig. 15 and Fig. 16. 458 Since water control was performed via both wells in an early stage of gas reservoir development, 459 the pressure profiles of the gas reservoirs of Experiments 2-1 and 2-2 decreased rapidly and 460 simultaneously. The residual pressure was low, which was significantly different from that of 461 Experiment 1-1, as plotted in Fig. 8. The high pressure drop funnel that formed in the immediate 462 vicinity of Well 1 in Experiment 1-1 was not observed in Experiments 2-1 and 2-2. This shows that 463 for a fractured WDGR, multiwell water control conducted in the early stage could effectively 464 prevent the aquifer water from intruding the gas reservoir, protect the entire gas reservoir, and 465 greatly improve the R and balance of the gas reservoir development. 466 Dynamic Pressure Drop 457 The dynamic pressure drop profiles of Experiments 2-1 and 2-2 are included in Fig. 15 and Fig. 16. 458 Since water control was performed via both wells in an early stage of gas reservoir development, 459 the pressure profiles of the gas reservoirs of Experiments 2-1 and 2-2 decreased rapidly and 460 simultaneously. The residual pressure was low, which was significantly different from that of 461 Experiment 1-1, as plotted in Fig. 8. The high pressure drop funnel that formed in the immediate 462 vicinity of Well 1 in Experiment 1-1 was not observed in Experiments 2-1 and 2-2. This shows that 463 for a fractured WDGR, multiwell water control conducted in the early stage could effectively 464 prevent the aquifer water from intruding the gas reservoir, protect the entire gas reservoir, and 465 greatly improve the R and balance of the gas reservoir development. 466 16 As Well 2 was deployed at a different position, the order of development of the remaining 467 reserves in different zones of the gas reservoir changed. In Experiment 2-1, Well 2 was at Point A, 468 in the middle of the LFZ connected to MZ 1. Distribution of Residual Water and Reserves 476 Comparing the experimental schemes, such as the large-fracture drainage Experiments 2-1 and 481 2-2, the average water saturation of the gas reservoir after production was 22.81% and 16.37%, 482 which were lower than 27.32% and 23.22% of the small-fracture drainage Experiments 2-3 and 2-4 483 and indicated that drainage in the large-fracture can more greatly restrain water invasion. 484 The production parameters in Table 6 show that the drainage well located in a large-fracture 485 (i.e., Experiments 2-1 and 2-2) will produce water earlier and more frequently. Based on the above 486 analysis of Fig. 14, Well 2 in Experiments 2-1 and 2-2 produces water earlier and faster than that in 487 Experiments 2-3 and 2-4, so its θ~R curve dips earlier and its dip amplitude is larger, implying a 488 stronger drainage effect. Both Table 6 and Fig. 14 show that this finding is reliable and accurate, 489 since the drainage effect of large fractures is better and more effective at avoiding water intrusion 490 into the deeper part of the gas reservoir. 491 The production parameters in Table 6 show that the drainage well located in a large-fracture 485 (i.e., Experiments 2-1 and 2-2) will produce water earlier and more frequently. Based on the above 486 analysis of Fig. 14, Well 2 in Experiments 2-1 and 2-2 produces water earlier and faster than that in 487 Experiments 2-3 and 2-4, so its θ~R curve dips earlier and its dip amplitude is larger, implying a 488 stronger drainage effect. Both Table 6 and Fig. 14 show that this finding is reliable and accurate, 489 since the drainage effect of large fractures is better and more effective at avoiding water intrusion 490 into the deeper part of the gas reservoir. 491 When the water is drained at the near-aquifer location in the FZ (Experiments 2-2 and 2-4), the 492 increment of water saturation in the gas reservoir is small, approximately 5%-7% lower than that 493 drained in the middle of the FZ (Experiments 2-1 and 2-3, far from the aquifer). This shows that 494 whether the reservoir permeability is high or low, the drainage of water near the aquifer can better 495 prevent water invasion. Impact of Drainage Positions 386 MZ 1 supplied the most gas to Well 2 through the 469 fracture, and the average pressure in that zone dropped rapidly, close to the rate of pressure drop in 470 16 As Well 2 was deployed at a different position, the order of development of the remaining 467 reserves in different zones of the gas reservoir changed. In Experiment 2-1, Well 2 was at Point A, 468 in the middle of the LFZ connected to MZ 1. MZ 1 supplied the most gas to Well 2 through the 469 fracture, and the average pressure in that zone dropped rapidly, close to the rate of pressure drop in 470 16 As Well 2 was deployed at a different position, the order of development of the remaining 467 reserves in different zones of the gas reservoir changed. In Experiment 2-1, Well 2 was at Point A, 468 in the middle of the LFZ connected to MZ 1. MZ 1 supplied the most gas to Well 2 through the 469 fracture, and the average pressure in that zone dropped rapidly, close to the rate of pressure drop in 470 16 MZ 2 connected to Well 1. After producing for 20 mins, the residual pressure almost reached 471 vanished. In Experiment 2-2, Well 2 was closer to the aquifer, and its drainage capacity was higher, 472 so the pressure in the other areas of the gas reservoir, including the SFZ and the MZ in the upper 473 part of the gas reservoir, was simultaneously reduced, which protected the entire gas reservoir more 474 effectively. 475 MZ 2 connected to Well 1. After producing for 20 mins, the residual pressure almost reached 471 vanished. In Experiment 2-2, Well 2 was closer to the aquifer, and its drainage capacity was higher, 472 so the pressure in the other areas of the gas reservoir, including the SFZ and the MZ in the upper 473 part of the gas reservoir, was simultaneously reduced, which protected the entire gas reservoir more 474 effectively. 475 Distribution of Residual Water and Reserves 476 Table 7 provides the average water saturation of the gas reservoir in Group II, which was 22.4% 477 when production ended and significantly lower than that in Experiment 1-1 (41.46% water). 478 Therefore, in the early stage, performing multiwell joint drainage and gas production can effectively 479 prevent water invasion. 480 Distribution of Residual Water and Reserves 476 This indicated that, on the one hand, 503 due to multiwell drainage, the MZ was basically unaffected by water invasion; on the other hand, 504 as the number of gas wells increased, the gas supply distance from the MZ to the gas wells was 505 greatly reduced, so the reserves could be developed more easily (Table 8). 506 occurred. In Experiment 2-2, the water content of the distal MZ 2 was increased by only 1.91%. 501 In general, although the MZ had poor physical properties and low permeability, its R on the 502 whole was higher than that of the FZ connected to the aquifer. This indicated that, on the one hand, 503 due to multiwell drainage, the MZ was basically unaffected by water invasion; on the other hand, 504 as the number of gas wells increased, the gas supply distance from the MZ to the gas wells was 505 greatly reduced, so the reserves could be developed more easily (Table 8). 506 In Experiments 2-1 and 2-2, which drained water along the large fractures, the R of the 507 reserves in LFZ 2 was greater than that in SFZ 1, which was far from Well 2. However, in 508 Experiments 2-3 and 2-4, which drained water along the small fractures, the R in SFZ 1 was better 509 than that in LFZ 2, which was far from Well 2. 510 In Experiments 2-1 and 2-2, which drained water along the large fractures, the R of the 507 reserves in LFZ 2 was greater than that in SFZ 1, which was far from Well 2. However, in 508 Experiments 2-3 and 2-4, which drained water along the small fractures, the R in SFZ 1 was better 509 than that in LFZ 2, which was far from Well 2. 510 Distribution of Residual Water and Reserves 476 496 17 Notably, due to the high-efficiency drainage in the early stage of the experiments, all four 497 experiments successfully protected the low-permeability MZ 1 surrounded by fractures and the low- 498 permeability MZ 2 at the toe of the well. In the large-fracture drainage Experiments 2-1 and 2-2, 499 the water saturation of MZ 1 was increased by only approximately 5%, and almost no water invasion 500 17 Notably, due to the high-efficiency drainage in the early stage of the experiments, all four 497 experiments successfully protected the low-permeability MZ 1 surrounded by fractures and the low- 498 permeability MZ 2 at the toe of the well. In the large-fracture drainage Experiments 2-1 and 2-2, 499 the water saturation of MZ 1 was increased by only approximately 5%, and almost no water invasion 500 17 occurred. In Experiment 2-2, the water content of the distal MZ 2 was increased by only 1.91%. 501 In general, although the MZ had poor physical properties and low permeability, its R on the 502 whole was higher than that of the FZ connected to the aquifer. This indicated that, on the one hand, 503 due to multiwell drainage, the MZ was basically unaffected by water invasion; on the other hand, 504 as the number of gas wells increased, the gas supply distance from the MZ to the gas wells was 505 greatly reduced, so the reserves could be developed more easily (Table 8). 506 In Experiments 2-1 and 2-2, which drained water along the large fractures, the R of the 507 reserves in LFZ 2 was greater than that in SFZ 1, which was far from Well 2. However, in 508 Experiments 2-3 and 2-4, which drained water along the small fractures, the R in SFZ 1 was better 509 than that in LFZ 2, which was far from Well 2. 510 occurred. In Experiment 2-2, the water content of the distal MZ 2 was increased by only 1.91%. 501 In general, although the MZ had poor physical properties and low permeability, its R on the 502 whole was higher than that of the FZ connected to the aquifer. Impact of Drainage Timing 511 However, by adding a 546 new gas well, Well 2, in the high-permeability zone of the gas reservoir, the water-sealed reserves 547 in the gas reservoir could be unlocked, which significantly improved the gas R (38.45% and 34.6%). 548 In addition, the later the drainage timing of the drainage well, the more serious the water invasion, 549 the more difficult the development of the water-sealed reserves, and the lower the R of the gas 550 reservoirs. 551 Experiments show that when a single well (Well 1) was put into production, once it was flooded, 545 its production would decrease rapidly and could not be recovered by itself. However, by adding a 546 new gas well, Well 2, in the high-permeability zone of the gas reservoir, the water-sealed reserves 547 in the gas reservoir could be unlocked, which significantly improved the gas R (38.45% and 34.6%). 548 In addition, the later the drainage timing of the drainage well, the more serious the water invasion, 549 the more difficult the development of the water-sealed reserves, and the lower the R of the gas 550 reservoirs. 551 Compared with the outstanding stimulation effect of adding Well 2 in Experiments 3-1 and 3- 552 2, the gas and Qw in the second stage of Experiment 3-3 were extremely low. The R was only 553 5.4%, and the cumulative R of the two stages was 68.7%, which was far lower than that achieved 554 in Experiments 3-1 and 3-2. Well 1 in the gas reservoir of Experiment 3-3 was directly connected 555 to the FZ via a high-conductivity fault zone, where the energy in the gas reservoir was largely 556 consumed in the first stage (the residual relative apparent pressure in Experiment 1-3 was only 0.25, 557 in Fig. 6), which was not enough to produce gas from the water-sealed zone; thus, it is not possible 558 to significantly increase the gas production in such gas reservoirs by adding gas wells. 559 Compared with the outstanding stimulation effect of adding Well 2 in Experiments 3-1 and 3- 552 2, the gas and Qw in the second stage of Experiment 3-3 were extremely low. The R was only 553 5.4%, and the cumulative R of the two stages was 68.7%, which was far lower than that achieved 554 in Experiments 3-1 and 3-2. Impact of Drainage Timing 511 As shown in Table 2, Experiments 3-1 and 3-2 were conducted with Geological Model 2. At the 512 beginning of Experiments 3-1 and 3-2, only a single well, Well 1, was put into production. Once 513 Well 1 reached the abandonment production rate, Well 2 was immediately opened in Experiment 3- 514 1. The drainage well was set in the middle of the LFZ, and it was produced simultaneously with 515 Well 1. In Experiment 3-2, Well 1 was first shut in and then opened to produce gas 16 hours later. 516 When the production of Well 1 decreased to the abandonment production rate again, drainage Well 517 2 was set at Point A in the middle of the LFZ and was put on production. The production process 518 lasted until the production of both wells was reduced to the abandonment production rate. 519 Experiment 3-3 was implemented with Geological Model 3. Well 1 was directly connected to the 520 fracture zone (FZ) through the high-conductivity fault zone. Its drainage timing was the same as 521 that of Experiment 3-1. 522 The above experimental process reflects that the first stage of production in Experiments 3-1 523 and 3-2 was identical to that in Experiment 1-2 (with the same gas reservoir) for single-well (Well 524 1) production, except that in the second stage, the increases in Well 2 were added. Similarly, the 525 first stage of Experiment 3-3 was identical to that in Experiment 1-3 of single-well (Well 1) 526 production (for the same gas reservoir). Therefore, the production situation of the first stage of 527 Group III is not repeated. 528 Production Performance 529 The production curve and the production parameters after adding Well 2 in the second stage of 530 18 The production curve and the production parameters after adding Well 2 in the second stage of 530 18 production of Group III are shown in Fig. 17 and Table 9, respectively. 531 As shown in Table 9, the gas production and Wp in the original production well, the remote 532 Well 1, were extremely small in the second stage of each of the three experiments. By the end of 533 production, the R of Well 1 was less than 3%, and the Wp was less than 4 mL, which can be 534 neglected. The main gas production could be attributed to the later addition of Well 2. Impact of Drainage Timing 511 535 As shown in Table 9, the gas production and Wp in the original production well, the remote 532 Well 1, were extremely small in the second stage of each of the three experiments. By the end of 533 production, the R of Well 1 was less than 3%, and the Wp was less than 4 mL, which can be 534 neglected. The main gas production could be attributed to the later addition of Well 2. 535 In the second stage of Experiment 3-1, the R of Well 2 reached 37.6%. The cumulative R of 536 the two stages was 79.9%. 537 In the second stage of Experiment 3-1, the R of Well 2 reached 37.6%. The cumulative R of 536 the two stages was 79.9%. 537 After the wells were shut in for 16 hours in Experiment 3-2, Well 1 was initiated first, in the 538 stage with a R of 2.9%. The water produced was only 3.6 mL. This showed that the water invasion 539 into the gas reservoir formed a strong water seal, making it difficult for gas and water to flow into 540 Well 1. It was difficult to effectively resume the production of Well 1 after a long-term shut in. After 541 that, Well 2 was added, and the R sharply reached 31.7%, which implies effective stimulation. In 542 the two stages, the cumulative R increased to 76%, which was slightly lower than that in 543 Experiment 3-1. 544 After the wells were shut in for 16 hours in Experiment 3-2, Well 1 was initiated first, in the 538 stage with a R of 2.9%. The water produced was only 3.6 mL. This showed that the water invasion 539 into the gas reservoir formed a strong water seal, making it difficult for gas and water to flow into 540 Well 1. It was difficult to effectively resume the production of Well 1 after a long-term shut in. After 541 that, Well 2 was added, and the R sharply reached 31.7%, which implies effective stimulation. In 542 the two stages, the cumulative R increased to 76%, which was slightly lower than that in 543 Experiment 3-1. 544 Experiments show that when a single well (Well 1) was put into production, once it was flooded, 545 its production would decrease rapidly and could not be recovered by itself. Dynamic Pressure Drop The dynamic pressure drop profiles of the second stage of Experiment 3-1 are shown in Fig. 18 (for 561 the first stage, see Experiment 1-2, shown in Fig. 9). 562 In the second stage of Experiment 3-1, after adding Well 2 and producing from the two wells 563 for 5 mins, the pressure drop profile changed. When Well 2 was put into production, the residual 564 reserves in MZ 1 were quickly produced. Since the gas production at Well 2 was stable, a large 565 amount of gas and water was produced. Specifically, the peripheral areas that were difficult to 566 exploit from Well 1 in the first stage were developed by Well 2 (Cores 1-3). Well 2 continued to 567 produce for 184 mins, and the pressure drop profile at the end of the experiment (Fig. 18(d)) showed 568 that the gas reservoir was relatively balanced and effectively developed. 569 At the beginning of the second stage of Experiment 3-2, Well 1 was produced first, and the 570 yield stimulation effect was poor, so the pressure drop profile did not change considerably. After 571 adding drainage Well 2, the change in the pressure drop profile was almost the same as that of 572 Experiment 3-1. To avoid repetition, those results are not described in this paper. 573 At the beginning of the second stage of Experiment 3-2, Well 1 was produced first, and the 570 yield stimulation effect was poor, so the pressure drop profile did not change considerably. After 571 adding drainage Well 2, the change in the pressure drop profile was almost the same as that of 572 Experiment 3-1. To avoid repetition, those results are not described in this paper. 573 Since the drainage scheme in the second stage of Experiment 3-3 almost failed, the relative 574 formation pressure was unchanged from that of Experiment 1-3, and the pressure profile was also 575 stabilized after the first stage of production. 576 Since the drainage scheme in the second stage of Experiment 3-3 almost failed, the relative 574 formation pressure was unchanged from that of Experiment 1-3, and the pressure profile was also 575 stabilized after the first stage of production. 576 20 Distribution of Residual Water and Reserves 577 The water saturations for different zones after gas reservoir production are presented in Table 10. Dynamic Pressure Drop 578 Compared with the four experiments in Group II, in which two wells were combined to control 579 water in the early stage of the experiments, Group III showed that the average water saturation 580 increased to more than 30% (from the 22.4% observed for Group II) due to the delay in the drainage 581 well initiation. This indicates that a delayed drainage led to an increase in the net water influx in the 582 gas reservoir. 583 In Experiments 3-1 and 3-2, after the second stage of production, the average water content of 584 the gas reservoir decreased to 31.22% and 33.77%, respectively, which were 11.52% and 8.97% 585 lower than that of the first stage (Experiment 1-2, 42.74%), respectively. Thus, by increasing the 586 effective drainage of the drainage well, the water saturation of the reservoir was reduced and the 587 flow resistance was reduced, which laid the foundation for the residual reserves to be developed. 588 Compared with Experiment 3-1, in Experiment 3-2, Well 1 was shut in, the drainage was 589 Impact of Drainage Timing 511 569 At the beginning of the second stage of Experiment 3-2, Well 1 was produced first, and the 570 yield stimulation effect was poor, so the pressure drop profile did not change considerably. After 571 adding drainage Well 2, the change in the pressure drop profile was almost the same as that of 572 Experiment 3-1. To avoid repetition, those results are not described in this paper. 573 Since the drainage scheme in the second stage of Experiment 3-3 almost failed, the relative 574 formation pressure was unchanged from that of Experiment 1-3, and the pressure profile was also 575 stabilized after the first stage of production. 576 Distribution of Residual Water and Reserves 577 The water saturations for different zones after gas reservoir production are presented in Table 10. 578 Compared with the four experiments in Group II, in which two wells were combined to control 579 water in the early stage of the experiments, Group III showed that the average water saturation 580 Dynamic Pressure Drop mpared with Experiment 3-1, in Experiment 3-2, Well 1 was shut in, the drainage was Impact of Drainage Timing 511 Well 1 in the gas reservoir of Experiment 3-3 was directly connected 555 to the FZ via a high-conductivity fault zone, where the energy in the gas reservoir was largely 556 consumed in the first stage (the residual relative apparent pressure in Experiment 1-3 was only 0.25, 557 in Fig. 6), which was not enough to produce gas from the water-sealed zone; thus, it is not possible 558 to significantly increase the gas production in such gas reservoirs by adding gas wells. 559 19 Dynamic Pressure Drop 560 The dynamic pressure drop profiles of the second stage of Experiment 3-1 are shown in Fig. 18 (for 561 the first stage, see Experiment 1-2, shown in Fig. 9). 562 In the second stage of Experiment 3-1, after adding Well 2 and producing from the two wells 563 for 5 mins, the pressure drop profile changed. When Well 2 was put into production, the residual 564 reserves in MZ 1 were quickly produced. Since the gas production at Well 2 was stable, a large 565 amount of gas and water was produced. Specifically, the peripheral areas that were difficult to 566 exploit from Well 1 in the first stage were developed by Well 2 (Cores 1-3). Well 2 continued to 567 produce for 184 mins, and the pressure drop profile at the end of the experiment (Fig. 18(d)) showed 568 that the gas reservoir was relatively balanced and effectively developed. 569 Dynamic Pressure Drop 560 The dynamic pressure drop profiles of the second stage of Experiment 3-1 are shown in Fig. 18 (for 561 the first stage, see Experiment 1-2, shown in Fig. 9). 562 In the second stage of Experiment 3-1, after adding Well 2 and producing from the two wells 563 for 5 mins, the pressure drop profile changed. When Well 2 was put into production, the residual 564 reserves in MZ 1 were quickly produced. Since the gas production at Well 2 was stable, a large 565 amount of gas and water was produced. Specifically, the peripheral areas that were difficult to 566 exploit from Well 1 in the first stage were developed by Well 2 (Cores 1-3). Well 2 continued to 567 produce for 184 mins, and the pressure drop profile at the end of the experiment (Fig. 18(d)) showed 568 that the gas reservoir was relatively balanced and effectively developed. The production of Experiments 3-1, 4-1 and 4-2 are shown in Fig. 19 and Fig. 20, and the production 618 parameters are shown in Table 12. 619 Distribution of Residual Water and Reserves 603 Therefore, the proportion of residual reserves in MZ 2 in Experiment 3-2 was significantly greater 604 than that in Experiment 3-1 (Table 11). 605 In Experiment 3-3, since the second stage of water drainage had little effect, there is no 606 significant difference in the distribution of residual reserves between the second stage and the first 607 stage (Experiment 1-3). 608 In Experiment 3-3, since the second stage of water drainage had little effect, there is no 606 significant difference in the distribution of residual reserves between the second stage and the first 607 stage (Experiment 1-3). 608 In Experiment 3-3, since the second stage of water drainage had little effect, there is no 606 significant difference in the distribution of residual reserves between the second stage and the first 607 stage (Experiment 1-3). 608 Distribution of Residual Water and Reserves 593 In Experiment 3-3, at the end of the first stage (Experiment 1-3) of production, the average 594 water content was 32.33%. In the second stage, the water drainage had little effect, as only a small 595 amount of gas and water was produced. The average water saturation dropped by only 1.54%. 596 In Experiment 3-3, at the end of the first stage (Experiment 1-3) of production, the average 594 water content was 32.33%. In the second stage, the water drainage had little effect, as only a small 595 amount of gas and water was produced. The average water saturation dropped by only 1.54%. 596 Compared with Experiment 3-1, the residual reserves of MZ 2 in the near-well area of Well 1 597 in Experiment 3-2 were increased due to the late timing of the drainage, and the gas and water in 598 the gas reservoir were rebalanced within 160 mins after Well 1 was shut in. Under the condition of 599 an enormous pressure difference in the near-well area, the gas and water continuously flowed into 600 MZ 2, which caused the gas and water contents to greatly increase, and the average residual pressure 601 increased by 4.0 MPa. The increase in the water saturation led to an increase in the gas and water 602 seepage resistance, and the difficulty of the development of the remaining reserves increased. 603 Therefore, the proportion of residual reserves in MZ 2 in Experiment 3-2 was significantly greater 604 than that in Experiment 3-1 (Table 11). 605 Compared with Experiment 3-1, the residual reserves of MZ 2 in the near-well area of Well 1 597 in Experiment 3-2 were increased due to the late timing of the drainage, and the gas and water in 598 the gas reservoir were rebalanced within 160 mins after Well 1 was shut in. Under the condition of 599 an enormous pressure difference in the near-well area, the gas and water continuously flowed into 600 MZ 2, which caused the gas and water contents to greatly increase, and the average residual pressure 601 increased by 4.0 MPa. The increase in the water saturation led to an increase in the gas and water 602 seepage resistance, and the difficulty of the development of the remaining reserves increased. Distribution of Residual Water and Reserves Distribution of Residual Water and Reserves 577 The water saturations for different zones after gas reservoir production are presented in Table 10. 578 Compared with the four experiments in Group II, in which two wells were combined to control 579 water in the early stage of the experiments, Group III showed that the average water saturation 580 increased to more than 30% (from the 22.4% observed for Group II) due to the delay in the drainage 581 well initiation. This indicates that a delayed drainage led to an increase in the net water influx in the 582 gas reservoir. 583 The water saturations for different zones after gas reservoir production are presented in Table 10. 578 Compared with the four experiments in Group II, in which two wells were combined to control 579 water in the early stage of the experiments, Group III showed that the average water saturation 580 increased to more than 30% (from the 22.4% observed for Group II) due to the delay in the drainage 581 well initiation. This indicates that a delayed drainage led to an increase in the net water influx in the 582 gas reservoir. 583 In Experiments 3-1 and 3-2, after the second stage of production, the average water content of 584 the gas reservoir decreased to 31.22% and 33.77%, respectively, which were 11.52% and 8.97% 585 lower than that of the first stage (Experiment 1-2, 42.74%), respectively. Thus, by increasing the 586 effective drainage of the drainage well, the water saturation of the reservoir was reduced and the 587 flow resistance was reduced, which laid the foundation for the residual reserves to be developed. 588 20 delayed, and the average water saturation of the gas reservoir was increased by 2.5%. The maximum 590 increase was in MZ 2 (where Well 1 located), indicating that during the shut in of Well 1, the gas 591 and water were rebalanced under the enormous pressure difference in the near-well area and 592 continued to flow to the near-well area. 593 delayed, and the average water saturation of the gas reservoir was increased by 2.5%. The maximum 590 increase was in MZ 2 (where Well 1 located), indicating that during the shut in of Well 1, the gas 591 and water were rebalanced under the enormous pressure difference in the near-well area and 592 continued to flow to the near-well area. Impact of the Aquifer Size 609 619 21 Comparing the gas production performance, in the first stage, Well 1 in the MZ in Experiment 620 3-1 (finite aquifer) exhibited stable production for only 30 min. After 49 mins of production, the 621 abandonment production rate was reached, and the R of the single well was 41.4% (42.9% in total, 622 combined with the second stage). However, for the infinite aquifer in Experiment 4-1, due to the 623 high energy of the aquifer, the water invasion was faster and more serious, which led to a shortened 624 (only 23 mins) stable production period of Well 1 in the MZ. After 35 mins of production, the 625 abandonment production rate was met, and the single-well R was only 33.4% (35.4% in total 626 combined with the second stage). 627 Comparing the gas production performance, in the first stage, Well 1 in the MZ in Experiment 620 3-1 (finite aquifer) exhibited stable production for only 30 min. After 49 mins of production, the 621 abandonment production rate was reached, and the R of the single well was 41.4% (42.9% in total, 622 combined with the second stage). However, for the infinite aquifer in Experiment 4-1, due to the 623 high energy of the aquifer, the water invasion was faster and more serious, which led to a shortened 624 (only 23 mins) stable production period of Well 1 in the MZ. After 35 mins of production, the 625 abandonment production rate was met, and the single-well R was only 33.4% (35.4% in total 626 combined with the second stage). 627 620 In the second stage, the same drainage measures were utilized for the same drainage timing 628 used for Experiments 3-1 and 4-1. In Experiment 3-1, the finite aquifer drained from Well 2 with a 629 stable production for 14 mins with a single-well R of 37.6%. In Experiment 4-1, the water drainage 630 was fully developed in Well 2 for the infinite aquifer, and the average drainage rate was 12 times 631 larger than that of the finite aquifer (according to Table 12), so only 10 mins of stable production 632 were observed. The single-well R was only 29.1%, which was 8.5% lower than that of Well 2 for 633 Experiment 3-1. Impact of the Aquifer Size 609 As shown in Table 2, Experiments 4-1 and 4-2 and Experiment 3-1 were conducted using Geological 610 Model 2 under conditions of an infinite aquifer, no aquifer, and an aquifer 15 times greater than the 611 reservoir. In the early stage of Experiments 4-1 and 3-1, only distal Well 1 was produced. After 612 production in Well 1 was reduced to the abandonment production rate, Well 2, in the middle of the 613 LFZ, was initiated, and the production was combined with Well 1. Once both wells reached the 614 abandonment production rate, the production was stopped. Since Experiment 4-2 considered a 615 volumetric gas reservoir, it was developed only by Well 1. 616 As shown in Table 2, Experiments 4-1 and 4-2 and Experiment 3-1 were conducted using Geological 610 Model 2 under conditions of an infinite aquifer, no aquifer, and an aquifer 15 times greater than the 611 reservoir. In the early stage of Experiments 4-1 and 3-1, only distal Well 1 was produced. After 612 production in Well 1 was reduced to the abandonment production rate, Well 2, in the middle of the 613 LFZ, was initiated, and the production was combined with Well 1. Once both wells reached the 614 abandonment production rate, the production was stopped. Since Experiment 4-2 considered a 615 volumetric gas reservoir, it was developed only by Well 1. 616 As shown in Table 2, Experiments 4-1 and 4-2 and Experiment 3-1 were conducted using Geological 610 Model 2 under conditions of an infinite aquifer, no aquifer, and an aquifer 15 times greater than the 611 reservoir. In the early stage of Experiments 4-1 and 3-1, only distal Well 1 was produced. After 612 production in Well 1 was reduced to the abandonment production rate, Well 2, in the middle of the 613 LFZ, was initiated, and the production was combined with Well 1. Once both wells reached the 614 abandonment production rate, the production was stopped. Since Experiment 4-2 considered a 615 volumetric gas reservoir, it was developed only by Well 1. 616 The production of Experiments 3-1, 4-1 and 4-2 are shown in Fig. 19 and Fig. 20, and the production 618 parameters are shown in Table 12. 619 The production of Experiments 3-1, 4-1 and 4-2 are shown in Fig. 19 and Fig. 20, and the production 618 parameters are shown in Table 12. Impact of the Aquifer Size 609 The average Qw from the infinite aquifer was 5.1 mL/min, which was 653 approximately 12 times of that from the finite aquifer. The Wp was as high as 721.7 mL, which was 654 9.2 times that of the infinite aquifer. 655 production process was not depleted, the water production of drainage Well 2 rapidly increased and 650 then stabilized at approximately 5 mL/min, indicating that the water invasion rate and the drainage 651 rate reached an equilibrium state. Thereafter, Well 2 continued to produce water at this rate until the 652 end of production. The average Qw from the infinite aquifer was 5.1 mL/min, which was 653 approximately 12 times of that from the finite aquifer. The Wp was as high as 721.7 mL, which was 654 9.2 times that of the infinite aquifer. 655 production process was not depleted, the water production of drainage Well 2 rapidly increased and 650 then stabilized at approximately 5 mL/min, indicating that the water invasion rate and the drainage 651 rate reached an equilibrium state. Thereafter, Well 2 continued to produce water at this rate until the 652 end of production. The average Qw from the infinite aquifer was 5.1 mL/min, which was 653 approximately 12 times of that from the finite aquifer. The Wp was as high as 721.7 mL, which was 654 9.2 times that of the infinite aquifer. 655 In the experiments, drainage Well 2 could achieve rapid and large-scale drainage. After the 656 energy of the aquifer in the gas reservoir was released, it would not intrude toward the direction of 657 Well 1. By the end of production, Well 1 in the finite aquifer Experiment 3-1 produced almost no 658 water, and the infinite aquifer Experiment 4-1 also produced only 14.4 mL of water. 659 Fig. 21 shows the dynamic pressure profiles of Experiment 4-1 (Geometrical Model 2, infinite 661 aquifer). From the early stage of production of Well 1 until the stable production of the well ended, 662 the reserve development was mainly concentrated in the immediate vicinity of Well 1. The pressure 663 remained almost unchanged due to water invasion on the left side, far from the well area (Fig. 21(a)). 664 Well 2 produced a large amount of water, forming a pressure drop funnel around the bottom of LFZ 665 2 and the connected MZ 1. Impact of the Aquifer Size 609 As production progressed, the pressure drop funnel around Well 2 666 gradually expanded outward, allowing the undeveloped reserves of the area far from Well 1 (Cores 667 1-3) to be developed. Due to the infinite volume of the aquifer in this case, the residual pressure of 668 the formation remained high until the gas well was shut in. 669 Comparing the dynamic pressure drops of Experiments 3-1 and 4-1 (Fig. 18 and Fig. 21), it 670 can be seen that the aquifer size affects the R of the entire gas reservoir. At the end of the stable 671 production of Well 1 in the first stage of Experiments 3-1 and 4-1, the pressure drop funnels in the 672 near-well area (Core 4) were 0.75 MPa and 1.14 MPa, respectively, and the pressure gradient of the 673 infinite aquifer was clearly higher. Until the end of the water drainage process, the average residual 674 pressure of the infinite aquifer case remained higher than that of the finite aquifer case (Fig. 18(d) 675 and Fig. 21(d)). 676 Comparing the dynamic pressure drops of Experiments 3-1 and 4-1 (Fig. 18 and Fig. 21), it 670 can be seen that the aquifer size affects the R of the entire gas reservoir. At the end of the stable 671 production of Well 1 in the first stage of Experiments 3-1 and 4-1, the pressure drop funnels in the 672 near-well area (Core 4) were 0.75 MPa and 1.14 MPa, respectively, and the pressure gradient of the 673 infinite aquifer was clearly higher. Until the end of the water drainage process, the average residual 674 pressure of the infinite aquifer case remained higher than that of the finite aquifer case (Fig. 18(d) 675 and Fig. 21(d)). 676 Impact of the Aquifer Size 609 634 At the end of production, the cumulative gas recovery of the infinite aquifer case was 64.5%, 635 which was 15.95% less than that of the finite aquifer case. The single-well stable production period 636 of the volumetric gas reservoir in Experiment 4-2 was as long as 55 mins, which was more than 637 twice that in Experiment 3-1, and the R was over 98%. 638 The above experiments show that the aquifer size had a significant impact on gas recovery. For 639 the same geological and production conditions, the larger the aquifer was, the lower the R. Fang et 640 al. (2019) performed water invasion experiments with small fractured cores at different aquifer 641 scales. Their research conclusion is consistent with that from this work, which shows that this 642 conclusion may be universal for fractured gas reservoirs. 643 Comparing the water production performance, in Experiments 3-1 and 4-1, Well 2 was located 644 in the LFZ with high conductivity and directly connected with the aquifer. Therefore, after Well 2 645 was initiated, it could quickly produce gas and water. In the finite aquifer Experiment 3-1, after the 646 water production of drainage Well 2 increased sharply during the initial stage of the well startup, 647 the aquifer energy was gradually depleted. Meanwhile, the Qw gradually decreased to only 0.07 648 mL/min in the later stage. In infinite aquifer Experiment 4-1, since the aquifer’s energy in the 649 Comparing the water production performance, in Experiments 3-1 and 4-1, Well 2 was located 644 in the LFZ with high conductivity and directly connected with the aquifer. Therefore, after Well 2 645 was initiated, it could quickly produce gas and water. In the finite aquifer Experiment 3-1, after the 646 water production of drainage Well 2 increased sharply during the initial stage of the well startup, 647 the aquifer energy was gradually depleted. Meanwhile, the Qw gradually decreased to only 0.07 648 mL/min in the later stage. In infinite aquifer Experiment 4-1, since the aquifer’s energy in the 649 22 production process was not depleted, the water production of drainage Well 2 rapidly increased and 650 then stabilized at approximately 5 mL/min, indicating that the water invasion rate and the drainage 651 rate reached an equilibrium state. Thereafter, Well 2 continued to produce water at this rate until the 652 end of production. Conclusions 691 The following conclusions were drawn according to the above-mentioned work: 692 The following conclusions were drawn according to the above-mentioned work: 692 1. When the fracture scale is appropriate and the permeability of a matrix zone is low, a 693 production well should be deployed in an area that is close to the fractured zone. Although the 694 fractured zone may lead to a fast water breakthrough, it will also result in a higher capacity for gas 695 production and draining water, which can effectively avoid water invasion from occurring in the 696 low-permeability zone. 697 1. When the fracture scale is appropriate and the permeability of a matrix zone is low, a 693 production well should be deployed in an area that is close to the fractured zone. Although the 694 fractured zone may lead to a fast water breakthrough, it will also result in a higher capacity for gas 695 production and draining water, which can effectively avoid water invasion from occurring in the 696 low-permeability zone. 697 1. When the fracture scale is appropriate and the permeability of a matrix zone is low, a 693 production well should be deployed in an area that is close to the fractured zone. Although the 694 fractured zone may lead to a fast water breakthrough, it will also result in a higher capacity for gas 695 production and draining water, which can effectively avoid water invasion from occurring in the 696 low-permeability zone. 697 2. In different zones of a gas reservoir, the water invasion mechanisms will be different. In a 698 matrix area surrounded by fractures, the water saturation is considerably lower than that in the 699 peripheral fractured zones after production. It is confirmed that the main reason for the water 700 invasion in the matrix area is the imbibition caused by capillary forces. 701 2. In different zones of a gas reservoir, the water invasion mechanisms will be different. In a 698 matrix area surrounded by fractures, the water saturation is considerably lower than that in the 699 peripheral fractured zones after production. It is confirmed that the main reason for the water 700 invasion in the matrix area is the imbibition caused by capillary forces. 701 2. In different zones of a gas reservoir, the water invasion mechanisms will be different. Distribution of Residual Water and Reserves 677 After water drainage was conducted with the newly added Well 2 in Experiments 3-1 and 4-1, the 678 23 water saturations of the gas reservoirs were similar, at 31.22% and 34.01%, respectively. The 679 difference is mainly due to LFZ 2, where drainage Well 2 was located. In Experiment 4-1, the water 680 content in the area was as high as 46.51%, which was 7.65% higher than the same area of Experiment 681 3-1, indicating that the large fractures were the main channels of the water invasion (Table 13). 682 water saturations of the gas reservoirs were similar, at 31.22% and 34.01%, respectively. The 679 difference is mainly due to LFZ 2, where drainage Well 2 was located. In Experiment 4-1, the water 680 content in the area was as high as 46.51%, which was 7.65% higher than the same area of Experiment 681 3-1, indicating that the large fractures were the main channels of the water invasion (Table 13). 682 water saturations of the gas reservoirs were similar, at 31.22% and 34.01%, respectively. The 679 difference is mainly due to LFZ 2, where drainage Well 2 was located. In Experiment 4-1, the water 680 content in the area was as high as 46.51%, which was 7.65% higher than the same area of Experiment 681 3-1, indicating that the large fractures were the main channels of the water invasion (Table 13). 682 The proportions of the residual reserves in different zones of the gas reservoir after gas 683 production in the two types of aquifers of Experiments 3-1 and 4-1 are shown in Table 14. 684 In Experiments 3-1 and 4-1, compared with other zones, MZ 2 had the highest R of the 685 reservoir; conversely, SFZ 1 had the lowest R in the reservoir. Although MZ 2 had poor physical 686 properties and low permeability, it was farthest from the aquifer but nearest to Well 1, so it had the 687 highest R of the gas reservoir. However, although SFZ 1 had favorable physical properties, it had 688 the worst R because it was directly connected to the aquifer and was the farthest from Well 1 and 689 Well 2. 690 Conclusions 691 In a 698 matrix area surrounded by fractures, the water saturation is considerably lower than that in the 699 peripheral fractured zones after production. It is confirmed that the main reason for the water 700 invasion in the matrix area is the imbibition caused by capillary forces. 701 3. The function of a drainage well varies with its location. A shorter distance between the well 702 location and the aquifer induces a higher drainage capacity and a lower gas production capacity. 703 3. The function of a drainage well varies with its location. A shorter distance between the well 702 location and the aquifer induces a higher drainage capacity and a lower gas production capacity. 703 4. In the process of development, a gas reservoir will be seriously flooded due to untimely 704 drainage. If the remaining energy of this type of reservoir is insufficient, it is difficult to break the 705 gas reservoir seal, even if more gas wells are drilled later. 706 4. In the process of development, a gas reservoir will be seriously flooded due to untimely 704 drainage. If the remaining energy of this type of reservoir is insufficient, it is difficult to break the 705 gas reservoir seal, even if more gas wells are drilled later. 706 4. In the process of development, a gas reservoir will be seriously flooded due to untimely 704 drainage. If the remaining energy of this type of reservoir is insufficient, it is difficult to break the 705 gas reservoir seal, even if more gas wells are drilled later. 706 5. The larger an aquifer is, the lower its R , and the larger the water drainage required to 707 maintain gas production, resulting in a lower production benefit. 708 24 Nomenclature 709 Ak= Core cross-sectional area measured at each measuring point, cm2. 710 BW= Formation water volume coefficient. 711 Bgi= Original natural gas volumetric coefficient. 712 G= Natural gas reserves, 104m 3. 713 Gp= Cumulative gas production, 104m3. 714 Lk=Core length measured by each measuring point, cm. 715 P= Current formation pressure, MPa. 716 Pi=Initial reservoir pressure, MPa. 717 Pk= Average formation pressure of the core measured at each measuring point, MPa. 718 Prk= Residual pressure in the cores measured at each measuring point, MPa. 719 P̅= Average formation pressure of the gas reservoir, MPa. 720 R= recovery factor, fraction. 721 R= recovery factor, fraction. 721 Sgk= Residual gas saturation measured in the core, fraction. 722 We= Cumulative water influx, 104m3. 723 We= Cumulative water influx, 104m3. 723 Wp= Cumulative water production, 104m3. 724 Z= Natural gas deviation coefficient at pressure P. 725 Zi= Natural gas deviation coefficient at pressure Pi. 726 θ= Core porosity measured by each measuring point, fraction. 727 ω= Water invasion volume coefficient. 728 ∅k= Core porosity measured at each measuring point, fraction. 729 Subscripts 730 𝑘= property at each measuring point 731 𝑖= initial condition 732 Acknowledgment 733 Z= Natural gas deviation coefficient at pressure P. 725 Zi= Natural gas deviation coefficient at pressure Pi. 726 θ= Core porosity measured by each measuring point, fraction. 727 ω= Water invasion volume coefficient. 728 ∅k= Core porosity measured at each measuring point, fraction. 729 25 This work was supported by the National Natural Science Foundation of China [grant numbers 734 51704326]; and the Major Science and Technology Projects of CNPC [grant numbers 2016E-0607], 735 the contribution of which is gratefully acknowledged. 736 Abdul-Majeed, G. 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Effect of Anisotropy on Water Invasion in Edge-Water 776 Drive Reservoirs. Presented at the Canadian International Petroleum Conference, Calgary, Alberta, 13-15 June. 777 PETSOC-2006-199. https://doi.org/10.2118/2006-199. 778 Jiao, C. Y., Liu, H. X., Liu, P. F. et al. 2019. Nomenclature 709 Similarity Criterion of the Physical Simulating Experiment for the 779 Development Performances of Low-Permeability Tight Gas Reservoirs. Pet. Geology and Oilfield Development in 780 Daqing. 38(1): 155-161. https://doi.org/10. 19597/J. ISSN. 1000-3754. 2018.08.008. 781 Jin, L., and Wojtanowicz, A. K. 2010. Coning Control and Recovery Improvement Using In-Situ Water 782 Drainage/Injection in Bottom/Water/Drive Reservoir. Presented at the SPE Improved Oil Recovery Symposium, 783 Tulsa, Oklahoma, 24-28 April. SPE-129663-MS. https://doi.org/10.2118/129663-MS. 784 Kabir CS., Parekh B. and Mustafa MA. 2015. Material-balance Analysis of Gas Reservoirs with Diverse Drive 785 Mechanisms. Presented at the SPE Annual Technical Conference and Exhibition, Houston, Texas, 28-30 786 September. SPE-175005-MS. https://doi.org/10.2118/175005-MS. 787 Lakatos, I. J., Bodi, T. and Lakatos-Szabo, J. 2009. Water Induced Formation Damage in Unconventional Ga 788 Reservoirs. Presented at the 8th European Formation Damage Conference, Scheveningen, The Netherlan 789 at the SPE EUROPEC/EAGE Annual Conference and Exhibition, Vienna, Austria, 23-26 May. SPE-143483-MS. 792 28 28 Li, S., Pan Y., and Sun, L. 2011. Thoughts and Suggestions for the Improvement of the Efficiency and Development 794 Level of Complex Gas Fields. Nat. Gas Industry. 31(12): 76-80. https://doi.org/10.3787/j.issn.1000- 795 0976.2011.12.013. 796 Liu, H. X., Ren, D., Gao, S. S. et al. 2015. Water Influx Mechanism and Development Strategy of Gas Reservoirs with 797 Edge and Bottom Water. Nat. Gas Industry. 35 (2): 47-53 https://doi.org/10.3787/j.issn.1000-0976.2015.02.007. 798 Liu, S. Z., Sun, A. Y., Huang, B. G. et al. 1999. The Prediction Method of Water Influx and Formation Pressure for A 799 Water Drive Gas Reservoir. Pet. Exploration and Development. 6 (2): 79-81. https://doi.org/10.3321/j.issn:1000- 800 0747.1999.02.021. 801 Mattey, P., Varshney, M., Daksh, P. V. et al. 2017. A Successful Water Shut Off Treatment in Gas Well of Bassein 802 Field by Polymeric Gel System. Presented at the SPE Oil and Gas India Conference and Exhibition, Mumbai, 803 India, 4–6 April. SPE-185371-MS. https://doi.org/10.2118/185371-MS. 804 Rezaee, M., Rostami, B., Zadeh, M. et al. 2013. Experimental Determination of Optimised Production Rate and Its 805 Upscaling Analysis in Strong Water Drive Gas Reservoirs. Presented at the International Petroleum Technology 806 Conference, Beijing, China, 26-28 March. IPTC-16938-Abstract. https://doi.org/10.2523/IPTC-16938-Abstract. 807 Sait I, Özkaya. 2019. Fracture Modeling from Borehole Image Logs and Water Invasion in Carbonate Reservoirs with 808 Layer-Bound Fractures and Fracture Corridors. J. Pe. Sci. Eng. 179(April): 199-209. 809 https://doi.org/10.1016/j.petrol.2019.04.052. 810 Siddiqui, F., Waqas, G. M. and Khan, M. N. 2010. Application of General Material Balance on Gas Condensate 811 Reservoirs GIIP Estimation. Nomenclature 709 Effictiveness Analysis of Drainage Water in Well Chi 27 and Resolvent 826 of Boundary Water Invasion in Carboniferous Reservoirs in East SiChuan. Drilling and Production Technol. 26(s1): 827 60-65. https://doi.org/10.3969/j.issn.1006-768X.2003.z1.012. 828 Yang, G., Honglan, Z., He, L. et al. 2013. Technique of Water Control and Oil Recovery Based on Water Plugging 829 Combined with Fracturing in Low Permeability and High Water Cut Oilfield. Presented at the SPE Asia Pacific Oil 830 and Gas Conference and Exhibition, Jakarta, Indonesia, 22-24 October. SPE-165863-MS. 831 https://doi.org/10.2118/165863-MS. 832 Appendix A—Detail of the Water Invasion Volume Coefficient Method 833 Currently, the main methods of water invasion degree identification were developed based on the 834 gas reservoir material balance equation, so generally, the evaluation results of the three methods for 835 Xia, C. S. 2002. Ways and Methods of Enhancing Recovery in Various Water-Carrying Gas Reservoirs. Nat. Gas 816 Industry. 22(s1):73-77. https://doi.org/10.3321/j.issn:1000-0976.2002.z1.018. 817 Xu, G., Yin, H., Yuan, H. et al.2020. Decline curve analysis for multiple-fractured horizontal wells in tight oil 818 reservoirs. Adv. Geo-Energy Res. 4(3): 296-304. https://doi.org/10.46690/ager.2020.03.07. 819 Xu, X., Liu, X. W., Yang, Z. M. et al. 2012. An Experimental Study on Single-Phase Seepage Characteristics with a 820 Large-Scale Model Made of Ultra-Low Permeability Sandstone Outcrops. Acta Pet. Sinica. 33(3): 453-458. 821 60-65. https://doi.org/10.3969/j.issn.1006-768X.2003.z1.012. Yang, G., Honglan, Z., He, L. et al. 2013. Technique of Water Control and Oil Recovery Based on Water Plug 829 Combined with Fracturing in Low Permeability and High Water Cut Oilfield. Presented at the SPE Asia Pacific Oil 830 and Gas Conference and Exhibition, Jakarta, Indonesia, 22-24 October. SPE-165863-MS. 831 Nomenclature 709 Presented at the SPE/PAPG Annual Technical Conference, Islamabad, Pakistan, 10- 812 11 November. SPE-142847-MS. https://doi.org/10.2118/142847-MS. 813 Li, S., Pan Y., and Sun, L. 2011. Thoughts and Suggestions for the Improvement of the Efficiency and Development 794 Level of Complex Gas Fields. Nat. Gas Industry. 31(12): 76-80. https://doi.org/10.3787/j.issn.1000- 795 Liu, H. X., Ren, D., Gao, S. S. et al. 2015. Water Influx Mechanism and Development Strategy of Gas Reservoirs with 797 Edge and Bottom Water. Nat. Gas Industry. 35 (2): 47-53 https://doi.org/10.3787/j.issn.1000-0976.2015.02.007. Field by Polymeric Gel System. Presented at the SPE Oil and Gas India Conference and Exhibition, Mumb 803 India, 4–6 April. SPE-185371-MS. https://doi.org/10.2118/185371-MS. 804 Conference, Beijing, China, 26-28 March. IPTC-16938-Abstract. https://doi.org/10.2523/IPTC-16938-Abstra 807 Sait I, Özkaya. 2019. Fracture Modeling from Borehole Image Logs and Water Invasion in Carbonate Reservoirs with 808 Layer-Bound Fractures and Fracture Corridors. J. Pe. Sci. Eng. 179(April): 199-209. 809 Layer-Bound Fractures and Fracture Corridors. J. Pe. Sci. Eng. 179(April): 199-209. 809 https://doi.org/10.1016/j.petrol.2019.04.052. 810 Siddiqui, F., Waqas, G. M. and Khan, M. N. 2010. Application of General Material Balance on Gas Condensa 811 Reservoirs GIIP Estimation. Presented at the SPE/PAPG Annual Technical Conference, Islamabad, Pakistan, 10- 812 Reservoirs GIIP Estimation. Presented at the SPE/PAPG Annual Technical Conference, Islamabad, Pakistan, 10- 812 Reservoirs GIIP Estimation. Presented at the SPE/PAPG Annual Technical Conference, Islamabad, Pakistan, 10- 812 11 November. SPE-142847-MS. https://doi.org/10.2118/142847-MS. 813 11 November. SPE-142847-MS. https://doi.org/10.2118/142847-MS. 813 29 30 Warren JE. and Root PJ. 1963. The behavior of naturally fractured reservoirs. SPE J. 3(3): 245-255. SPE-426-PA. 814 https://doi.org/10.2118/426-PA. 815 Xia, C. S. 2002. Ways and Methods of Enhancing Recovery in Various Water-Carrying Gas Reservoirs. Nat. Gas 816 Industry. 22(s1):73-77. https://doi.org/10.3321/j.issn:1000-0976.2002.z1.018. 817 Xu, G., Yin, H., Yuan, H. et al.2020. Decline curve analysis for multiple-fractured horizontal wells in tight oil 818 reservoirs. Adv. Geo-Energy Res. 4(3): 296-304. https://doi.org/10.46690/ager.2020.03.07. 819 Xu, X., Liu, X. W., Yang, Z. M. et al. 2012. An Experimental Study on Single-Phase Seepage Characteristics with a 820 Large-Scale Model Made of Ultra-Low Permeability Sandstone Outcrops. Acta Pet. Sinica. 33(3): 453-458. 821 https://doi.org/10.7623/syxb201203016. 822 Xu, X., Wan, Y.J., Chen, Y. L. et al. 2019. Physical Simulation of Water Invasion and Water Control for the Fractured 823 Water-Bearing Gas Reservoirs. Nat. Gas Geoscience. 30(10):1508–1518. https://doi.org/10.11764/j.issn.1672- 824 1926.2019.10.014. 825 Yang, J. H., Shao, Y., Luo, Y. P. et al. 2003. Appendix A—Detail of the Water Invasion Volume Coefficient Method 3 Currently, the main methods of water invasion degree identification were developed based on the 834 gas reservoir material balance equation, so generally, the evaluation results of the three methods for 835 Currently, the main methods of water invasion degree identification were developed based on the 834 gas reservoir material balance equation, so generally, the evaluation results of the three methods for 835 30 the same production data are quite similar, as Siddiqui et al. (2010) noted in their work on the 836 application of the general material balance equation. 837 31 Two parameters, namely, the gas recovery and the relative apparent pressure of the formation, 838 are utilized in the water invasion volume coefficient method, which is equivalent to the normalized 839 pressure drop curve method. The water invasion volume coefficient method is more suitable for a 840 comparative analysis of water invasion with different gas reserves and formation pressures. 841 However, the water influx volume coefficient method requires more gas reservoir parameters, such 842 as the original gas reserves, compared to the other two methods. As such, the water invasion volume 843 coefficient method was utilized to analyze the water invasion and development performance of the 844 gas reservoir in the experiments. 845 The water invasion volume coefficient is defined as the ratio of the net water amount invading 846 into the gas-bearing area to the primary reserves of the gas reservoir, namely, 847 ω= We-WpBW GBgi . …………………………………………………………..……..………… (A-1) 848 The pressure drop equation of a normal pressure WDGR can also be expressed as 849 P Z = Pi Zi ( G-Gp G-ωG ). ……………….…………………………….…………….………………....(A-2) 850 The R of reserves is defined as 851 R= 𝐺𝑝 𝐺. ………………………….………………………………...…………………..…...(A-3) 852 The relative apparent pressure of formation is defined as 853 θ= P/Z Pi/Zi. ………………………………….……………………………………………....(A-4) 854 From the above relationship, 855 θ= 1-R 1-ω. …………………………….……………………………………………….……(A-5) 856 For non-water drive gas reservoirs, ω = 0, and Eq. (5) becomes 857 θ=1-R. ………………………………….………………………………………………...(A-6) 858 Eq. (5) shows that the angle between the θ~R curve and the longitudinal axis is greater than 859 45° for WDGRs since the water invasion volume coefficient ω < 1. In contrast, for volumetric gas 860 reservoirs, the angle between the θ~R curve and the longitudinal axis is 45°. 861 It is difficult to obtain an accurate average formation pressure during actual production. Appendix A—Detail of the Water Invasion Volume Coefficient Method 3 …………………………….………………….……………………..….…(A-7) 870 At the same time, due to the large pressure variation range in the gas reservoir during the gas 871 At the same time, due to the large pressure variation range in the gas reservoir during the gas 871 reservoir development process, the value of the nitrogen deviation factor under different pressures 872 should be considered when calculating the apparent formation pressure P/Z. 873 Compared with the pressure drawdown curve method, the water invasion volume coefficient 874 method requires a more accurate and reliable estimation of the initial in-place reserves of the gas 875 reservoirs, in addition to accurate average formation pressure, to calculate the R of the gas 876 reservoirs, posing additional challenges for gas reservoir developers. In the experiment, the 877 reservoir pore volume and the initial reservoir pressure are already known, so the primary reserves 878 can be accurately obtained. 879 Appendix B—Figures 880 881 Fig. 1—Schematic diagram and production history of Chi 27-39 well in the gas field in eastern Sichuan, China. 882 Appendix B—Figures 880 Modified according to Gou et al. (2002). 883 Appendix A—Detail of the Water Invasion Volume Coefficient Method 3 866 According to the experiments, the pressure and physical reservoir parameters of the different 867 zones of the gas reservoir are different, so the average formation pressure of the gas reservoir is 868 calculated by the weighted average values of the physical parameters related to the reserves: 869 P̅= ∑ pkLkAk∅k n k=1 ∑ LkAk∅k n k=1 . …………………………….………………….……………………..….…(A-7) 870 At the same time, due to the large pressure variation range in the gas reservoir during the gas 871 reservoir development process, the value of the nitrogen deviation factor under different pressures 872 should be considered when calculating the apparent formation pressure P/Z. 873 Compared with the pressure drawdown curve method, the water invasion volume coefficient 874 method requires a more accurate and reliable estimation of the initial in-place reserves of the gas 875 reservoirs, in addition to accurate average formation pressure, to calculate the R of the gas 876 reservoirs, posing additional challenges for gas reservoir developers. In the experiment, the 877 reservoir pore volume and the initial reservoir pressure are already known, so the primary reserves 878 can be accurately obtained. 879 formation pressure of the entire gas reservoir. This is beneficial to analyzing the degree of water 865 invasion in the gas reservoir. 866 formation pressure of the entire gas reservoir. This is beneficial to analyzing the degree of water 865 invasion in the gas reservoir. 866 formation pressure of the entire gas reservoir. This is beneficial to analyzing the degree of water 865 invasion in the gas reservoir. 866 According to the experiments, the pressure and physical reservoir parameters of the different 867 zones of the gas reservoir are different, so the average formation pressure of the gas reservoir is 868 calculated by the weighted average values of the physical parameters related to the reserves: 869 zones of the gas reservoir are different, so the average formation pressure of the gas reservoir is 868 calculated by the weighted average values of the physical parameters related to the reserves: 869 P̅= ∑ pkLkAk∅k n k=1 ∑ LkAk∅k n k=1 . …………………………….………………….……………………..….…(A-7) 870 At the same time, due to the large pressure variation range in the gas reservoir during the gas 871 i d l h l f h i d i i f d diff 872 P̅= ∑ pkLkAk∅k n k=1 ∑ LkAk∅k n k=1 . Appendix A—Detail of the Water Invasion Volume Coefficient Method 3 In the 862 experiments, the internal pressures of the gas reservoir are accurately measured in real time at more 863 than 20 measurement points, which provides a sound foundation for accurately obtaining the average 864 Two parameters, namely, the gas recovery and the relative apparent pressure of the formation, 838 are utilized in the water invasion volume coefficient method, which is equivalent to the normalized 839 pressure drop curve method. The water invasion volume coefficient method is more suitable for a 840 comparative analysis of water invasion with different gas reserves and formation pressures. 841 However, the water influx volume coefficient method requires more gas reservoir parameters, such 842 as the original gas reserves, compared to the other two methods. As such, the water invasion volume 843 coefficient method was utilized to analyze the water invasion and development performance of the 844 gas reservoir in the experiments. 845 The water invasion volume coefficient is defined as the ratio of the net water amount invading 846 into the gas-bearing area to the primary reserves of the gas reservoir, namely, 847 The water invasion volume coefficient is defined as the ratio of the net water amount invading 846 into the gas-bearing area to the primary reserves of the gas reservoir, namely, 847 (A-1) The R of reserves is defined as The relative apparent pressure of formation is defined as 853 For non-water drive gas reservoirs, ω = 0, and Eq. (5) becomes .(A-6) Eq. (5) shows that the angle between the θ~R curve and the longitudinal axis is greater than 859 45° for WDGRs since the water invasion volume coefficient ω < 1. In contrast, for volumetric gas 860 reservoirs, the angle between the θ~R curve and the longitudinal axis is 45°. 861 31 It is difficult to obtain an accurate average formation pressure during actual production. In the 862 experiments, the internal pressures of the gas reservoir are accurately measured in real time at more 863 than 20 measurement points, which provides a sound foundation for accurately obtaining the average 864 31 formation pressure of the entire gas reservoir. This is beneficial to analyzing the degree of water 865 invasion in the gas reservoir. Appendix B—Figures 880 Fig. 1—Schematic diagram and production history of Chi 27-39 well in the gas field in eastern Sichuan, China Fig. 1—Schematic diagram and production history of Chi 27-39 well in the gas field in eastern Sichuan, China. 882 32 884 Fig. 2—Multi-well joint water-control geological model for fractured water drive gas reservoirs (three geological 885 models) 886 884 Fig. 2—Multi-well joint water-control geological model for fractured water drive gas reservoirs (three geological 885 models). 886 884 Fig. 2—Multi-well joint water-control geological model for fractured water drive gas reservoirs (three geological 885 models). 886 887 Fig. 3—Schematic diagram of the joint water-control experiments (three geological models, Well 2 at points 888 A/B/C/D). 889 Fig. 2—Multi-well joint water-control geological model for fractured water drive gas reservoirs (three geological 885 Fig. 2—Multi-well joint water-control geological model for fractured water drive gas reservoirs (three geological 885 models) 886 models). 886 887 Fig. 3—Schematic diagram of the joint water-control experiments (three geological models, Well 2 at points 888 A/B/C/D). 889 890 Fig. 4—Joint water-control experimental setup for fractured water drive gas reservoirs. 891 887 Fig. 3—Schematic diagram of the joint water-control experiments (three geological models, Well 2 at points 888 A/B/C/D). 889 887 Fig. 3—Schematic diagram of the joint water-control experiments (three geological models, Well 2 at points 888 A/B/C/D). 889 887 Fig. 3—Schematic diagram of the joint water-control experiments (three geological models, Well 2 at points 888 A/B/C/D). 889 890 Fig. 4—Joint water-control experimental setup for fractured water drive gas reservoirs. 891 Fig. 3—Schematic diagram of the joint water-control experiments (three geological models, Well 2 at points A/B/C/D). Fig. 3—Schematic diagram of the joint water-control experiments (three geological models, Well 2 at points 888 A/B/C/D). 889 890 Fig. 4—Joint water-control experimental setup for fractured water drive gas reservoirs. 891 890 Fig. 4—Joint water-control experimental setup for fractured water drive gas reservoirs. 891 890 Fig. 4—Joint water-control experimental setup for fractured water drive gas reservoirs. 891 33 5—Production performance of the Group I : (a) Gas production, and (b) Water production of Experiment Fig. 5—Production performance of the Group I : (a) Gas production, and (b) Water production of Experiment 893 1-3. 894 895 Fig. 6—Relationship between relative apparent pressure of the formation and R. 896 895 Fig. 6—Relationship between relative apparent pressure of the formation and R. 896 Fig. 6—Relationship between relative apparent pressure of the formation and R. 896 34 897 Fig. Appendix B—Figures 880 7—Pressure distributions at different stages of Experiment 4-2: (a) Early stage of production – 10 mins, 898 (b) Early stage of production – 30 mins, (c) End of the stabilized period – 55 mins, and (d) Production halts – 899 88 mins. 900 Fig. 7—Pressure distributions at different stages of Experiment 4-2: (a) Early stage of production – 10 mins, 898 (b) Early stage of production – 30 mins, (c) End of the stabilized period – 55 mins, and (d) Production halts – 899 900 Fig. 7—Pressure distributions at different stages of Experiment 4-2: (a) Early stage of production – 10 mins Fig. 7—Pressure distributions at different stages of Experiment 4-2: (a) Early stage of production – 10 mins, 898 (b) Early stage of production – 30 mins, (c) End of the stabilized period – 55 mins, and (d) Production halts – 899 88 mins. 900 (b) Early stage of production – 30 mins, (c) End of the stabilized period – 55 mins, and (d) Production halts – 899 88 mins. 900 901 Fig. 8—Pressure distributions at different stages of Experiment 1-1: (a) Early stage of production – 10 mins, 902 (b) Early stage of production – 20 mins, (c) End of the stabilized period – 30 mins, and (d) Production halts -76 903 mins. 904 Fig. 8—Pressure distributions at different stages of Experiment 1-1: (a) Early stage of production – 10 mins, 902 (b) Early stage of production – 20 mins, (c) End of the stabilized period – 30 mins, and (d) Production halts -76 903 mins. 904 35 905 Fig. 9—Pressure distributions at different stages of Experiment 1-2: (a) Early stage of production – 10 mins, 906 (b) Early stage of production – 20 mins, (c) End of the stabilized period – 30 mins, and (d) Production halts -49 907 mins. 908 905 Fig. 9—Pressure distributions at different stages of Experiment 1-2: (a) Early stage of production – 10 mins Fig. 9—Pressure distributions at different stages of Experiment 1-2: (a) Early stage of production – 10 mins, 906 (b) Early stage of production – 20 mins, (c) End of the stabilized period – 30 mins, and (d) Production halts -49 907 mins. 908 909 Fig. Appendix B—Figures 880 10—Pressure distributions at different stages of Experiment 1-3: (a) Early stage of production – 10 mins, 910 (b) Early stage of production – 20 mins, (c) End of the stabilized period – 32 mins, and (d) Production halts - 911 115 mins. 912 Fig. 10—Pressure distributions at different stages of Experiment 1-3: (a) Early stage of production – 10 mins, 910 (b) Early stage of production – 20 mins, (c) End of the stabilized period – 32 mins, and (d) Production halts - 911 115 mins. 912 36 913 Fig. 11—Average pressure gradients at different times in the near-wellbore zone of the Group I. 914 915 Fig. 12—Gas production of two wells in Experiments 2-1 and 2-2: (a) Gas production of Well 1 located 916 913 Fig. 11—Average pressure gradients at different times in the near-wellbore zone of the Group I. 914 913 Fi 11 A di diff i i h llb f h G I 914 913 Fig. 11—Average pressure gradients at different times in the near-wellbore zone of the Group I. 914 915 Fig. 12—Gas production of two wells in Experiments 2-1 and 2-2: (a) Gas production of Well 1 located in the 916 MZ, and (b) Gas production of Well 2 located in the FZ. 917 Fig. 11—Average pressure gradients at different times in the near-wellbore zone of the Group I. 914 Fig. 12—Gas production of two wells in Experiments 2-1 and 2-2: (a) Gas production of Well 1 located in the Fig. 12—Gas production of two wells in Experiments 2-1 and 2-2: (a) Gas production of Well 1 located in the 916 MZ, and (b) Gas production of Well 2 located in the FZ. 917 Fig. 12—Gas production of two wells in Experiments 2-1 and 2-2: (a) Gas production of Well 1 located in the 916 MZ, and (b) Gas production of Well 2 located in the FZ. 917 37 37 918 Fig. 13—Water productions of Well 2 in Experiments 2-1 and 2-2. 919 920 Fig. 14—Relationship between relative apparent pressure of the formation and R: (a) Drainage Experiments 921 1 and 2-2 in the LFZ, and (b) Drainage Experiments 2-3 and 2-4 in the SFZ. 922 918 Fig. 13—Water productions of Well 2 in Experiments 2-1 and 2-2. 919 Fig. 13—Water productions of Well 2 in Experiments 2-1 and 2-2. 919 920 Fig. Appendix B—Figures 880 14—Relationship between relative apparent pressure of the formation and R: (a) Drainage Experiments 2- 921 1 and 2-2 in the LFZ, and (b) Drainage Experiments 2-3 and 2-4 in the SFZ. 922 920 920 Fig. 14—Relationship between relative apparent pressure of the formation and R: (a) Drainage Experiments 2- 921 1 and 2-2 in the LFZ, and (b) Drainage Experiments 2-3 and 2-4 in the SFZ. 922 Fig. 14—Relationship between relative apparent pressure of the formation and R: (a) Drainage Experiments 2- 921 1 and 2-2 in the LFZ, and (b) Drainage Experiments 2-3 and 2-4 in the SFZ. 922 38 923 Fig. 15—Pressure distributions at different stages of Experiment 2-1 (Well 2 is at Point A): (a) Produced for 10 924 mins, (b) Produced for 20 mins, (c) Produced for 30 mins, and (d) Gas reservoir production halted at 135 mins. 925 Fig. 15—Pressure distributions at different stages of Experiment 2-1 (Well 2 is at Point A): (a) Produced for 10 924 mins, (b) Produced for 20 mins, (c) Produced for 30 mins, and (d) Gas reservoir production halted at 135 mins. 925 ig. 15—Pressure distributions at different stages of Experiment 2-1 (Well 2 is at Point A): (a) Produced for 10 mins, (b) Produced for 20 mins, (c) Produced for 30 mins, and (d) Gas reservoir produc 925 mins, (b) Produced for 20 mins, (c) Produced for 30 mins, and (d) Gas reservoir production halted at 135 mins. 926 Fig. 16—Pressure distributions at different stages of Experiment 2-2 (Well 2 is at Point B): (a) Produced for 10 927 mins, (b) Produced for 20 mins, (c) Produced for 30min, and (d) Gas reservoir production halted at 105 mins. 928 Fig. 16—Pressure distributions at different stages of Experiment 2-2 (Well 2 is at Point B): (a) Produced for 10 927 mins, (b) Produced for 20 mins, (c) Produced for 30min, and (d) Gas reservoir production halted at 105 mins. 928 39 Production of Gas Well 2 in the second stage of Group III: (a) Gas production of the gas Well 2, and production of the gas Well 2. Fig. 17—Production of Gas Well 2 in the second stage of Group III: (a) Gas production of the gas Well 2, and 930 (b) Water production of the gas Well 2. 931 932 932 40 40 Fig. Appendix B—Figures 880 18—Pressure distributions at different times in the second stage of Experiment 3-1 (after drainage): (a) 933 Well 2 started up – 5 mins, (b) End of the stabilized period of Well 2 – 14 mins, (c) Late production period -40 934 mins, and (d) Gas well production halted – 184 mins. 935 Fig. 18—Pressure distributions at different times in the second stage of Experiment 3-1 (after drainage): (a) 933 Well 2 started up – 5 mins, (b) End of the stabilized period of Well 2 – 14 mins, (c) Late production period -40 934 mins, and (d) Gas well production halted – 184 mins. 935 Fig. 18—Pressure distributions at different times in the second stage of Experiment 3-1 (after drainage): (a) 933 Well 2 started up – 5 mins, (b) End of the stabilized period of Well 2 – 14 mins, (c) Late production period -40 934 mins, and (d) Gas well production halted – 184 mins. 935 936 Fig. 19—Gas production performance of Experiments 3-1 and 4-1: (a)Gas production of Gas Well 1 in the MZ, 937 and (b) Gas production of the Gas Well 2 at point A. 938 6 Fig. 19—Gas production performance of Experiments 3-1 and 4-1: (a)Gas production of Gas Well 1 in the MZ, 937 and (b) Gas production of the Gas Well 2 at point A. 938 41 939 Fig. 20—Water production of Gas Well 2 in the FZ of experiments 3-1 and 4-1. 940 941 Fig. 21—Pressure distributions at different stages of Experiment 4-1 (Model 2, infinite aquifer): (a) Stable 942 production of Well 1 ended -23 mins, (b) End of the stabilized period of Well 2 – 45 mins, (c) Late production 943 period -70 mins, and (d) Gas well production halted-142 mins. 944 939 939 Fig. 20—Water production of Gas Well 2 in the FZ of experiments 3-1 and 4-1. 940 Fig. 20—Water production of Gas Well 2 in the FZ of experiments 3-1 and 4-1. 940 941 941 Fig. 21—Pressure distributions at different stages of Experiment 4-1 (Model 2, infinite aquifer): (a) Stable 942 production of Well 1 ended -23 mins, (b) End of the stabilized period of Well 2 – 45 mins, (c) Late production 943 period -70 mins, and (d) Gas well production halted-142 mins. 944 945 Fig. Appendix B—Figures 880 21—Pressure distributions at different stages of Experiment 4-1 (Model 2, infinite aquifer): (a) Stable 942 production of Well 1 ended -23 mins, (b) End of the stabilized period of Well 2 – 45 mins, (c) Late production 943 period -70 mins, and (d) Gas well production halted-142 mins. 944 945 42 43 Appendix C—Tables 946 947 Core No. Reservoir types Length , cm Diameter , cm Porosity , % Permeability ,mD 1 Through-going fracture 50 2.52 5.42 9.68 2 Pore type 50 2.51 4.54 0.67 3 Through-going fracture 50 2.53 5.63 18.08 4 Pore type 50/25/0 2.51 5.91 2.54 Table 1—Basic physical parameters of the four groups of cores used in the experiments 948 949 Group No. Experimental No. Factors Experimental characteristics Location of Gas Well 2 Geological Model Aquifer I 1-1 Location relationship between gas wells and fracture zones Single well production in Gas Well 1 The distance from Well 1 to the fractured zones are different n/a Model 1 Finite aquifer (15 times) 1-2 n/a Model 2 1-3 n/a Model 3 II 2-1 Drainage position Gas Well 1 and 2 producing at the same time Well 2 is set at a different position of the fractured zone Middle of large fracture-A Model 1 Finite aquifer (15 times) 2-2 Near water end of large fracture-B Model 1 2-3 Middle of small fracture-C Model 1 2-4 Near water end of small fracture - D Model 1 III 3-1 Drainage timing Well 2 was started up immediately after production was stopped in Well 1 Middle of large fracture-A Model 2 Finite aquifer (15 times) Appendix C—Tables 946 Appendix C—Tables 946 947 Core No. Reservoir types Length , cm Diameter , cm Porosity , % Permeability ,mD 1 Through-going fracture 50 2.52 5.42 9.68 2 Pore type 50 2.51 4.54 0.67 3 Through-going fracture 50 2.53 5.63 18.08 4 Pore type 50/25/0 2.51 5.91 2.54 Table 1—Basic physical parameters of the four groups of cores used in the experiments 948 949 Table 1—Basic physical parameters of the four groups of cores used in the experiments Table 1—Basic physical parameters of the four groups of cores used in the experiments 948 949 Table 1 Basic physical parameters of the four groups of cores used in the experiments 48 49 Group No. Experimental No. Factors Experimental characteristics Location of Gas Well 2 Geological Model Aquifer I 1-1 Location relationship between gas wells and fracture zones Single well production in Gas Well 1 The distance from Well 1 to the fractured zones are different n/a Model 1 Finite aquifer (15 times) 1-2 n/a Model 2 1-3 n/a Model 3 II 2-1 Drainage position Gas Well 1 and 2 producing at the same time Well 2 is set at a different position of the fractured zone Middle of large fracture-A Model 1 Finite aquifer (15 times) 2-2 Near water end of large fracture-B Model 1 2-3 Middle of small fracture-C Model 1 2-4 Near water end of small fracture - D Model 1 III 3-1 Drainage timing Well 2 was started up immediately after production was stopped in Well 1 Middle of large fracture-A Model 2 Finite aquifer (15 times) 43 3-2 Well 2 was started up in 16 hours after production in Well 1 was stopped Middle of large fracture-A Model 2 3-3 Well 2 was started immediately after production was stopped in Well 1 Middle of large fracture-A Model 3 IV 4-1 Aquifer size Well 2 was started immediately after production was stopped in Well 1 Different Aquifer Middle of large fracture-A Model 2 Infinite aquifer 4-2 n/a Model 2 n/a V 5-1 Basic experiment No aquifer, only Well 1 was producing n/a Model 1 n/a 5-2 n/a Model 3 n/a Table 2—Contents and parameters of different groups of experiments 950 951 Experimental No. Appendix C—Tables 946 Length of Core 4, cm Stable production period, min R during stabilized period, % Production period, min R, % Breakthrough time, min Wp, ml 1-1 50 30 34.2 76 37.9 74 0.2 1-2 25 30 41.4 49 41.4 47 0.05 1-3 0 32 51.7 115 62.5 22 51.7 4-2 25 55 91.7 88 98.6 n/a n/a Table 3—Statistics on the production of the Group I and Experiment 4-2 952 953 Experimental No. SFZ 1 - Core 1 MZ 1 - Core 2 LFZ 2 - Core 3 MZ 2 - Core 4 Average in the Gas Reservoir 1-1 46.34 27.60 47.47 44.43 41.46 1-2 53.11 21.45 50.73 45.65 42.74 1-3 45.86 9.56 41.56 n/a 32.33 Table 4—Water saturation increment in different zones at the end of Group I (%) 954 955 3-2 Well 2 was started up in 16 hours after production in Well 1 was stopped Middle of large fracture-A Model 2 3-3 Well 2 was started immediately after production was stopped in Well 1 Middle of large fracture-A Model 3 IV 4-1 Aquifer size Well 2 was started immediately after production was stopped in Well 1 Different Aquifer Middle of large fracture-A Model 2 Infinite aquifer 4-2 n/a Model 2 n/a V 5-1 Basic experiment No aquifer, only Well 1 was producing n/a Model 1 n/a 5-2 n/a Model 3 n/a Table 2—Contents and parameters of different groups of experiments 950 3-2 Table 2—Contents and parameters of different groups of experiments 950 951 Table 3—Statistics on the production of the Group I and Experiment 4-2 952 953 Experimental No. SFZ 1 - Core 1 MZ 1 - Core 2 LFZ 2 - Core 3 MZ 2 - Core 4 Average in the Gas Reservoir 1-1 46.34 27.60 47.47 44.43 41.46 1-2 53.11 21.45 50.73 45.65 42.74 1-3 45.86 9.56 41.56 n/a 32.33 Table 4—Water saturation increment in different zones at the end of Group I (%) 954 44 Experimental No. SFZ 1 - Core 1 MZ 1 - Core 2 LFZ 2 - Core 3 MZ 2 - Core 4 1-1 27.93 32.08 28.51 11.48 1-2 26.93 38.05 29.56 5.46 1-3 29.48 39.69 30.83 n/a Table 5—Percentage of residual reserves at the end of Group I (%) 957 958 Experi mental No. Appendix C—Tables 946 Gas Well 1 in the distal MZ Gas Well 2 in the FZ Whole gas reservoir Stable production period, min Abando nment time, min Single well R, % Breakthrough time, min Stable production period, min Abandonment time, min Single well R, % Production period, min Cumulative water production, mL R, % 2-1 27 29 37.46 5 17 135 35.44 135 83.9 72.79 2-2 40 105 50.29 5 19 44 28.58 105 103.8 78.87 2-3 25 34 33.53 11 23 144 44.44 144 80.2 78.08 2-4 26 65 35.44 6 14 175 44.78 175 86.9 80.33 Table 6—Statistics on the production of Group II 959 960 Experimental No. SFZ 1 - Core 1 MZ 1 - Core 2 LFZ 2 - Core 3 MZ 2 - Core 4 Average water content 2-1 31.99 4.46 31.32 23.46 22.81 2-2 33.01 6.78 23.78 1.91 16.37 2-3 38.09 22.91 33.51 14.78 27.32 2-4 23.27 13.49 41.12 14.99 23.22 Table 7—Water saturation increment in different zones at the end of Group II (%) 961 962 Experimental SFZ 1 C 1 MZ 1 C 2 LFZ 2 C 3 MZ 2 C 4 Table 7—Water saturation increment in different zones at the end of Group II (%) 961 962 Experimental No. SFZ 1 - Core 1 MZ 1 - Core 2 LFZ 2 - Core 3 MZ 2 - Core 4 2-1 48.62 0.61 36.47 14.30 2-2 42.68 25.76 25.29 6.27 2-3 21.37 28.09 40.49 10.05 2-4 26.13 27.46 35.10 11.30 Table 8—Percentage of residual reserves in different zones at the end of Group II (%) 963 Table 8—Percentage of residual reserves in different zones at the end of Group II (%) 964 45 Experime ntal No. First stage R, % The Joint production of two wells in the second stage Cumulative R of the two stages, % Gas Well 2 in the FZ Matrix Well 1 Stable production period, min Abandonment time, min R,% Wp, mL Stable production period, min Abandonment time, min R,% Wp, mL 3-1 41.4 14 184 37.6 78.6 0 30 0.9 1.1 79.9 3-2 41.4 10 168 31.7 56.1 0 20 2.9 3.6 76.0 3-3 62.5 1 25 5.4 0.33 0 12 0.8 2.5 68.7 Table 9—Statistics on the production of Group III 965 966 Experimental No. Table 12—Statistics on the production of Group IV and Experiment 3-1 971 Appendix C—Tables 946 SFZ 1 - Core 1 MZ 1 - Core 2 LFZ 2 - Core 3 MZ 2 - Core 4 Average in the gas reservoir 3-1 37.42 19.74 38.86 28.87 31.22 3-2 40.11 17.74 36.02 41.20 33.77 3-3 45.33 8.05 38.99 n/a 30.79 Table 10—Water saturation increment in different zones at the end of Group III (%) 967 968 Experimental No. SFZ 1 - Core 1 MZ 1 - Core 2 LFZ 2 - Core 3 MZ 2 - Core 4 3-1 41.07 23.08 26.95 8.90 3-2 40.03 14.58 27.93 17.45 3-3 35.64 34.56 29.80 n/a Table 11—Distribution of residual reserves in different zones at the end of Group III (%) 969 970 Experim ental No. Gas Well 2 in the FZ Gas Well 1 in the distal MZ Cumulative R of the reservoir, % Stable production period, min Downtime , min Single well R, % Wp, mL Stable production period, min First abandonment time, min Downtime , min Single well R, % Wp,mL 3-1 14 184 37.6 78.6 30 49 184 42.9 1.1 80.45 4-1 10 142 29.1 721.7 23 35 142 35.4 14.4 64.50 4-2 Dry well 55 88 88 98.6 0 98.6 Table 12—Statistics on the production of Group IV and Experiment 3-1 971 972 The Joint production of two wells in the second stage 46 Experimental No. SFZ 1 - Core 1 MZ 1 - Core 2 LFZ 2 - Core 3 MZ 2 - Core 4 Average in the gas reservoir 3-1 37.42 19.74 38.86 28.87 31.22 4-1 35.16 19.67 46.51 34.68 34.01 Table 13—Water saturation increment (%) in different zones at the ends of Experiments 3-1 and 4-1 973 974 Experimental No. SFZ 1 - Core 1 MZ 1 - Core 2 LFZ 2 - Core 3 MZ 2 - Core 4 3-1 41.07 23.08 26.95 8.90 4-1 43.48 21.93 26.64 7.95 Table 14—Distribution of residual reserves (%) in different zones at the ends of Experiments 3-1 and 4-1 975 Table 14—Distribution of residual reserves (%) in different zones at the ends of Experiments 3-1 and 4-1 Xuan Xu is a senior engineer at PetroChina Research Institute of Petroleum Exploration & 977 Development. His research interests include Seepage mechanics, Gas reservoir 978 engineering and Petrophysics. He has authored or coauthored more than 30 technical 979 papers and holds 12 patents. Xuan Xu holds a Ph.D. Appendix C—Tables 946 in Fluid Mechanics from the Graduate 980 School of Chinese Academy of Sciences. 981 Xuan Xu is a senior engineer at PetroChina Research Institute of Petroleum Exploration & 977 Development. His research interests include Seepage mechanics, Gas reservoir 978 engineering and Petrophysics. He has authored or coauthored more than 30 technical 979 papers and holds 12 patents. Xuan Xu holds a Ph.D. in Fluid Mechanics from the Graduate 980 School of Chinese Academy of Sciences. 981 Xizhe Li is a senior engineer at PetroChina Research Institute of Petroleum Exploration & 982 Development. His research interests include Gas reservoir development and Petrophysics. 983 He has authored or coauthored more than 32 technical papers and holds 12 patents. Xizhe 984 Li holds a Ph.D. in Oil-Gas Field Development Engineering from University of Petroleum 985 (East China). 986 Yong Hu is a senior engineer at PetroChina Research Institute of Petroleum Exploration & 987 Development. His research interests include Gas reservoir development and Petrophysics. 988 He has authored or coauthored more than 31 technical papers and holds 13 patents. Yong 989 Hu holds a Ph.D. in Oil-Gas Field Development Engineering from Northeast Petroleum 990 University. 991 Yu Shi is an associate professor at Xi'an Shiyou University. His research interests include 992 gas reservoir development, phase behavior, and reservoir numerical simulation. He has 993 Yu Shi is an associate professor at Xi'an Shiyou University. His research interests include 992 gas reservoir development, phase behavior, and reservoir numerical simulation. He has 993 47 authored or coauthored more than 30 technical papers. Yu Shi holds a Ph.D. in Fluid 994 Mechanics from the Graduate School of Chinese Academy of Sciences and a Ph.D. in 995 Petroleum Systems Engineering from University of Regina, respectively. 996 Qingyan Mei is a senior engineer at the Research Institute of Southwest Oil & Gas Field 997 Company, PetroChina. Her research interests include gas reservoir development. She has 998 authored or coauthored more than 6 technical papers and holds 2 patents. Qingyan Mei 999 holds a Master's degree in Petroleum Engineering from Southwest Petroleum University. 1000 Chunyan Jiao is a senior engineer at PetroChina Research Institute of Petroleum 1001 Exploration & Development. Her research interests include Gas reservoir engineering and 1002 Petrophysics. She has authored or coauthored more than 26 technical papers and holds 1003 10 patents. Chunyan Jiao holds a Ph.D. in Oil-Gas Field Development Engineering from 1004 China University of Petroleum. Appendix C—Tables 946 1005 1006 authored or coauthored more than 30 technical papers. Yu Shi holds a Ph.D. in Fluid 994 Mechanics from the Graduate School of Chinese Academy of Sciences and a Ph.D. in 995 Petroleum Systems Engineering from University of Regina, respectively. 996 Qingyan Mei is a senior engineer at the Research Institute of Southwest Oil & Gas Field 997 Company, PetroChina. Her research interests include gas reservoir development. She has 998 authored or coauthored more than 6 technical papers and holds 2 patents. Qingyan Mei 999 holds a Master's degree in Petroleum Engineering from Southwest Petroleum University. 1000 Chunyan Jiao is a senior engineer at PetroChina Research Institute of Petroleum 1001 Exploration & Development. Her research interests include Gas reservoir engineering and 1002 Petrophysics. She has authored or coauthored more than 26 technical papers and holds 1003 10 patents. Chunyan Jiao holds a Ph.D. in Oil-Gas Field Development Engineering from 1004 China University of Petroleum. 1005 48 Figures Figure 1 Schematic diagram and production history of Chi 27-39 well in the gas ¦eld in eastern Sichuan, China. Modi¦ed according to Gou et al. (2002). Joint water-control experimental setup for fractured water drive gas reservoirs. Figure 1 Figure 1 Schematic diagram and production history of Chi 27-39 well in the gas ¦eld in eastern Sichuan, China. Modi¦ed according to Gou et al. (2002). Modi¦ed according to Gou et al. (2002). Figure 2 Multi-well joint water-control geological model for fractured water drive gas reservoirs (three geological models). Figure 3 Schematic diagram of the joint water-control experiments (three geological models, Well 2 at points A/B/C/D). Figure 3 Schematic diagram of the joint water-control experiments (three geological models, Well 2 at points A/B/C/D). Schematic diagram of the joint water-control experiments (three geological models, Well 2 at points Figure 4 Joint water-control experimental setup for fractured water drive gas reservoirs. Figure 4 Figure 4 Joint water-control experimental setup for fractured water drive gas reservoirs. Joint water-control experimental setup for fractured water drive gas reservoirs. gure 5 roduction performance of the Group I : (a) Gas production, and (b) Water production of Experiment 1-3 Figure 5 Production performance of the Group I : (a) Gas production, and (b) Water production of Experiment 1-3. Figure 5 Production performance of the Group I : (a) Gas production, and (b) Water production of Experiment 1-3. Production performance of the Group I : (a) Gas production, and (b) Water production of Experiment 1-3. Figure 6 Relationship between relative apparent pressure of the formation and "R". Figure 6 Relationship between relative apparent pressure of the formation and "R". Relationship between relative apparent pressure of the formation and "R". Figure 7 Pressure distributions at different stages of Experiment 4-2: (a) Early stage of production – 10 mins, (b) Early stage of production – 30 mins, (c) End of the stabilized period – 55 mins, and (d) Production halts – 88 mins. Figure 7 Figure 7 Pressure distributions at different stages of Experiment 4-2: (a) Early stage of production – 10 mins, (b) Early stage of production – 30 mins, (c) End of the stabilized period – 55 mins, and (d) Production halts – 88 mins. Figure 8 Pressure distributions at different stages of Experiment 1-1: (a) Early stage of production – 10 mins, (b) Early stage of production – 20 mins, (c) End of the stabilized period – 30 mins, and (d) Production halts -76 mins. Figure 8 Pressure distributions at different stages of Experiment 1-1: (a) Early stage of production – 10 mins, (b) Early stage of production – 20 mins, (c) End of the stabilized period – 30 mins, and (d) Production halts -76 mins. Figure 9 Pressure distributions at different stages of Experiment 1-2: (a) Early stage of production – 10 mins, (b) Early stage of production – 20 mins, (c) End of the stabilized period – 30 mins, and (d) Production halts -49 mins. Figure 9 Pressure distributions at different stages of Experiment 1-2: (a) Early stage of production – 10 mins, (b) Early stage of production – 20 mins, (c) End of the stabilized period – 30 mins, and (d) Production halts -49 mins. igure 10 ressure distributions at different stages of Experiment 1-3: (a) Early stage of production – 10 mins, (b) arly stage of production – 20 mins, (c) End of the stabilized period – 32 mins, and (d) Production halts 15 mins. re 10 Figure 10 Figure 10 Pressure distributions at different stages of Experiment 1-3: (a) Early stage of production – 10 mins, (b) Early stage of production – 20 mins, (c) End of the stabilized period – 32 mins, and (d) Production halts -115 mins. Pressure distributions at different stages of Experiment 1-3: (a) Early stage of production – 10 mins, (b) Early stage of production – 20 mins, (c) End of the stabilized period – 32 mins, and (d) Production halts -115 mins. Figure 11 Average pressure gradients at different times in the near-wellbore zone of the Group I. Figure 11 Figure 11 Average pressure gradients at different times in the near-wellbore zone of the Group I. gure 12 as production of two wells in Experiments 2-1 and 2-2: (a) Gas production of Well 1 locate nd (b) Gas production of Well 2 located in the FZ. Figure 12 Gas production of two wells in Experiments 2-1 and 2-2: (a) Gas production of Well 1 located in the MZ, and (b) Gas production of Well 2 located in the FZ. Figure 12 Gas production of two wells in Experiments 2-1 and 2-2: (a) Gas production of Well 1 located in the M and (b) Gas production of Well 2 located in the FZ. Figure 12 Gas production of two wells in Experiments 2-1 and 2-2: (a) Gas production of Well 1 located in the MZ, and (b) Gas production of Well 2 located in the FZ. Figure 13 Water productions of Well 2 in Experiments 2-1 and 2-2. Figure 13 Water productions of Well 2 in Experiments 2-1 and 2-2. Water productions of Well 2 in Experiments 2-1 and 2-2. re 14 tionship between relative apparent pressure of the formation and "R" : (a) Drainage Exper 2-2 in the LFZ, and (b) Drainage Experiments 2-3 and 2-4 in the SFZ. Figure 14 Relationship between relative apparent pressure of the formation and "R" : (a) Drainage Experiments 2-1 and 2-2 in the LFZ, and (b) Drainage Experiments 2-3 and 2-4 in the SFZ. Figure 14 Relationship between relative apparent pressure of the formation and "R" : (a) Drainage Experiments 2-1 and 2-2 in the LFZ, and (b) Drainage Experiments 2-3 and 2-4 in the SFZ. gure 15 ressure distributions at different stages of Experiment 2-1 (Well 2 is at Point A): (a) Produced for 10 ins, (b) Produced for 20 mins, (c) Produced for 30 mins, and (d) Gas reservoir production halted at 13 ins. Figure 15 Pressure distributions at different stages of Experiment 2-1 (Well 2 is at Point A): (a) Produced for 10 mins, (b) Produced for 20 mins, (c) Produced for 30 mins, and (d) Gas reservoir production halted at 135 mins Figure 15 Pressure distributions at different stages of Experiment 2-1 (Well 2 is at Point A): (a) Produced for 10 mins, (b) Produced for 20 mins, (c) Produced for 30 mins, and (d) Gas reservoir production halted at 135 mins. Figure 11 Pressure distributions at different stages of Experiment 2-1 (Well 2 is at Point A): (a) Produced for 10 mins, (b) Produced for 20 mins, (c) Produced for 30 mins, and (d) Gas reservoir production halted at 135 mins. Figure 16 Pressure distributions at different stages of Experiment 2-2 (Well 2 is at Point B): (a) Produced for 10 mins, (b) Produced for 20 mins, (c) Produced for 30min, and (d) Gas reservoir production halted at 105 mins. gure 16 Figure 16 Figure 16 Pressure distributions at different stages of Experiment 2-2 (Well 2 is at Point B): (a) Produced for 10 mins, (b) Produced for 20 mins, (c) Produced for 30min, and (d) Gas reservoir production halted at 105 mins. gure 17 roduction of Gas Well 2 in the second stage of Group III: (a) Gas production of the gas Well t d ti f th W ll 2 Figure 17 Production of Gas Well 2 in the second stage of Group III: (a) Gas production of the gas Well 2, and (b) Water production of the gas Well 2. Figure 17 Production of Gas Well 2 in the second stage of Group III: (a) Gas production of the gas Well 2, and (b) Water production of the gas Well 2. Figure 18 Pressure distributions at different times in the second stage of Experiment 3-1 (after drainage): (a) Well 2 started up – 5 mins, (b) End of the stabilized period of Well 2 – 14 mins, (c) Late production period -40 mins, and (d) Gas well production halted – 184 mins. Figure 18 Figure 18 Pressure distributions at different times in the second stage of Experiment 3-1 (after drainage): (a) Well 2 started up – 5 mins, (b) End of the stabilized period of Well 2 – 14 mins, (c) Late production period -40 mins, and (d) Gas well production halted – 184 mins. Pressure distributions at different times in the second stage of Experiment 3-1 (after drainage): (a) Well 2 started up – 5 mins, (b) End of the stabilized period of Well 2 – 14 mins, (c) Late production period -40 mins, and (d) Gas well production halted – 184 mins. gure 19 as production performance of Experiments 3-1 and 4-1: (a)Gas production of Gas Well 1 in Figure 19 Gas production performance of Experiments 3-1 and 4-1: (a)Gas production of Gas Well 1 in the MZ, and (b) Gas production of the Gas Well 2 at point A. Figure 19 Gas production performance of Experiments 3-1 and 4-1: (a)Gas production of Gas Well 1 in the MZ, and (b) Gas production of the Gas Well 2 at point A. Figure 20 Water production of Gas Well 2 in the FZ of experiments 3-1 and 4-1. Figure 20 Pressure distributions at different stages of Experiment 4-1 (Model 2, in¦nite aquifer): (a) Stable production of Well 1 ended -23 mins, (b) End of the stabilized period of Well 2 – 45 mins, (c) Late production period -70 mins, and (d) Gas well production halted-142 mins. Figure 20 Water production of Gas Well 2 in the FZ of experiments 3-1 and 4-1. Figure 21 Pressure distributions at different stages of Experiment 4-1 (Model 2, in¦nite aquifer): (a) Stable production of Well 1 ended -23 mins, (b) End of the stabilized period of Well 2 – 45 mins, (c) Late production period -70 mins, and (d) Gas well production halted-142 mins. Pressure distributions at different stages of Experiment 4-1 (Model 2, in¦nite aquifer): (a) Stable production of Well 1 ended -23 mins, (b) End of the stabilized period of Well 2 – 45 mins, (c) Late production period -70 mins, and (d) Gas well production halted-142 mins.
https://openalex.org/W4220985030	https://www.frontiersin.org/articles/10.3389/fphar.2022.863403/pdf	English	null	Screening of Bioactive Fraction of Radix Paeoniae Alba and Enhancing Anti-Allergic Asthma by Stir-Frying Through Regulating PI3K/AKT Signaling Pathway	Frontiers in pharmacology	2,022	cc-by	10,475	Abbreviations: BALF, bronchoalveolar lavage ﬂuid; BCA, bicinchoninic acid; DCM, dichloromethane fraction of RPA; EA, ethyl acetate fraction of RPA; ECL, enhanced chemiluminescence; FDCM, dichloromethane fraction of FRPA; FEA, ethyl acetate fraction of FRPA; Fn-BuOH, n-butanol fraction of FRPA; FPE, petroleum ether fraction of FRPA; FRPA, stir-frying Radix Paeoniae Alba with honey and bran; FW, water fraction of FRPA; H&E, hematoxylin and eosin staining; HD, DCM high-dose group; HFD, FDCM high-dose group; LD, DCM low-dose group; LFD, FDCM low-dose group; n-BuOH, n-butanol fraction of RPA; one-way ANOVA, one-way analysis of variance; OVA, ovalbumin; PBS, phosphate-buffered saline; PBST, phosphate-buffered saline with Tween-20; PE, petroleum ether fraction of RPA; PVDF, polyvinylidene ﬂuoride; RA, rheu- matoid arthritis; RPA, Radix Paeoniae Alba; SD, standard deviation; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; W, water fraction of RPA. ORIGINAL RESEARCH published: 31 March 2022 doi: 10.3389/fphar.2022.863403 Keywords: Radix Paeoniae Alba, stir frying Radix Paeoniae Alba, PI3K, AKT, allergic asthma Edited by: Jian Gao, Edited by: Jian Gao, Shanghai Children’s Medical Center, China Edited by: Jian Gao, Shanghai Children’s Medical Center, China Allergic asthma is a common respiratory inﬂammation disease. The crude Radix Paeoniae Alba (RPA) and its processed products have been used frequently as antipyretic and anti- inﬂammatory agents in traditional medicine. To evaluate the effect of honey and bran processing, different fractions of RPA were used for treating anti-allergic asthma in the ovalbumin (OVA)-induced mice model, and then, the most effective fraction of RPA and stir-frying Radix Paeoniae Alba with honey and bran (FRPA) for treating anti-allergic asthma were compared mutually for pharmacological effects. The results showed that the treatment of the dichloromethane fraction of RPA signiﬁcantly improved the pathological condition of lung tissues, decreased the number of eosinophils and other cells in bronchoalveolar lavage ﬂuid (BALF), and the increased the expression of various inﬂammatory factors. Furthermore, the study discovered that the lung pathological conditions, compared with the high dose of dichloromethane RPA fraction, could be ameliorated by high dose of dichloromethane FRPA fraction treatment. Moreover, the expression of inﬂammatory factors and the phosphorylation of the PI3K/AKT signaling pathway could be diminished by FRPA. Finally, the contents of compounds with a signiﬁcant difference in the FRPA dichloromethane fraction were paeoniﬂorin, ethyl gallate, pentagalloylglucose, galloylpaeoniﬂorin, and others by UPLC/Q-TOF-MS analysis. These ﬁndings suggest that the dichloromethane fraction of FRPA has an enhancement effect on anti-allergic asthma and provide the experimental basis for exploring the processed mechanism of RPA. Reviewed by: Beibei Cao, Zhejiang University, China Guanghai Yan, Yanbian University Medical College, China Pei Gang, Hunan University of Chinese Medicine, China Reviewed by: Beibei Cao, Zhejiang University, China Guanghai Yan, Yanbian University Medical College, China Pei Gang, Hunan University of Chinese Medicine, China Correspondence: Gang Cao caogang33@163.com †These authors have contributed equally to this work Correspondence: Gang Cao caogang33@163.com †These authors have contributed equally to this work Specialty section: This article was submitted to Inﬂammation Pharmacology, a section of the journal Frontiers in Pharmacology Received: 27 January 2022 Accepted: 07 March 2022 Published: 31 March 2022 Specialty section: This article was submitted to Inﬂammation Pharmacology, a section of the journal Frontiers in Pharmacology Received: 27 January 2022 Accepted: 07 March 2022 Published: 31 March 2022 Citation: Citation: Sang X, Ying J, Wan X, Han X, Shan Q, Lyu Q, Yang Q, Wang K, Hao M, Liu E and Cao G (2022) Screening of Bioactive Fraction of Radix Paeoniae Alba and Enhancing Anti-Allergic Asthma by Stir-Frying Through Regulating PI3K/AKT Signaling Pathway. Front. Pharmacol. 13:863403. doi: 10.3389/fphar.2022.863403 March 2022 \| Volume 13 \| Article 863403 1 Frontiers in Pharmacology \| www.frontiersin.org Processed RPA Enhances Anti-Allergic Asthma Sang et al. 2 MATERIALS AND METHODS 2.1 Preparation of Radix Paeoniae Alba and Fried Radix Paeoniae Alba Extracts RPA and FRPA were purchased from Zhejiang University of Chinese Medicine Chinese Herbal Pieces Co., Ltd. Hangzhou City, Zhejiang. The dried herbs (7 kg) were exhaustively extracted two times with 70% (v/v, 5 × 10 L) ethanol by reﬂuxing for 2 h. The supernatant was collected and concentrated under reduced pressure, using a rotatory evaporator, yielding crude RPA and FRPA extract. The extract of RPA was then dispersed in water and successively partitioned with 4 L of petroleum ether fraction (PE), dichloromethane fraction (DCM), ethyl acetate fraction (EA), n-butanol fraction (n-BuOH), and water fraction (W), respectively. The extraction time of each extractant was controlled at 3 h. In addition, the abbreviation of FRPA for each fraction is as follows FPE, FDCM, FEA, Fn-BuOH, and FW. Decompressing and concentrating the fractions of each fraction and the water fraction obtained the extract concretes of RPA and FRPA. The weight and yield of RPA and FRPA fractions are shown in Figure 1A. ) p ( g ) Modern research indicates that monoterpenes, ﬂavonoids, tannins, triterpenes, and polysaccharides are the main components of RPA related to the pharmacological activities of Paeoniae Radix (Ye et al., 2020; Gu et al., 2021). Monoterpene glycosides as the major characteristic compounds of RPA display a wide pharmacological effect, including anti-inﬂammatory and antitumor activities. Total glucosides of peony were isolated from RPA, which had been approved for treating rheumatoid arthritis (RA) by the State Food and Drug Administration of China. Galloylglucose is an important compound with signiﬁcant biomedical beneﬁts, such as being a free radical sink, an anti- inﬂammatory agent, anti-diabetic agent, and enzymatic resistant properties (Patnaik et al., 2019). Paeonol is one of the main polyphenolic compounds in RPA, and its injection has been successfully applied in China for nearly 50 years for inﬂammation/pain-related indications (Zhang et al., 2019). The extensive biological activities made RPA a therapeutic agent to be widely used in clinical therapy. Modern pharmacological studies show that RPA has been suggested to play an important role in the treatment of asthma (Wang et al., 2021). 1 INTRODUCTION regulators, such as half-skin acid leukotriene 1 receptor blockers and 5-lipoxygenase inhibitors, are widely used to treat allergic asthma (Hamid et al., 2003; Han and Wang, 2021). Radix Paeoniae Alba (RPA), the dried root of Paeonia lactiﬂora Pall, is a well-known natural medicine for more than 1,200 years in China. Processing of RPA is a pharmaceutical technique that transforms medicinal raw materials into decoction pieces for use in clinical application. There are many processing methods of RPA, including wine-made RPA, FRPA, stir-frying RPA, and wine–bran-made RPA. In the long-term clinical use, FRPA can relieve pain, strengthen the spleen, and soften the liver. Over the past few decades, there has been a rapid growth in the information available on the pharmacological activities of RPA. Studies have shown that RPA displays a wide spectrum of pharmacological effects, including anti-inﬂammation, pain easing, liver protection, and anti-oxidation (Ye et al., 2020; Gu et al., 2021). These pharmacological effects laid the foundation for RPA being a potential therapeutic agent for the treatment of several diseases, such as MPTP-induced experimental parkinsonism (Zhang et al., 2019), atopic dermatitis (Jo et al., 2018), asthma (Wang et al., 2021), depression (Zhang et al., 2020), and hepatic disorders (Wang et al., 2012). RPA is already widely available for the management of respiratory conditions, mainly regarding inﬂammation and immune function symptoms. In this study, the research aimed to evaluate the effects of each fraction from RPA for anti-allergic asthma by using the OVA-sensitized mice model and further investigated whether the most active fraction from processed products possesses the enhancement of the pharmacological effects after stir-frying with honey and bran. At the same time, the inﬂammatory factors and signaling pathways of the processed products increased pharmacological actions, and the comparison about the contents of the main components in the active fraction from RPA and FRPA for anti-allergic asthma were studied. Frontiers in Pharmacology \| www.frontiersin.org 2.2 Establishment and Drug Treatment of Allergic Asthma Model in Mice Asthma is a common heterogenic disease caused by chronic inﬂammation in the lower respiratory tract. Its characteristics include variability of airway obstruction and high reactivity of the bronchus. Asthma often occurs in children younger than 10 years, and the prevalence rate is about 10%. Patients with asthma are characterized by repeated cough, wheezing, shortness of breath, chest tightness, etc., and lung cancer, which affects tens of thousands of people all over the world, has a great correlation with asthma (Mims, 2015; Qu et al., 2017). As one of the most common asthma types, allergic asthma has airway inﬂammation and changing choroidal structure manifestation (Yılmaz et al., 2021), whose sensitization mainly affects mast cells and histamine, trypsin, etc., which are treated by a variety of therapies, such as inhaled corticosteroids, budesonide, and ﬂuticasone in the clinic. In addition, β2 receptor agonists, such as salbutamol, pirbuterol, and formoterol, and leukotriene g All animal protocols involved in this experiment were approved by the Laboratory Animal Research Center of Zhejiang Chinese Medicine University, and the experiments were conducted in strict accordance with the “Guide for the Care and Use of Laboratory Animals” issued by the U.S. National Institute of Health. Female BALB/c mice were purchased from SLAC Laboratory Animal Co., Ltd. (Shanghai, China). OVA was a common allergen, which was often used to establish an allergic asthma model (Chen et al., 2021). The overview of animal experiments is given in Figure 1B. The research established the OVA model for the ﬁrst batch; sixty female mice, six to 8 weeks old and weighing about 22 g, were sensitized with an OVA prescription containing 0.2 mg/ml OVA in an aluminum hydroxide gel, and 0.2 ml suspension was intraperitoneally injected into the abdominal cavity at day 1 and day March 2022 \| Volume 13 \| Article 863403 Frontiers in Pharmacology \| www.frontiersin.org 2 Processed RPA Enhances Anti-Allergic Asthma Sang et al. FIGURE 1 \| Weight, yield and outline of experimental design. (A) Weight and yield of RPA and FRPA fraction; the small letters of pe, dcm, ea, n-BuOH, and w are short forms for petroleum ether, dichloromethane, ethyl acetate, n-butanol, and water fraction, respectively. (B) Outline of experimental design for establishing the OVA- sensitized allergic asthma model, and an illustration of critical points during the experimental timeline. FIGURE 1 \| Weight, yield and outline of experimental design. 2.4 Serum Cytokine Assay by Enzyme-Linked Immunosorbent Assay The research established the OVA model for the second batch to further study; sixty female mice were used in this batch, and ﬁfty sensitized female mice were divided into ﬁve groups, with ten mice per group: OVA-sensitized model, high DCM dose (HD, 57.33 mg/kg), low DCM dose (LD, 28.67 mg/kg), high FDCM dose (HFD, 127.34 mg/kg), and low FDCM dose (LFD, 63.67 mg/kg). The remaining methods of the establishment of the OVA model were the same as the ﬁrst batch. y y According to the manufacturer’s instructions, the quantiﬁcation analysis included IL-17, IL-4, and IgE, which were monitored using the mouse enzyme-linked immunosorbent assay kit (MEIMIAN). The determination results were substituted into the regression equation of the standard curve and ﬁnally multiplied by the dilution multiple to obtain the contents of each fraction. 2.2 Establishment and Drug Treatment of Allergic Asthma Model in Mice (A) Weight and yield of RPA and FRPA fraction; the small letters of pe, dcm, ea, n-BuOH, and w are short forms for petroleum ether, dichloromethane, ethyl acetate, n-butanol, and water fraction, respectively. (B) Outline of experimental design for establishing the OVA- sensitized allergic asthma model, and an illustration of critical points during the experimental timeline. 0.4 ml phosphate-buffered saline (PBS). Then 1% glacial acetic acid was used to dilute the aliquot of the lavage ﬂuid samples, and the microscope with cell counting slides was used to determine the total leukocyte numbers. To research further, the BALF samples were loaded onto the slides and dried to stain with the Wright–Giemsa stain. Finally, the optical microscope was used to count the number of total monocytes, eosinophils, and neutrophils at high magniﬁcation. 14. For seven uninterrupted days from day 28 to day 34, the sixty sensitized female mice were divided into six groups, with ten mice per group: OVA-sensitized model, PE treatment (6.84 mg/kg), DCM treatment (54.72 mg/kg), EA treatment (20.06 mg/kg), n-BuOH treatment (104.88 mg/kg), and W treatment (1,550.40 mg/kg). Each group, except the OVA-sensitized model group, received the treatment, respectively, which was administered by using the oral gavage method with each fraction from RPA. Then, sixty mice were allowed to inhale 5% atomized OVA for 30 min in an exposure cage. In addition, another ten non-sensitized female BALB/c mice were assigned to the normal control group. After 7 days, the relevant index was observed. 2.5 Pathological Analysis of Lung Tissue in Mice 2.3 Collection and Analysis of Mouse Bronchoalveolar Lavage Fluid and Blood The mice’s blood was taken from the heart after anesthesia and then the blood was centrifuged at 3,000 rpm for 10 min to gain serum samples. After blood collection, the mice were dissected, the mice lung was accurately found, and the right lung was ligated. The left lung was lavaged three times with Mice The mice lung was removed by dissection and ﬁxed in 4% paraformaldehyde. Then parafﬁn was used to embed the lung tissues, and they were cut into sections. Next, the mice tissue sections were stained with hematoxylin and eosin (H&E) to analyze histopathology. After staining, the optical microscope The mice’s blood was taken from the heart after anesthesia and then the blood was centrifuged at 3,000 rpm for 10 min to gain serum samples. After blood collection, the mice were dissected, the mice lung was accurately found, and the right lung was ligated. The left lung was lavaged three times with March 2022 \| Volume 13 \| Article 863403 Frontiers in Pharmacology \| www.frontiersin.org 3 Processed RPA Enhances Anti-Allergic Asthma Sang et al. was used to analyze the degree of inﬂammatory inﬁltration in the tissue sections. standards and the compounds reported in the references. Finally, the research summarized some compounds with signiﬁcantly different contents after stir-frying with honey and bran. was used to analyze the degree of inﬂammatory inﬁltration in the tissue sections. standards and the compounds reported in the references. Finally, the research summarized some compounds with signiﬁcantly different contents after stir-frying with honey and bran. The elution conditions were as follows: ACQUITY UPLC®BEH C18 column (2.1 mm × 50 mm, 1.7 μm); the mobile phase: A was 0.1% formic acid water, and B was acetonitrile; The ﬂow rate was 0.3 ml/min, and the injection volume was 1 μl: column temperature: 30°C; gradient elution: 0→0.6 min, 95% A; 0.6→0.8 min, 95%→89% A; 0.8→2.0 min, 89% A; 2.0→4.0 min, 89%→82% A; 4.0→4.5 min, 82%→81% A; 4.5→7.0 min, 81% A; 7.0→8.5 min, 81%→5% A; 8.5→9.0 min, 5% A; 9.0→9.5 min, 5%→95% A; 9.5→12.0 min, 95% A. The elution conditions were as follows: ACQUITY UPLC®BEH C18 column (2.1 mm × 50 mm, 1.7 μm); the mobile phase: A was 0.1% formic acid water, and B was acetonitrile; The ﬂow rate was 0.3 ml/min, and the injection volume was 1 μl: column temperature: 30°C; gradient elution: 0→0.6 min, 95% A; 0.6→0.8 min, 95%→89% A; 0.8→2.0 min, 89% A; 2.0→4.0 min, 89%→82% A; 4.0→4.5 min, 82%→81% A; 4.5→7.0 min, 81% A; 7.0→8.5 min, 81%→5% A; 8.5→9.0 min, 5% A; 9.0→9.5 min, 5%→95% A; 9.5→12.0 min, 95% A. Mice 2.6 Western Blotting Explored the Regulation of Inﬂammatory Factors and Signaling Pathway in Fried Radix Paeoniae Alba That Enhances Anti-Allergic Asthma In this research, the lung tissues were lysed with RIPA Lysis Buffer and used protein extraction reagent to extract the total protein of tissues. Then, the homogenates were centrifuged at 12,000 rpm for 10 min at 4°C, the supernatants were collected, and a bicinchoninic acid (BCA) protein assay was used to measure the protein concentration. The proteins were separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and 5% concentrated glue, and the proteins were transferred onto polyvinylidene ﬂuoride (PVDF) membranes at appropriate electric current and time. Then, the PVDF membranes were blocked in 5% skim milk for 2 h at about 20°C. After blocking, the membranes were washed with phosphate-buffered saline with Tween-20 (PBST) for 30 min and then incubated with appropriate primary antibodies overnight on the shaking bed of 4°C refrigerators. The primary antibodies used were as follows: IL-1β, IL-6, p-PI3K, PI3K, p-AKT, AKT, and GAPDH. The next day, the membranes were washed with PBST and incubated with a 1:10,000–1: 5,000 dilution of appropriate secondary antibody for 1–2 h on a shaker at 20°C. After incubating, the membranes were washed with PBST three times, and then the enhanced chemiluminescence (ECL) was used to visualize the proteins of membranes under the Western blotting detection system. The protein bands were analyzed using ImageJ. 2.9 Statistical Analysis All experimental data were analyzed by GraphPad Prism 8.0.2 (GraphPad InStat Software, San Diego, CA, United States). One- way analysis of variance (one-way ANOVA) or t-test was used to compare each group. All experimental data were expressed by mean ± standard deviation (SD). Meanwhile, the value of p < 0.05 was statistically signiﬁcant. 3.1 Effects of Each Fraction in Radix Paeoniae Alba on the Total Number of Monocytes and the Number of Eosinophils and Neutrophils in Bronchoalveolar Lavage Fluid In this section, the effects of each fraction of RPA on the total number of monocytes, the number of eosinophils, and neutrophils in the BALF of OVA-sensitized mice were determined in the experiment. The results of the Wright–Giemsa staining are shown in Figure 2A. The experimental results of cell count are shown in Figure 2B: the BALF cell counts were compared with the control, the BALF total number of monocytes, the number of eosinophils, and neutrophils were signiﬁcantly increased in the OVA-sensitized allergic asthma model group (p < 0.001), and the number of eosinophils indicated that the OVA-sensitized mice in allergic asthma experiment were successful. The mice treated with DCM, EA, and W had signiﬁcantly lower BALF total number of monocytes, the number of eosinophils, and neutrophils (p < 0.001) than the OVA-induced allergic asthma model group, especially DCM. These experimental results indicated that DCM was the most active fraction in RPA to anti-allergic asthma. 2.7 Immunoﬂuorescence Analysis of Fried Radix Paeoniae Alba Lung Tissue Sections The lung tissue was cut to a thickness of 5 µm by using a cryomicrotome. The sections were subsequently blocked with 5% goat serum for 30 min and incubated overnight with the p-AKT (1: 500 dilution) antibody at 4°C. The next day, after washing three times with PBS, the sections were incubated with appropriate secondary antibodies (1:500 dilution) at room temperature for 2 h. Then, the sections were washed three times with PBS and ﬁnally mounted with UltraCruz mounting media containing DAPI. The confocal ﬂuorescence microscope was used in this experiment. 3.2 Effect of Each Fraction in Radix Paeoniae Alba on Lung Tissue Pathology in OVA-Sensitized Allergic Asthma g After H&E staining, the pathological sections of mice were observed by using an optical microscope. The pathological section results of each fraction in RPA are shown in Figure 3A. The results of the pathological sections showed that the alveolar size of the mice in the control was normal, and there was no signiﬁcant inﬁltration of inﬂammatory cells. However, in the OVA-sensitized model group, The samples of the most active fraction of anti-allergic asthma in RPA and FRPA would be analyzed by UPLC/Q-TOF-MS to detect the liquid chromatograms of the characteristic peaks. Meanwhile, the components and contents of the compounds were identiﬁed by UPLC/MS software and the supports of the Pharmacopoeia March 2022 \| Volume 13 \| Article 863403 Frontiers in Pharmacology \| www.frontiersin.org 4 Processed RPA Enhances Anti-Allergic Asthma Sang et al. Sang et al. FIGURE 2 \| Wright–Giemsa staining and cell count results. (A) Results of Wright–Giemsa staining by using a microscope. Scale bar, 100 μm. (B) Total number of monocytes, eosinophils, and neutrophils in BALF of each fraction in RPA (n = 5). The data were analyzed using one-way ANOVA and are presented as the mean ± SD. p < 0.05 was statistically signiﬁcant. Compared with control, p < 0.05, p < 0.01, p < 0.001; #compared with model, #p < 0.05, ##p < 0.01, ###p < 0.001, ns, not signiﬁcant. FIGURE 2 \| Wright–Giemsa staining and cell count results. (A) Results of Wright–Giemsa staining by using a microscope. Scale bar, 100 μm. (B) Total number of monocytes, eosinophils, and neutrophils in BALF of each fraction in RPA (n = 5). The data were analyzed using one-way ANOVA and are presented as the mean ± SD. p < 0.05 was statistically signiﬁcant. Compared with control, p < 0.05, p < 0.01, p < 0.001; #compared with model, #p < 0.05, ##p < 0.01, ###p < 0.001, ns, not signiﬁcant. increased mildly in the serum (p < 0.01), whereas in OVA- sensitized mice fed different fractions of each fraction from RPA, IL-17 was differently diminished in the serum, and DCM had a vigorous reducing effect (p < 0.001). the bronchus of the lung tissue was inﬁltrated deeply by inﬂammatory cells and could be seen that the alveolar area was signiﬁcantly enlarged, etc. 3.2 Effect of Each Fraction in Radix Paeoniae Alba on Lung Tissue Pathology in OVA-Sensitized Allergic Asthma The administration of each fraction from RPA had different degrees of improvement about the inﬂammation in lung tissue, which included the degree of inﬂammatory inﬁltration by inﬂammatory cells, and the degree of moderation about the pathological structure of lung tissue. The results of the asthma inﬂammation score are shown in Figure 3B, the scores of the control were about 1 point, and the OVA-sensitized model group was about 4 points. The lower scores the group got, the better improvement it had. The experimental results showed that DCM and EA had a good improvement effect (p < 0.001), but DCM was better. The results in this section veriﬁed that DCM was the most active fraction in RPA to anti-allergic asthma once again. IL-4 was produced by CD4 T cells stimulated by antigen or mitogen, which was closely related to the occurrence of allergic asthma. The OVA-sensitized mice had obviously enhanced IL-4 levels in the serum (p < 0.001). In OVA-sensitized mice treated with different fractions from RPA, there were varying degrees of the signiﬁcantly decreased IL-4 levels in the serum, and DCM played the best inhibition effect compared with other treatment groups (p < 0.001). IgE was produced by the plasma cells in the lamina propria of the respiratory and digestive tract mucosa and was one of the immune features that produce allergic asthma. After OVA- sensitized, the IgE levels in the serum were slightly increased compared with the control (p < 0.01), whereas in the OVA- sensitized mice gaining each fraction from RPA treatment, to a certain extent, only DCM could reduce IgE levels (p < 0.05). 3.4 Effects of DCM and FDCM on the Total Number of Monocytes and the Number of Eosinophils and Neutrophils in Bronchoalveolar Lavage Fluid HD, HFD, and LFD had signiﬁcantly a lower BALF total number of monocytes, the number of eosinophils, and neutrophils (p < 0.001) than the OVA-sensitized allergic asthma model group, especially HFD. These experimental results indicated that the dichloromethane fraction from FRPA had a stronger effect on anti-allergic asthma than from RPA. g In the study of screening the most active fraction from RPA for anti-allergic asthma, the results conﬁrmed that the most active fraction of RPA of anti-allergic asthma was DCM. And in this section, the experiments of the effects of the high and low doses from DCM (HD, LD) and FDCM (HFD, LFD) on the total number of monocytes and the number of eosinophils and neutrophils in BALF of OVA-sensitized mice were determined to explore the enhancement for anti-allergic asthma effects by FDCM. The results of the Wright–Giemsa staining are shown in Figure 4A. The experimental results of cell count are shown in Figure 4B: the BALF cell counts were compared with the control, the BALF total number of monocytes, the number of eosinophils, and neutrophils were sharply increased in the OVA-induced model group (p < 0.001), and the number of eosinophils manifested that the OVA-sensitized mice in the allergic asthma experiment were successful. The mice treated with 3.3 Effect of Each Fraction in Radix Paeoniae Alba on the Expression Levels of IL-17, IL-4, and IgE in OVA-Sensitized Allergic Asthma The cytokine expression levels of IL-17, IL-4, and IgE are shown in Figure 3C. These results also proved that DCM was the most active fraction in the treatment of anti-allergic asthma in RPA. The occurrence of allergic asthma was related to the mediation of IL-17. In OVA-sensitized mice without any treatment, IL-17 was March 2022 \| Volume 13 \| Article 863403 Frontiers in Pharmacology \| www.frontiersin.org 5 Processed RPA Enhances Anti-Allergic Asthma Sang et al. FIGURE 3 \| H&E staining, asthma inﬂammation score and ELISA results. (A) Pathological changes of mice lung tissue treated with each fraction from RPA. H&E staining was used to stain lung sections (n = 5). Scale bar, 100 μm. (B) The results of asthma inﬂammation score about each fraction from RPA. (C) Expression levels of cytokines in the serum (n = 3). The data were analyzed by using one-way ANOVA and are presented as mean ± SD. p < 0.05 was statistically signiﬁcant. Compared with control, p < 0.05, p < 0.01, p < 0.001; #compared with model, #p < 0.05, ##p < 0.01, ###p < 0.001, ns, not signiﬁcant. FIGURE 3 \| H&E staining, asthma inﬂammation score and ELISA results. (A) Pathological changes of mice lung tissue treated with each fraction from RPA. H&E staining was used to stain lung sections (n = 5). Scale bar, 100 μm. (B) The results of asthma inﬂammation score about each fraction from RPA. (C) Expression levels of FIGURE 3 \| H&E staining, asthma inﬂammation score and ELISA results. (A) Pathological changes of mice lung tissue treated with each fraction from RPA. H&E staining was used to stain lung sections (n = 5). Scale bar, 100 μm. (B) The results of asthma inﬂammation score about each fraction from RPA. (C) Expression levels of cytokines in the serum (n = 3). The data were analyzed by using one-way ANOVA and are presented as mean ± SD. p < 0.05 was statistically signiﬁcant. Compared with control, p < 0.05, p < 0.01, p < 0.001; #compared with model, #p < 0.05, ##p < 0.01, ###p < 0.001, ns, not signiﬁcant. 3.5 Effect of DCM and FDCM on Lung Tissue Pathology in OVA-Sensitized Allergic Asthma s in Pharmacology \| www frontiersin org March 2022 \| Volume 13 \| Article 863403 7 FIGURE 4 \| Wright–Giemsa staining and cell count results. (A) Results of Wright–Giemsa staining by using a microscope. Scale bar, 100 μm. (B) Total number of monocytes, eosinophils, and neutrophils in BALF of high and low doses from DCM and FDCM (n = 5). The data were analyzed by using one-way ANOVA and are presented as mean ± SD. p < 0.05 was statistically signiﬁcant. p < 0.05 was statistically signiﬁcant. Compared with control, p < 0.05, p < 0.01, *p < 0.001; #compared with model, #p < 0.05, ##p < 0.01, ###p < 0.001. FIGURE 4 \| Wright–Giemsa staining and cell count results. (A) Results of Wright–Giemsa staining by using a microscope. Scale bar, 100 μm. (B) Total number of monocytes, eosinophils, and neutrophils in BALF of high and low doses from DCM and FDCM (n = 5). The data were analyzed by using one-way ANOVA and are presented as mean ± SD. p < 0.05 was statistically signiﬁcant. p < 0.05 was statistically signiﬁcant. Compared with control, p < 0.05, p < 0.01, *p < 0.001; #compared with model, #p < 0.05, ##p < 0.01, ###p < 0.001. FIGURE 5 \| H&E staining and asthma inﬂammation score. (A) Pathological changes of mice lung tissues treated with high and low doses from DCM and FDCM. H&E staining was used to stain lung sections (n = 5). Scale bar, 50 μm. (B) The results of asthma inﬂammation score about high and low doses from DCM and FDCM. The data were analyzed by using One-way ANOVA and are presented as mean ± SD. p < 0.05 was statistically signiﬁcant. Compared with control, p < 0.05, p < 0.01, p < 0.001; #compared with model, #p < 0.05, ##p < 0.01, ###p < 0.001. FIGURE 5 \| H&E staining and asthma inﬂammation score. (A) Pathological changes of mice lung tissues treated with high and low doses from DCM and FDCM. H&E staining was used to stain lung sections (n = 5). Scale bar, 50 μm. (B) The results of asthma inﬂammation score about high and low doses from DCM and FDCM. The data were analyzed by using One-way ANOVA and are presented as mean ± SD. p < 0.05 was statistically signiﬁcant. 3.5 Effect of DCM and FDCM on Lung Tissue Pathology in OVA-Sensitized Allergic Asthma Furthermore, the histopathology of OVA-sensitized mice lungs sections was examined by H&E. The pathological section results are shown in Figure 5A. The results of pathological sections showed that there was no signiﬁcant inﬁltration of inﬂammatory cells in the control, and the mice’s alveolar size was normal. Nevertheless, the bronchus in the lung tissue of the OVA-sensitized model group was inﬁltrated extremely by the inﬂammatory cells and could be seen that the alveolar area was markedly enlarged. The administration of each group had different degrees of improvement about the March 2022 \| Volume 13 \| Article 863403 Frontiers in Pharmacology \| www.frontiersin.org 6 Processed RPA Enhances Anti-Allergic Asthma Sang et al. FIGURE 4 \| Wright–Giemsa staining and cell count results. (A) Results of Wright–Giemsa staining by using a microscope. Scale bar, 100 μm. (B) Total number of monocytes, eosinophils, and neutrophils in BALF of high and low doses from DCM and FDCM (n = 5). The data were analyzed by using one-way ANOVA and are presented as mean ± SD. p < 0.05 was statistically signiﬁcant. p < 0.05 was statistically signiﬁcant. Compared with control, p < 0.05, p < 0.01, p < 0.001; #compared with model, #p < 0.05, ##p < 0.01, ###p < 0.001. RE 4 \| Wright–Giemsa staining and cell count results. (A) Results of Wright–Giemsa staining by using a microscope. Scale bar, 100 μm. (B) Total number of ocytes, eosinophils, and neutrophils in BALF of high and low doses from DCM and FDCM (n = 5). The data were analyzed by using one-way ANOVA and are ented as mean ± SD. p < 0.05 was statistically signiﬁcant. p < 0.05 was statistically signiﬁcant. Compared with control, p < 0.05, p < 0.01, *p < 0.001; pared with model, #p < 0.05, ##p < 0.01, ###p < 0.001. RE 5 \| H&E staining and asthma inﬂammation score. (A) Pathological changes of mice lung tissues treated with high and low doses from DCM and FDCM. staining was used to stain lung sections (n = 5). Scale bar, 50 μm. (B) The results of asthma inﬂammation score about high and low doses from DCM and FDCM. The were analyzed by using One-way ANOVA and are presented as mean ± SD. p < 0.05 was statistically signiﬁcant. Compared with control, p < 0.05, *p < 0.01, < 0.001; #compared with model, #p < 0.05, ##p < 0.01, ###p < 0.001. 3.8 Effect of DCM and FDCM on the Expression Level of p-AKT in Lung Tissues by Immunoﬂuorescence Analysis in OVA-Sensitized Allergic Asthma inﬂammation in lung tissue, which included the degree of moderation about the pathological structure of lung tissues and the degree of inﬂammatory inﬁltration by inﬂammatory cells. The results of the asthma inﬂammation score are shown in Figure 5B, the scores of the control were about 1 point, and the OVA-sensitized model group was about 4 points. The higher scores the group got, the worse improvement it was. The experimental results showed that HD, HFD, and LFD had a good improvement effect (p < 0.001), but HFD was best. In general, the experimental results conﬁrmed that the dichloromethane fraction from FRPA had a better effect on anti-allergic asthma than from RPA once again. Immunoﬂuorescence staining of p-AKT further veriﬁed the results of the Western blotting, and the immunoﬂuorescence results are presented in Figure 6E. The immunoﬂuorescence analysis of frozen sections of FRPA lung tissues with p-AKT showed that HD and HFD could inhibit the expression of AKT phosphorylation in the lung tissue of OVA-induced allergic asthma mice, but HFD had a better inhibition effect. The experimental results also proved that the FDCM had a signiﬁcant anti-allergic effect on asthma, and the effect was better than that of DCM. 3.9 Changed Contents of Main Compounds in FDCM Compared With DCM Through UPLC/MS software analysis, the experimental results conﬁrmed that the contents of many components in FDCM were changed after stir-frying by honey and bran. And the compounds were identiﬁed by the pharmacopoeia standards, the reported compounds, and mass spectrometry. The results are summarized in Table 1. Subsequently, the research performed signiﬁcant difference analysis on the average values of their peak area by using a t-test, and at last, the experimental results manifested that the contents of many compounds in FDCM were signiﬁcantly increased with a statistical difference after stir-frying with honey and bran. The bar charts of comparisons of the contents of some compounds are presented in Figure 7B. 3.7 Effect of DCM and FDCM on the Activation of PI3K/AKT Signaling Pathway in OVA-Sensitized Allergic Asthma g The PI3K/AKT signaling pathway was one of the common signaling pathways in immunosuppression and inﬂammatory diseases. Also in order to explore the signaling pathway of FDCM in OVA-sensitized allergic asthma, the expression of PI3K and AKT was detected by Western blotting. The primary antibodies of p-PI3K, PI3K, p-AKT, AKT, and GAPDH were used to detect the protein expression levels. The protein expression results of the relevant bands are shown in Figure 6C. The results indicated that OVA could enhance the levels of p-PI3K and p-AKT, while these effects could be decreased by treatment of high and low doses of DCM and FDCM. In brief, the HFD could prevent the phosphorylation of PI3K and AKT when compared with the OVA-sensitized model group (p < 0.001). The band analysis results are presented in Figure 6D. These ﬁndings suggested that the dichloromethane fraction from FRPA could enhance the effect of anti-allergic asthma under OVA-sensitized by targeting the PI3K/AKT signaling pathway. 3.5 Effect of DCM and FDCM on Lung Tissue Pathology in OVA-Sensitized Allergic Asthma Compared with control, p < 0.05, p < 0.01, *p < 0.001; #compared with model, #p < 0.05, ##p < 0.01, ###p < 0.001. March 2022 \| Volume 13 \| Article 863403 7 Processed RPA Enhances Anti-Allergic Asthma Sang et al. FIGURE 6 \| Western blotting and Immunoﬂuorescence staining results. (A) Representative Western blotting analysis for expressions of IL-1β and IL-6. Densitometric values were normalized against GAPDH. (B) Relative protein expression of IL-1β and IL-6 in OVA-sensitized mice with the treatment of high and low doses of DCM and FDCM. (C) Representative Western blotting analysis for expressions of p-PI3K, PI3K, p-AKT and AKT. Densitometric values were normalized against GAPDH. (D) Relative protein expression of p-PI3K and p-AKT OVA-sensitized mice with the treatment of high and low doses of DCM and FDCM. (E) Immunoﬂuorescence staining of p-AKT. Scale bar, 200 µm. Protein bands were analyzed by ImageJ (n = 3). GAPDH was used as internal controls. The data were analyzed by using one-way ANOVA and presented as mean ± SD. p < 0.05 was statistically signiﬁcant. Compared with control, p < 0.05, p < 0.01, p < 0.001; #Compared with model, #p < 0.05, ##p < 0.01, ###p < 0.001. Sang et al. Processed RPA Enhances Anti Allergic Asthm FIGURE 6 \| Western blotting and Immunoﬂuorescence staining results. (A) Representative Western blotting analysis for expressions of IL-1β and IL-6. Densitometric values were normalized against GAPDH. (B) Relative protein expression of IL-1β and IL-6 in OVA-sensitized mice with the treatment of high and low doses of DCM and FDCM. (C) Representative Western blotting analysis for expressions of p-PI3K, PI3K, p-AKT and AKT. Densitometric values were normalized against GAPDH. (D) Relative protein expression of p-PI3K and p-AKT OVA-sensitized mice with the treatment of high and low doses of DCM and FDCM. (E) Immunoﬂuorescence staining of p-AKT. Scale bar, 200 µm. Protein bands were analyzed by ImageJ (n = 3). GAPDH was used as internal controls. The data were analyzed by using one-way ANOVA and presented as mean ± SD. p < 0.05 was statistically signiﬁcant. Compared with control, p < 0.05, p < 0.01, p < 0.001; #Compared with model, #p < 0.05, ##p < 0.01, ###p < 0.001. March 2022 \| Volume 13 \| Article 863403 Frontiers in Pharmacology \| www.frontiersin.org 8 Sang et al. Processed RPA Enhances Anti-Allergic Asthma 3.9 Changed Contents of Main Compounds in FDCM Compared With DCM IL-1β and IL-6 were two inﬂammatory factors related to allergic asthma. For the sake of exploring the regulation of inﬂammatory factors of FDCM in OVA-sensitized allergic asthma, the expression of IL-1β and IL-6 was detected by Western blotting. The primary antibody of IL-1β, IL-6, and GAPDH was used to detect the protein expression levels. The protein expressions of associated bands are shown in Figure 6A. The results illustrated that OVA could enhance the levels of IL-1β and IL-6, while the effects could be diminished by treatment about high and low doses of DCM and FDCM. In short, HFD could decrease the protein expression of IL-1β and IL-6 when compared with the OVA-sensitized model group (p < 0.001). The correlative bands analysis results were presented in Figure 6B. These ﬁndings asserted that the dichloromethane fraction from FRPA could enhance the effect of anti-allergic asthma under OVA-sensitized by regulating inﬂammatory factors IL-β and IL-6. in FDCM Compared With DCM The pharmacological experimental results conﬁrmed that FDCM had stronger pharmacological effects on anti-allergic asthma than DCM. In this section, the experiment analyzed further to explore the main components in DCM and FDCM and the changed contents of FDCM after stir-frying by honey and bran. Therefore, UPLC/Q-TOF-MS was used to analyze the samples of FDCM and DCM. The chromatograms of UPLC are shown in Figure 7A. Through UPLC/MS software analysis, the experimental results conﬁrmed that the contents of many components in FDCM were changed after stir-frying by honey and bran. And the compounds were identiﬁed by the pharmacopoeia standards, the reported compounds, and mass spectrometry. The results are summarized in Table 1. Subsequently, the research performed signiﬁcant difference analysis on the average values of their peak area by using a t-test, and at last, the experimental results manifested that the contents of many compounds in FDCM were signiﬁcantly increased with a statistical difference after stir-frying with honey and bran. The bar charts of comparisons of the contents of some compounds are presented in Figure 7B. The pharmacological experimental results conﬁrmed that FDCM had stronger pharmacological effects on anti-allergic asthma than DCM. In this section, the experiment analyzed further to explore the main components in DCM and FDCM and the changed contents of FDCM after stir-frying by honey and bran. Therefore, UPLC/Q-TOF-MS was used to analyze the samples of FDCM and DCM. The chromatograms of UPLC are shown in Figure 7A. 4 DISCUSSION Over the past decades, allergic asthma and airway inﬂammatory disease had exerted great impacts on human and animal health worldwide. At present, most medical researchers believed that the main factor causing asthma was the imbalance of Th1/Th2 and Th17/Treg. At present, most medical researchers believed that the main factor causing asthma is the imbalance of Th1/Th2 and Th17/Treg, and the regulation of PI3K/AKT and other signaling pathways can effectively control the progression of asthma. PI3K was a heterodimer protein consisting of a catalytic subunit and a regulatory subunit. Under basic conditions, regulatory proteins would bind, stabilize, and inhibit catalytic subunits. During cell activation, the regulatory proteins would contact the catalytic subunits with a lipid substrate on the membrane. PI3K would be activated by Ras, RTKs, and GPCRs, and after activation, the activated PI3K was able to phosphorylate PIP2 to produce PIP3, March 2022 \| Volume 13 \| Article 863403 Frontiers in Pharmacology \| www.frontiersin.org 9 Sang et al. Sang et al. Processed RPA Enhances Anti-Allergic Asthma FIGURE 7 \| UPLC analysis results and bar chart of compounds contents. (A) Chromatograms of UPLC on DCM and FDCM. (B) Bar charts about the signiﬁcantly increased contents of some compounds in FDCM compared with DCM, which included compound structures (n = 3). The data were analyzed using a T-test and presented as mean ± SD. p < 0.05 was statistically signiﬁcant. Compared with DCM, p < 0.05, p < 0.01, *p < 0.001. FIGURE 7 \| UPLC analysis results and bar chart of compounds contents. (A) Chromatograms of UPLC on DCM and FDCM. (B) Bar charts about the signiﬁcantly increased contents of some compounds in FDCM compared with DCM, which included compound structures (n = 3). The data were analyzed using a T-test and presented as mean ± SD. p < 0.05 was statistically signiﬁcant. Compared with DCM, p < 0.05, p < 0.01, **p < 0.001. March 2022 \| Volume 13 \| Article 863403 Frontiers in Pharmacology \| www.frontiersin.org 10 Processed RPA Enhances Anti-Allergic Asthma Sang et al. TABLE 1 \| Changed content of main compounds in FDCM compared with DCM. No. Name Formula tR (min) Ion mode Mass (m/z) Typical fragment ions (m/z) Peak area References RPA FRPA 1 Sucrose C12H22O11 0.45 [M−H]- 341.1078 119, 89, 71, 59 309,725 1,732,254 Ren et al. (2019) 2 Oxypaeoniﬂorin C23H28O12 1.99 [M+CH2O2−H]- 541.1553 495, 465, 333, 137 28 218 Ren et al. 4 DISCUSSION (2019) 3 Albiﬂorin C23H28O11 3.18 [M−H]- 479.1552 319, 197, 159, 129 169 4,654 Zheng et al. (2015) 4 Isomaltopaeoniﬂorin C29H38O16 3.20 [M+CH2O2−H]- 687.2143 593, 525, 323, 121 5,121 487,242 Tan et al. (2017) 5 Mudanpioside E C24H30O13 3.63 [M−H]− 525.1605 449, 327, 165, 121 1,757,345 11,362,228 Zheng et al. (2015) 6 Gallic acid C7H6O5 3.78 [M−H]− 169.0132 171, 153, 127, 109 2072 10,094 Zheng et al. (2015) 7 Ethyl gallate C9H10O5 3.79 [M−H]− 197.0444 197, 169, 125, 124 1,377,324 3,459,845 Yang et al. (2021) 8 Pyrogallol C6H6O3 3.81 [M−H]− 125.0240 125, 108, 95, 81 611 6,112 Ren et al. (2019) 9 Ellagic acid C14H6O8 4.37 [M−H]− 300.9988 300, 257, 229, 201 30,490 27,915 Ren et al. (2019) 10 Pentagalloylglucose C41H32O26 5.18 [M−H]− 939.1095 939, 769, 469, 451 975,683 1,414,974 Zheng et al. (2015) 11 Galloylpaeoniﬂorin C30H32O15 5.49 [M−H]− 631.1661 631, 601, 313, 169 26,455 564,717 Zheng et al. (2015) 12 Paeonol C9H10O3 5.86 [M−H]− 211.0601 211, 196, 166, 123 15,827 51,269 Zheng et al. (2015) 13 Paeoniﬂorin C23H28O11 6.21 [M−H]− 479.1554 525, 509, 479, 121 625 22,346 Zheng et al. (2015) 14 Lactiﬂorin C23H26O10 6.68 [M + CH2O2−H]− 507.1501 507, 339, 177, 121 525,724 7,703,073 Zheng et al. (2015) 15 Ethyl 3,4-dihydroxy-5-[(3,4,5- trihydroxybenzoyl)oxy]benzoate C16H14O9 7.60 [M−H]− 349.0577 197, 169, 124 51,757 17,656 Wu L. F. et al. (2020) 16 Pinen-10-yl vicianoside C21H34O10 7.98 [M + CH2O2−H]− 491.2123 445, 293, 233, 191 29,794 1,355,835 Tan et al. (2017) 17 Mudanpioside B C31H34O14 8.15 [M−H]− 629.1861 553, 431, 165, 121 8,176,472 13,453,885 Zheng et al. (2015) 18 Benzoylalbiﬂorin C30H32O12 8.17 [M−H]− 583.1816 583, 551, 431, 121 178,756 372,997 Zheng et al. (2015) 19 30-Norhederagenin C29H44O4 8.72 [M−H]− 455.3157 455, 409 319,396 563,943 Ren et al. (2019) 20 Hederagenin C30H48O4 8.90 [M−H]− 471.3465 471, 435, 407, 325 531,821 537,677 Zheng et al. (2015) TABLE 1 \| Changed content of main compounds in FDCM compared with DCM. causing the recruitment of PDK1 and AKT to the plasma membrane. Then, PDK1 and mTORC2 would phosphorylate AKT. Finally, the activated AKT was able to phosphorylate plenty of downstream effectors, which regulated the cell growth, cell survival, angiogenesis, and migration, or invasion. In the previous research, Wu et al. found through experiments that alpinetin had good anti-inﬂammatory activity against allergic asthma by regulating PI3K/AKT/NF-κB and HO-1 signaling pathways (Wu D. et al., 2020). Ma et al. 4 DISCUSSION Galloylpaeoniﬂorin can alleviate PM2.5-induced vascular endothelial cell injury on inﬂammatory responses, which can be achieved by activating the Nrf2 signaling pathway (Bi et al., 2020). In addition, there were also some compounds with other structures that showed a signiﬁcant increase, such as ethyl gallate and pentagalloylglucose. To a certain extent, ethyl gallate and pentagalloylglucose can protect the LPS- induced acute lung injury by inhibiting the number of polymorphonuclear leukocytes in BALF, reducing the wet/ dry ratio and protein concentration of the lung, and improving the permeability of the lung (Zhang et al., 2018). The research group is currently conducting research on their pharmacological activities and other aspects. Afterward, the dichloromethane fraction from FRPA was obtained according to the RPA-related extraction and the separation process to evaluate the enhancement of pharmacological effect after the processing with honey and bran. From the results of H&E staining, the high dose of the dichloromethane fraction from FRPA did exert a stronger pharmacological effect than the high dose of the dichloromethane fraction from RPA, and the pathological sections of the lung tissue showed the best inhibition degree of inﬂammatory inﬁltration compared with other groups. The cell count results of BALF also showed that HFD could reduce the number of eosinophils and neutrophils more effectively than HD. In addition, the research further studied the inﬂammatory factors and signaling pathways of FDCM against OVA-induced allergic asthma. Th2 cells secreted IL-6 to prevent allergic diseases (Jian et al., 2013). IL-1β can stimulate Th2 cells and increase the eosinophils in the lung inﬂammation site, thus mediating the development process of allergic asthma (Sobkowiak et al., 2017). In Western blotting analysis, it was found that HFD signiﬁcantly reduced the contents of IL-1β and IL-6, and HFD was better than HD. Furthermore, pathologies such as inﬂammatory cell inﬁltration can be suppressed by inhibiting the activation of PI3K and AKT in mouse models of allergic asthma (Wu D. et al., 2020). FDCM was Coronavirus disease 2019 (COVID-19) is a global infectious disease, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), making it one of the severe public health issues in recent decades, which had put a heavy toll on public health, lives, and the world economy. Hundreds of molecular tests and immunoassays were rapidly developed (Vandenberg et al., 2021), and several agents were being evaluated (Gavriatopoulou et al., 2021). 4 DISCUSSION Judging from the cell count after Wright–Giemsa staining of BALF, DCM can prevent the alveolar area from being inﬁltrated by inﬂammatory cells, especially eosinophils, to some extent. The experimental results were similar to those of pulmonary histopathological analysis, indicating that DCM had a good anti-allergic effect on asthma. T cells differentiated into Th1, Th2, and Th17 after antigen exposure and receptor stimulation. IL-17 produced by Th17 cells can stimulate bronchial epithelial cells in allergic asthma and had a certain effect on the production of cytokines and chemokines. IL-4 produced by Th2 cells played a central role in the development of asthma, in that it increased the expression level of inﬂammatory cytokines in the lung and ultimately stimulated the production of IgE. Inspired by the fact that IgE can mediate an allergic immune response, it was the main regulator of Th2 cytokines that controlled the progression of asthma (Aghajani et al., 2019; Wu D. et al., 2020). The results of ELISA showed that DCM could effectively reduce the expression levels of IgE, IL-17, and IL-4 in serum. The aforementioned experimental results showed that DCM was the most active effective fraction for anti-allergic asthma in RPA. found to be capable of inhibiting the expression levels of p-PI3K and p-AKT. At the same time, according to the analysis results of immunoﬂuorescence, it was found that HFD could effectively inhibit the expression of AKT phosphorylation in the lung tissue. These experimental results indicated that FDCM had potential pharmacological mechanisms of action to exert anti-allergic asthma by targeting the PI3K/AKT signaling pathway. Furthermore, the better pharmacological action of the dichloromethane fraction of FRPA was due to the increased contents of its main components after processing with honey and bran, which might be because the carbohydrate-rich honey played a protective role in the structure of the main chemical components of the traditional Chinese medicine during the high-temperature processing (Ao et al., 2020). The main components of the dichloromethane fraction from FRPA were generally paeoniﬂorin and its structure analogous compounds. The experimental results indicated that the components of paeoniﬂorin and the structure analogous compounds of paeoniﬂorin like galloylpaeoniﬂorin were signiﬁcantly increased in the dichloromethane fraction from FRPA after processing. Paeoniﬂorin can reduce the expression levels of IL-5, IL-13, IL-17, and eotaxin in the OVA-induced allergic asthma mice model, which may be concerned with the blocking of the MAPK pathway activation (Zhou et al., 2020). 4 DISCUSSION mentioned in the study that allergic asthma would activate the mTOR signaling pathway, which led to the upregulation of p-PI3K, p-AKT, and p-mTOR. The general pathway can be simply described as PI3K was activated by external stimulation and inﬂammation, and after activation, p-PI3K was formed, which would phosphorylate AKT into p-AKT and ﬁnally activate mTOR, translate protein, and grow cells (Ma et al., 2021). Chen Xi et al., through experiments, found that surfactant protein A can inhibit allergic reactions of asthma mice induced by OVA by regulating the activity of JAK/ STAT (Chen et al., 2021). Vitex negundo Linn. extracts can regulate the AMPK/PI3K/AKT/p38-NF-κB and TGF-β/Smad/ Bcl2/caspase/LC3 signaling pathways to produce cascade reaction and activate macrophages, thus alleviating allergic asthma and lung infection induced by OVA (Tirpude et al., 2021). causing the recruitment of PDK1 and AKT to the plasma membrane. Then, PDK1 and mTORC2 would phosphorylate AKT. Finally, the activated AKT was able to phosphorylate plenty of downstream effectors, which regulated the cell growth, cell survival, angiogenesis, and migration, or invasion. In the previous research, Wu et al. found through experiments that alpinetin had good anti-inﬂammatory activity against allergic asthma by regulating PI3K/AKT/NF-κB and HO-1 signaling pathways (Wu D. et al., 2020). Ma et al. mentioned in the study that allergic asthma would activate the mTOR signaling pathway, which led to the upregulation of p-PI3K, p-AKT, and p-mTOR. The general pathway can be simply described as PI3K was activated by external stimulation and inﬂammation, and after activation, p-PI3K was formed, which would phosphorylate AKT into p-AKT and ﬁnally activate mTOR, translate protein, and grow cells (Ma et al., 2021). Chen Xi et al., through experiments, found that surfactant protein A can inhibit allergic reactions of In this study, the most effective fraction of RPA that exerts the anti-allergic asthma effect was evaluated, and after processing honey and bran, the pharmacological effect, pharmacological mechanisms of action, and material basis of increasing the anti-allergic asthma effect of the effective active fraction in FRPA were further explored. First, RPA was extracted, and the drug components of each fraction were obtained by using different polar extracts. Subsequently, the research evaluated the most active fraction for anti-allergic asthma by OVA sensitization of female BALB/c mice to model and administer drugs to each fraction of RPA. 4 DISCUSSION March 2022 \| Volume 13 \| Article 863403 Frontiers in Pharmacology \| www.frontiersin.org 11 Processed RPA Enhances Anti-Allergic Asthma Sang et al. Sang et al. According to the experimental results of histopathological analysis, the lung tissues of OVA-induced BALB/c mice showed obvious inﬂammatory inﬁltration, and the alveolar structure was also signiﬁcantly damaged. Based on the results of H&E staining, DCM, the dichloromethane fraction of RPA, could signiﬁcantly reduce the degree of inﬂammatory inﬁltration of lung tissue. On the other hand, in the process of airway inﬂammation, there were many inﬂammatory cells involved, including eosinophils and neutrophils. Eosinophils were the main participants in the process of allergic asthma. Judging from the cell count after Wright–Giemsa staining of BALF, DCM can prevent the alveolar area from being inﬁltrated by inﬂammatory cells, especially eosinophils, to some extent. The experimental results were similar to those of pulmonary histopathological analysis, indicating that DCM had a good anti-allergic effect on asthma. T cells differentiated into Th1, Th2, and Th17 after antigen exposure and receptor stimulation. IL-17 produced by Th17 cells can stimulate bronchial epithelial cells in allergic asthma and had a certain effect on the production of cytokines and chemokines. IL-4 produced by Th2 cells played a central role in the development of asthma, in that it increased the expression level of inﬂammatory cytokines in the lung and ultimately stimulated the production of IgE. Inspired by the fact that IgE can mediate an allergic immune response, it was the main regulator of Th2 cytokines that controlled the progression of asthma (Aghajani et al., 2019; Wu D. et al., 2020). The results of ELISA showed that DCM could effectively reduce the expression levels of IgE, IL-17, and IL-4 in serum. The aforementioned experimental results showed that DCM was the most active effective fraction for anti-allergic asthma in RPA. According to the experimental results of histopathological analysis, the lung tissues of OVA-induced BALB/c mice showed obvious inﬂammatory inﬁltration, and the alveolar structure was also signiﬁcantly damaged. Based on the results of H&E staining, DCM, the dichloromethane fraction of RPA, could signiﬁcantly reduce the degree of inﬂammatory inﬁltration of lung tissue. On the other hand, in the process of airway inﬂammation, there were many inﬂammatory cells involved, including eosinophils and neutrophils. Eosinophils were the main participants in the process of allergic asthma. 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AUTHOR CONTRIBUTIONS adjuvants in the treatment of early/mild common ﬂu in otherwise healthy adults within the context of COVID-19. adjuvants in the treatment of early/mild common ﬂu in otherwise healthy adults within the context of COVID-19. This study was conceived and designed by XS and JY. XW, XH, QS, QL, QY, KW, MH, and EL conducted experiments and analyzed the data results. The manuscript was written by JY. XS and GC revised the manuscript. 4 DISCUSSION However, most of the current guidelines did not speciﬁcally advise on how to treat pneumonia and acute respiratory distress syndrome, which were the major complications of COVID-19, such as cough and inﬂammation (Anka et al., 2021). RPA was a well-known natural medicine in China already widely available for the management of respiratory conditions, mainly regarding the symptoms (inﬂammation and immune function). Our work suggests that RPA had evidence to the discussion about its potential use as March 2022 \| Volume 13 \| Article 863403 Frontiers in Pharmacology \| www.frontiersin.org 12 Processed RPA Enhances Anti-Allergic Asthma Sang et al. FUNDING This work was supported by the National Natural Science Foundation of China (No. 81903803), the Chinese Medicine Research Program of Zhejiang Province, China (No. 2022ZB094), and the Youth Natural Science Program of Zhejiang Chinese Medical University (No. 2021JKZKTS025B). DATA AVAILABILITY STATEMENT The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author. ETHICS STATEMENT The animal study was reviewed and approved by the Zhejiang Chinese Medical University. The animal study was reviewed and approved by the Zhejiang Chinese Medical University. 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Zhang, Q., Nie, J., Chen, S. J., and Li, Q. (2018). Protective Effects of Ethyl Gallate and Pentagalloylglucose, the Active Components of Qingwen Baidu Decoction, against Lipopolysaccharide-Induced Acute Lung Injury in Rats. Drug Des. Devel Ther. 13, 71–77. doi:10.2147/DDDT.S186029 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. Zhang, L., Li, D. C., and Liu, L. F. (2019). Paeonol: Pharmacological Effects and Mechanisms of Action. Int. Immunopharmacol. 72, 413–421. doi:10.1016/j. Frontiers in Pharmacology \| www.frontiersin.org REFERENCES intimp.2019.04.033 Copyright © 2022 Sang, Ying, Wan, Han, Shan, Lyu, Yang, Wang, Hao, Liu and Cao. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Zhang, H., Zhang, S., Hu, M., Chen, Y., Wang, W., Zhang, K., et al. (2020). An Integrative Metabolomics and Network Pharmacology Method for Exploring the Effect and Mechanism of Radix Bupleuri and Radix Paeoniae Alba on Anti- depression. J. Pharm. Biomed. Anal. 189, 113435. doi:10.1016/j.jpba.2020.113435 Zheng, Z., Cao, G., Wu, X., Chen, X., and Cai, B. (2015). Ultra-performance Liquid Chromatography Coupled with High-Resolution Quadrupole Time-Of-Flight Mass Spectrometry Analysis of the Impact of Bran-Processing on the Chemical March 2022 \| Volume 13 \| Article 863403 Frontiers in Pharmacology \| www.frontiersin.org 14
https://openalex.org/W1919597550	https://bmcecolevol.biomedcentral.com/counter/pdf/10.1186/1471-2148-6-53	English	null	Highly variable sperm precedence in the stalk-eyed fly, Teleopsis dalmanni	BMC evolutionary biology	2,006	cc-by	7,094	BioMed Central BioMed Central This article is available from: http://www.biomedcentral.com/1471-2148/6/53 This article is available from: http://www.biomedcentral.com/1471-2148/6/53 © 2006 Corley 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: When females mate with different males, competition for fertilizations occurs after insemination. Such sperm competition is usually summarized at the level of the population or species by the parameter, P2, defined as the proportion of offspring sired by the second male in double mating trials. However, considerable variation in P2 may occur within populations, and such variation limits the utility of population-wide or species P2 estimates as descriptors of sperm usage. To fully understand the causes and consequences of sperm competition requires estimates of not only mean P2, but also intra-specific variation in P2. Here we investigate within-population quantitative variation in P2 using a controlled mating experiment and microsatellite profiling of progeny in the multiply mating stalk-eyed fly, Teleopsis dalmanni. Results: We genotyped 381 offspring from 22 dam-sire pair families at four microsatellite loci. The mean population-wide P2 value of 0.40 was not significantly different from that expected under random sperm mixing (i.e. P2 = 0.5). However, patterns of paternity were highly variable between individual families; almost half of families displayed extreme second male biases resulting in zero or complete paternity, whereas only about one third of families had P2 values of 0.5, the remainder had significant, but moderate, paternity skew. Conclusion: Our data suggest that all modes of ejaculate competition, from extreme sperm precedence to complete sperm mixing, occur in T. dalmanni. Thus the population mean P2 value does not reflect the high underlying variance in familial P2. We discuss some of the potential causes and consequences of post-copulatory sexual selection in this important model species. BMC Evolutionary Biology Open Access dalmanni Laura S Corley1,2, Samuel Cotton1, Ellen McConnell1, Tracey Chapman3, Kevin Fowler1 and Andrew Pomiankowski1 Laura S Corley1,2, Samuel Cotton1, Ellen McConnell1, Tracey Kevin Fowler1 and Andrew Pomiankowski1 Address: 1Galton Laboratory, Department of Biology, University College London, Wolfson House, 4 Stephenson Way, London NW1 2HE, UK, 2Department of Entomology, Washington State University, Pullman WA 99164-6382, USA and 3Department of Biology, University College London, Darwin Building, Gower Street, London, WC1E 6BT, UK Email: Laura S Corley - corley@mail.wsu.edu; Samuel Cotton - s.cotton@ucl.ac.uk; Ellen McConnell - e.mcconnell@ucl.ac.uk; Tracey Chapman - t.chapman@ucl.ac.uk; Kevin Fowler - k.fowler@ucl.ac.uk; Andrew Pomiankowski* - ucbhpom@ucl.ac.uk * Corresponding author * Corresponding author Received: 10 March 2006 Accepted: 26 June 2006 Received: 10 March 2006 Accepted: 26 June 2006 Received: 10 March 2006 Accepted: 26 June 2006 Published: 26 June 2006 Published: 26 June 2006 BMC Evolutionary Biology 2006, 6:53 doi:10.1186/1471-2148-6-53 BMC Evolutionary Biology 2006, 6:53 doi:10.1186/1471-2148-6-53 Page 1 of 7 (page number not for citation purposes) Background behavioral, physiological, and morphological adapta- tions that influence sperm competition, such as mate guarding, increased copulation duration, seminal fluid induced reluctance of female re-mating, and the mechan- ical removal of sperm [1-3]. In addition, post-copulatory g When females copulate with more than one partner, com- petition for fertilizations occurs after insemination. Such post-copulatory sexual selection can be a potent evolu- tionary force, as is evidenced by the numerous male Page 1 of 7 (page number not for citation purposes) Page 1 of 7 (page number not for citation purposes) http://www.biomedcentral.com/1471-2148/6/53 http://www.biomedcentral.com/1471-2148/6/53 BMC Evolutionary Biology 2006, 6:53 sexual selection can enhance or diminish male ornament evolution if ornament size covaries positively or nega- tively, respectively, with sperm competitive ability [4-6]. the eyes on elongate protuberances on the side of the head capsule, a trait common to both sexes in all species [19,23]. In many species the eyespan of males is greater than that of females, the result of sexual selection through female mate choice [24-29] and male-male competition [30,31]. The most widely used metric for sperm competition that is used to infer patterns of paternity is the proportion of eggs sired by the second male in controlled double-mat- ing trials (P2; [7]). Species or population level studies of P2 have been used extensively to describe sperm competi- tion, particularly in insects [2] and birds [1]. However, considerable variation in P2 often occurs between popula- tions and individuals, and intra-specific values of P2 can range from zero to one [2,5,8]. Such variation can severely limit the utility of population-wide (or species) P2 esti- mates as descriptors of sperm usage, because it fails to account for variation in male performance (P2 is derived from the performance of both first and second males), or aspects of female morphology and behaviour, such as sperm storage, that may differ between individual females [9]. For example, within many Lepidopteran species, some females lay eggs fertilized almost exclusively by the first male to mate, whereas others show strong second male sperm precedence ([10]; see also [11] for an example in guppies); in these instances mean P2 values do not reflect the underlying bimodal distribution of male fertili- zation success. So in order to fully understand the causes and consequences of sperm competition it is necessary to estimate not only mean levels of sperm precedence, but also intra-specific variation around that mean. Background The Malaysian stalk-eyed fly, Teleopsis dalmanni (formerly known as Cyrtodiopsis dalmanni; [32]), exhibits extreme sexual dimorphism for eyespan resulting from strong intra- and inter-sexual selection on the trait in males (ibid.). Females form large harems on root hairs overhang- ing the eroded banks of streams and males compete to control these harems [19,33]. Both sexes are highly pro- miscuous and mate at high frequencies (~10 and 6 times per hour in the laboratory, for males and females respec- tively; [34,35]). There is also some evidence that males strategically allocate more ejaculate to larger, more pro- ductive females through the production and delivery of larger spermatophores [36]. However, T. dalmanni sper- matophores are small [37] and females store few sperm following a single mating (~140; [36]). Females are there- fore sperm-limited and must copulate repeatedly to attain maximal fertility [38]. This problem is exacerbated in large, highly fecund females despite being allocated more ejaculate, as they lay a lower proportion of fertilised eggs following a single copulation in comparison with less productive females [38]. Without measures of paternity however, the value of both mate choice and multiple mat- ing can only be inferred indirectly. Numerous factors have been shown to influence intra- specific variation in P2 including male size, sexual orna- mentation, mating history, reproductive organ size and diet [2,3,5]. There is also support for the hypothesis that males tailor their ejaculate in response to female factors such as size, mating status, age, fecundity and familiarity [12]. In addition, evidence is accumulating that, contrary to traditional views, ejaculates are in fact costly and can- not be produced in limitless quantities [12,13], and that male gamete availability can limit zygote formation. Under sperm-limitation, reproductive asymmetry between the sexes for variance in fertilisation success, and with it the advantage of sperm competition for available ova, will be reduced. Nonetheless, if a particular mating role is favoured (e.g. first rather than last), a male is still predicted to invest more in the favoured role [14]. Sperm- limitation phenotypes are most commonly seen in free- spawning external fertilizers at low density [15-17], although similar selective environments may be common in internal fertilisers when females receive insufficient sperm to fertilise all of their eggs [e.g. [12,18]]. Previous research describing patterns of paternity in a con- gener, T. whitei, provides ambiguous evidence about pat- terns of sperm use [39,40]. Results A total of 662 progeny were collected from the 22 dam- sire pair families (mean total brood size ± S.D. = 30.09 ± 16.09, range 8 – 63), of which 381 were genotyped suc- cessfully at four microsatellite loci (mean genotyped brood size ± S.D. = 17.32 ± 7.92, range 5 – 35). The mean value (± S.D.) of P2 across families was 0.40 ± 0.38. This average is not significantly different from a P2 of 0.5 (t = 1.16, d.f. = 21, P = 0.26). 0.65, second male thorax r = -0.11, d.f. = 21, P = 0.62). Relative male eyespan did not explain variation in P2 (tho- rax included in model: first male relative eyespan F = 2.91, d.f. 1,19, P = 0.10, second male relative eyespan F = 0.27, d.f. = 1,19, P = 0.61), and neither did the difference in morphology between first and second males explain vari- ation in P2 (eyespan difference r = -0.09, d.f. = 21, P = 0.68, thorax difference r = 0.16, d.f. = 21, P = 0.47). Female body size (thorax length) had no significant effect on P2 (r = -0.10, d.f. = 19, P = 0.66), and neither did female eye- span (r = -0.37, d.f. = 19, P = 0.10), although the latter did reveal a trend for females with larger eyespans to produce broods with fewer offspring sired by the second male. We found significant variation between the different fam- ilies in terms of their P2 (GH = 292.09, d.f. = 21, P < 0.001). The pattern is approximately tri-modal, with P2 peaks at approximately 0, 0.5 and 1 (Figure 1). Ten families (45%) exhibited extreme second male biases resulting in zero or complete paternity (n = 1 and 3, respectively; Fig. 1). These results were unlikely to be due entirely to one of the males being infertile in all three of their matings, as the incidence of P2 = 0 or 1 was greater than that expected given the level of male infertility (over three matings) in the population (22.7% vs. 12.5%, see Methods § χ2 = 9.14, d.f. = 1, P = 0.002). Moreover, heterogeneity of P2 was not entirely attributable to these families, as a repli- cated goodness-of-fit test using families with P2 ≠ 0 or 1 was still significant (GH = 60.55, d.f. = 11, P < 0.001). Background Using an irradiated sterile male technique, Lorch et al. [39] reported that T. whitei exhibits first male sperm precedence. Irradiated (I) and non-irradiated (N) males were mated sequentially to females in a reciprocal mating design, and evidence for first male precedence was inferred as NI families produced offspring more frequently than IN families. Lorch et al. [39] also concluded that sperm mixing was important since some IN crosses produced pupae. In another study [40], female T. whitei were mated to a male from two dif- ferent populations 24 hours apart, and offspring paternity was assigned using heritable inter-population differences in leg colour. Wilkinson and Fry [40] found no significant effect of mating order on patterns of paternity with both first and second males siring equal numbers of progeny, suggesting that sperm mixing (P2 ≈ 0.5) is the predomi- nant mode of sperm utilization. Wilkinson and Fry [40] also found some evidence for intra-specific variation in P2, as males carrying a meiotic drive chromosome produced fewer sperm than non-drive males, and hence suffered a reduction in their proportion of progeny sired. Stalk-eyed flies (Diptera:Diopsidae) are increasingly important model organisms for studies of sexual selection [19-22]. They are characterised by the lateral extension of Page 2 of 7 (page number not for citation purposes) http://www.biomedcentral.com/1471-2148/6/53 BMC Evolutionary Biology 2006, 6:53 As yet, there has been no effort to identify within-popula- tion quantitative variation in P2 in stalk-eyed flies. In this paper we investigated intra-specific, within population variation of P2 in T. dalmanni using controlled mating experiments and microsatellite profiling of progeny. We standardized male mating history, diet and body size in order to limit variation in male reproductive organ size, as we have shown that testes and accessory glands are reduced by mating [41] and scale positively with diet quality and body size [Rogers et al. in prep; Cotton & Pomiankowski unpublished]. We also chose sires with sim- ilar eyespan to each other to limit the potential effects on P2 of female mate choice based on male ornament size [25,27,28]. We show that even in the absence of these fac- tors there is significant variation in sperm precedence in T. dalmanni, with familial P2 values ranging from zero to one. We discuss the likely causes of this variation. The distrib among 22 dalmanni Figure 1 p p ( 2) g p y y p g The distribution of second male sperm precedence (P2) among 22 dam-sire pair families in the stalk-eyed fly, Teleopsis dalmanni. Open bars depict the frequency of families with P2 significantly different from 0.5 (using 2-tailed binomial tests); black bars denote those with P2 not significantly different from 0.5. The grey bar denotes the sperm precedence of a single family where the observed P2 of zero was not signifi- cantly different from P2 = 0.5. However, the brood size of this family was small (n = 5), so this result should be treated with caution. Results Eight families (36%) displayed patterns of random pater- nity (i.e. their P2 was not significantly different from 0.5; Figure 1). The remainder had significant moderate (but not extreme) paternity skew, reflecting both first and sec- ond male sperm precedence (Figure 1). Our data are unlikely to be affected by biased sampling of progeny within broods (with respect to male order) or variable brood sizes, as there was no correlation between the proportion of the brood sampled and P2 (r = 0.15, d.f. = 21, P = 0.51), or between total or genotyped brood size and P2 (total brood size r = -0.03, d.f. = 21, P = 0.89; gen- otyped brood size r = 0.03, d.f. = 21, P = 0.91). First and second males did not differ consistently from each other for eyespan (t = 1.21, d.f. = 21, P = 0.24) or tho- rax length (t = 0.00, d.f. = 21, P = 1.00). Male morphology did not explain variation in P2 (first male eyespan r = - 0.28, d.f. = 21, P = 0.20, second male eyespan r = -0.16, d.f. = 21, P = 0.47, first male thorax r = 0.10, d.f. = 21, P = Discussion (P2 = 0 or 1, respectively). Sperm precedence was not the only mode of sperm utilization, since the null model of random sperm mixing (i.e. P2 = 0.5) explained patterns of paternity in over one third of families. Furthermore, a number of families displayed first or second male sperm precedence in conjunction with varying degrees of sperm mixing, resulting in moderate, but significant, paternity biases (i.e. 0 <P2 < 0.5 and 0.5 <P2 < 1, respectively). Thus all modes of sperm usage were found in T. dalmanni, and P2 exhibited a tri-modal distribution (Figure 1). What are the causes of such variation in paternity? We can exclude some factors which our experimental design deliberately set out to minimise. It is unlikely that our data can be explained by (cryptic) female choice for male ornaments or other aspects of external morphology, a phenomenon seen in numerous other species [1-3,5], since first and second males did not differ in eyespan, the male sexual trait, or body size. As male eyespan is an accu- rate indicator of accessory gland and testis size in T. dal- manni [Rogers et al. in prep; Cotton & Pomiankowski unpublished], differences in reproductive organ size are also unlikely to be explanatory variables, even though we did not explicitly measure these traits. It is possible that males varied their ejaculate expenditure in relation to the reproductive value of females, in con- junction with their perceived mating status, as is observed frequently in other species [reviewed in [12]]. Since large eyespan females have higher fecundity ([36]; Cotton & Pomiankowski unpublished) and are also more sperm lim- ited [36], first males are predicted to invest relatively more in large eyespan, virgin females. We found some support for this in the form of a (albeit non-significant) negative correlation between female eyespan and the P2 of a brood; males mating first with a large eyespan female showed a tendency to sire more offspring that the subsequent male. Another possibility is that males varied in aspects of exter- nal morphology or behaviour, independent of male body and ornament size, which affected male fertility. For example, males may have differed in the size or shape of their intromittant organs [43] and/or their copulatory courtship behaviour and this may have altered their abil- ity in sperm competition, as has been reported in other insects [3,44,45]. Discussion Using controlled mating experiments, assigning paternity using microsatellite markers, we demonstrated that sperm usage is highly variable in the stalk-eyed fly T. dalmanni (Figure 1). Almost half of the dam-sire pair families geno- typed had extreme first or second male paternity biases Page 3 of 7 (page number not for citation purposes) Page 3 of 7 (page number not for citation purposes) http://www.biomedcentral.com/1471-2148/6/53 BMC Evolutionary Biology 2006, 6:53 each male [18]. Whilst this simulation study is not directly applicable to our experiment, it does point out that male infertility alone can generate a high frequency of P2 = 0 or 1. Even though infertility is quite common in T. dalmanni (estimated as 12.5% for males mated three times [38]), it is not high enough to explain the incidence of families with extreme paternity bias in our study (almost half had P2 = 0 or 1, which would require a rate of infertility equal to 22.7%). However, infertility seems a good explanation of a major part of the frequency of P2 = 0 or 1. In addition, partial infertility due to insemination failure of one or two of the three matings made by each male could account for a large fraction of the variation observed between 0 <P2 < 1. Another hypothesis that can generate a tri-modal distri- bution is "sloppy" sperm mixing. Harvey and Parker [42] report that when sperm from each male's ejaculate tend to clump together and females only use a small proportion of the sperm they receive to fertilise their eggs, high vari- ance and multi-modal P2 distributions will be common. There is no information about sperm usage in stalk-eyed flies, so it is difficult to evaluate this idea. Further experi- mental work is needed to evaluate the relative importance of these hypotheses in explaining our data. (P2 = 0 or 1, respectively). Sperm precedence was not the only mode of sperm utilization, since the null model of random sperm mixing (i.e. P2 = 0.5) explained patterns of paternity in over one third of families. Furthermore, a number of families displayed first or second male sperm precedence in conjunction with varying degrees of sperm mixing, resulting in moderate, but significant, paternity biases (i.e. 0 <P2 < 0.5 and 0.5 <P2 < 1, respectively). Thus all modes of sperm usage were found in T. dalmanni, and P2 exhibited a tri-modal distribution (Figure 1). Page 4 of 7 (page number not for citation purposes) Study animal h l b The laboratory population of T. dalmanni used in this experiment was derived from wild caught flies collected from Malaysia in 1993 by AP. Flies have been maintained in cage culture at 25°C on a 12:12 light/dark cycle, at high population size (>200 individuals) to minimize inbreed- ing. This population does not carry X-linked meiotic drive, which is found in natural populations of T. dal- manni [50]. The light regime included a 15-min 'dawn' period in which the culture room was illuminated by a single 60 W light bulb. All observations of mating behav- iour commenced at the start of the dawn period. Experi- mental flies were collected as eggs and reared under low larval density [51]. Emerging adults were segregated by sex and measured for eyespan and thorax length [51] to an accuracy of 0.01 mm using a monocular microscope and Mating design Individual males were anaesthetized on ice and their left middle tibia was removed 2 weeks prior to the mating tri- als, to yield DNA for pre-screening microsatellite geno- type (n = 96). Pilot mating trials showed that tibia removal had no effect on the ability of males to mount and mate with females. Males were genotyped at each of four microsatellite loci ([52], see below for details), and pairs were formed that maximised allelic differences between them, to give a total of n = 30 pairs. All of these male pairs differed by at least one allele for at least two loci. In the mating trials, a single virgin female was placed in a container with one randomly chosen male (male 1) from a pair of males, at artificial dawn (lights on). The female and male were observed until three matings of 30 seconds or longer had occurred (spermatophore transfer usually occurs after copulations of 30 seconds). The male was then removed. At dawn on the second day, the second male (male 2) from the pair was placed with the female, and observed until three matings of 30 seconds or longer had occurred. The second male was then removed. Three matings per male were used to reduce the incidence of complete infertility [38]. http://www.biomedcentral.com/1471-2148/6/53 http://www.biomedcentral.com/1471-2148/6/53 http://www.biomedcentral.com/1471-2148/6/53 BMC Evolutionary Biology 2006, 6:53 the number of females inseminated in a harem, and also the sperm load within multiply mated individual females. In the context of the current study, the latter may be par- ticularly important given inter-male competition and the high incidence of sperm mixing. the image analysis program NIH Image (Version 1.55; National Institute of Health, Bethesda, MD, USA). Flies were measured "blind" by a single person (EM). All indi- viduals used in this experiment were sexually mature (6– 8 weeks post eclosion) at the start of each experiment and fed high quality food (puréed corn) ad libitum. Variable P2 means that pre-copulatory mate choice is somewhat paradoxical both in terms of direct fertility ben- efits and indirect "good genes" benefits. Superficially it appears that the male that a female prefers to mate with is not necessarily the male that will sire her offspring. This paradox can be resolved however, with the observation that preferred males with large ornaments also have larger testes and accessory glands [Rogers et al. in prep.; Cotton & Pomiankowski unpublished]. Accessory gland size covaries both phenotypically [41,46] and genetically [47] with male mating frequency, and number of sperm stored in a female's spermathecae correlates positively with the testis size of her mate [48]. Therefore, the advantages of choice (both direct and indirect; Rogers et al. in prep; [49]) for exaggerated ornaments might be maintained because large eyespan males can minimise the uncertainty arising from variable paternity by mating more frequently and out numbering the sperm of other males. However, fur- ther experiments are required to specifically test these hypotheses. Conclusion We have shown that the pattern of sperm usage is highly variable in the stalk-eyed fly, T. dalmanni. This greatly lim- its the utility of population-based estimates of P2 as descriptors of sperm usage, and suggests that sperm prec- edence should be viewed as context-specific, rather than a general, constant, metric in stalk-eyed flies. The unex- plained variance in male fertilization success found by this study requires further investigation in order to evalu- ate potential causes and consequences. Mated females were provided with food and water ad libi- tum and eggs were collected every two days for the follow- ing 10 days. All females were frozen on the last day and their abdomens were used to obtain DNA for genotyping. To maximise survival, eggs were allowed to develop into pupae at low larval density [51] at which time they were sacrificed for genotype analysis. One cross was excluded from the analysis because one of the males failed to achieve 3 copulations within 2 hours. A further 7 crosses were excluded from the analysis because there was allele sharing between the female and one (or both) males which precluded assignment of offspring paternity. This left 22 families for which we could estimate P2. Page 5 of 7 (page number not for citation purposes) Discussion Male mating history is often associated with changes in male investment in current mating attempts, with con- comitant effects on patterns of paternity [12,13]. How- ever, all males were virgins at the start of the experiment and performed equal numbers of copulations, so varia- tion in mating experience did not differ between first and second males, and hence is not an explanation of our data. In addition, all males were maintained on a high quality diet, so variation in environmental conditions was minimised between pairs of sires. These factors (when var- iable) may well be important in determining stalk-eyed fly paternity and deserve further investigation, but they can- not explain the results reported in the current study. In contrast, female mating history was not constant across sires (only the first male mated with a virgin). In promis- cuous species, such as T. dalmanni, it is likely to be advan- tageous for a male to mate with a virgin female as they only risk defence, not offence, sperm competition, and theory suggests that males should on average invest more in their favoured mating role (i.e. first vs. second) when females are sperm limited [14]. However, this hypothesis can be refuted, as the average value of P2 was not signifi- cantly different from 0.5. The presence of such extreme variation in paternity means that post-copulatory sexual selection will have a profound influence on the effects of pre-mating biases, and may par- tially explain the high mating rate seen in T. dalmanni. Female T. dalmanni mate frequently with both the same and different males, and prefer to mate most often with large eyespan males [25,27,28]. Repeated mating is bene- ficial for females since it helps to alleviate sperm limita- tion and increases the number of fertile eggs [36,38]. A high mating rate is also beneficial for males as it increases Another possible explanation is that male infertility explains the variation in P2 seen in our study. A recent sim- ulation study showed that tri-modal distributions can be generated by male infertility in the range 10–30%, given that relative fertilization success is randomly allocated to Page 4 of 7 (page number not for citation purposes) Page 4 of 7 (page number not for citation purposes) Statistical analysis We therefore asked whether the incidence of these events were significantly greater than that expected given the standing level of functional infertility in the population for males mated 3 times (estimated as 12.5% by Baker et al. [38]). We note that this test is conservative as Baker et al. [38] defined males as infertile if their hatching success was < 10%. Correlates of sperm precedence were per- formed using arcsine-transformed data (arcsin ). Sta- P2 We tested for consistent differences in male size between first and second males in the mating trials using paired t- tests for absolute eyespan and thorax length. Second male sperm precedence (P2) was calculated as the proportion of offspring from a brood sired by the second male to have mated with the female (P2 = n2/(n1+n2), where ni equals the number of offspring from male i). We tested for heter- ogeneity in P2 among families using the GH statistic from a replicated goodness-of-fit test, compared against a χ2- distribution with number of families minus 1 degrees of freedom ([54], p. 715). The numbers of offspring sired by each male, rather than proportions, were used in the der- ivation of GH. Each family was also tested for its adherence to a null-hypothesis of P2 = 0.5 (i.e. paternity distributed at random) using 2-tailed binomial tests ([55], p. 533). Extreme values of P2 (1 or 0) may have arisen from func- tional infertility of the first or second male, respectively. We therefore asked whether the incidence of these events were significantly greater than that expected given the standing level of functional infertility in the population for males mated 3 times (estimated as 12.5% by Baker et al. [38]). We note that this test is conservative as Baker et al. [38] defined males as infertile if their hatching success was < 10%. Correlates of sperm precedence were per- formed using arcsine-transformed data (arcsin ). Sta- P2 Authors' contributions LSC conceived the study, contributed to the design and execution of the experiment, and helped to draft the man- uscript. SC conducted the statistical analysis and drafted the manuscript. EM performed the experiment and carried out the molecular genetic analyses. TC, KF and AP jointly conceived the study with LSC, participated in the design and coordination, and helped to draft the manuscript. All authors have read and approved the final manuscript. Microsatellite genotyping DNA isolation from male middle tibia, female abdomens, and whole pupae was conducted with slight modification to the protocol of Holehouse et al. [53]. Briefly, the tissue was ground in 45 of μl Tris-EDTA buffer (pH 8.0) with 10 μl of 20 mg/ml proteinase K (Sigma Aldrich) and then incubated for 30 minutes at 55°C and 10 minutes at 100°C. Multiplex PCR was performed according to Page 5 of 7 (page number not for citation purposes) Page 5 of 7 (page number not for citation purposes) http://www.biomedcentral.com/1471-2148/6/53 http://www.biomedcentral.com/1471-2148/6/53 BMC Evolutionary Biology 2006, 6:53 Wright et al. [52] using a 1:10 dilution of extracted DNA and 10 μM primers for ms-039, ms-090, ms-301A, and ms-402 (Table 1). PCR conditions were as follows: dena- turation at 94°C for 2 minutes followed by 30 cycles of 94°C for 30 sec, 55°C for 30 sec, 72°C for 65 sec, fol- lowed by 72°C for 10 minutes. Fluorescently labeled PCR products were separated on a 3100 DNA Analyzer (Applied Biosystems) and analyzed with Genescan 3.1.2 software (Applied Biosystems). Alleles from all 4 loci were scored for all individuals in the study. tistical analyses were performed using JMP software (version 5, SAS Institute, Gary, NC, USA). tistical analyses were performed using JMP software (version 5, SAS Institute, Gary, NC, USA). Acknowledgements This work was funded by a BBSRC research grant to TC, KF and AP, This work was funded by a BBSRC research grant to TC, KF and AP, This work was funded by a BBSRC research grant to TC, KF and AP, employing LSC, SC and EM. The authors thank G Wilkinson for providing the microsatellite primer sequences and PCR conditions. y g employing LSC, SC and EM. The authors thank G Wilkinson for providing the microsatellite primer sequences and PCR conditions. employing LSC, SC and EM. The authors thank G Wilkinson for providing the microsatellite primer sequences and PCR conditions. Statistical analysis Statistical analysis We tested for consistent differences in male first and second males in the mating trials u tests for absolute eyespan and thorax length sperm precedence (P2) was calculated as the offspring from a brood sired by the second mated with the female (P2 = n2/(n1+n2), wh the number of offspring from male i). We te ogeneity in P2 among families using the GH a replicated goodness-of-fit test, compared distribution with number of families minus freedom ([54], p. 715). The numbers of offs each male, rather than proportions, were us ivation of GH. Each family was also tested for to a null-hypothesis of P2 = 0.5 (i.e. paterni at random) using 2-tailed binomial tests ( Extreme values of P2 (1 or 0) may have arise tional infertility of the first or second male We therefore asked whether the incidence o were significantly greater than that expect standing level of functional infertility in th for males mated 3 times (estimated as 12.5 al. [38]). We note that this test is conservati al. [38] defined males as infertile if their hat was < 10%. Correlates of sperm preceden formed using arcsine-transformed data (arcs Table 1: Microsatellite loci used in this study; repe al. [52]. Locus Repeat Motif ms-039 [AC]2AA[AC]4GCW[CA]3A[AC]7AT[AC ms-090 [GT]11GA[GT]3GG[GT]4AT[GT]1 ms-301A [AC]8AT[AC]3TC[AC]2 ms-402A 1. [CAA]2ATA[CAA]8CAG[CAA]2 2. [AC]8 We tested for consistent differences in male size between first and second males in the mating trials using paired t- tests for absolute eyespan and thorax length. Second male sperm precedence (P2) was calculated as the proportion of offspring from a brood sired by the second male to have mated with the female (P2 = n2/(n1+n2), where ni equals the number of offspring from male i). We tested for heter- ogeneity in P2 among families using the GH statistic from a replicated goodness-of-fit test, compared against a χ2- distribution with number of families minus 1 degrees of freedom ([54], p. 715). The numbers of offspring sired by each male, rather than proportions, were used in the der- ivation of GH. Each family was also tested for its adherence to a null-hypothesis of P2 = 0.5 (i.e. paternity distributed at random) using 2-tailed binomial tests ([55], p. 533). Extreme values of P2 (1 or 0) may have arisen from func- tional infertility of the first or second male, respectively. References 1. Birkhead TR, Moller AP: Sperm competition in birds: evolutionary causes and consequences Academic Press: London; 1992. 1. Birkhead TR, Moller AP: Sperm competition in birds: evolutionary causes and consequences Academic Press: London; 1992. 2. Simmons LW, Siva-Jothy MT: Sperm competition in insects: mechanisms and the potential for selection. In Sperm competi- tion and sexual selection Edited by: Birkhead TR, Møller AP. San Diego, Calif: Academic Press; 1998:341-434. 3. Simmons LW: Sperm competition and its evolutionary consequences in the insects Princeton: Princeton University Press; 2001. 3. Simmons LW: Sperm competition and its evolutionary consequences in the insects Princeton: Princeton University Press; 2001. y 4. Doussard DE, Hands CA, Meinwald J, Eisner T: Pheromonal adver- tisement of a nuptial gift by a male moth (Utetheisa ornatrix). Proc Natl Acad Sci USA 1991, 88:9224-9227. y 4. Doussard DE, Hands CA, Meinwald J, Eisner T: Pheromonal adver- tisement of a nuptial gift by a male moth (Utetheisa ornatrix). 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Behav Ecol 1998, 9:525-533. 50. 54. Sokal RR, Rohlf FJ: Biometry 3rd edition. New York: Freeman; 1995. 55 Zar JH: Biostatistical analysis 3rd edition London: Prentice Hall; 1996 References [AC]8 R:AGGAAAGTGGATGCATTCGT Table 1: Microsatellite loci used in this study; repeat motif, allele size range in base pairs, and nucleotide sequence all from Wright et al. [52]. Locus Repeat Motif Product Size (bp) Primer Sequence ms-039 [AC]2AA[AC]4GCW[CA]3A[AC]7AT[AC]1TC[AC]2 147 F:FAM-AATCACAACGCTAACGAGTCA R:ATGCTTCAACGCTTACCTACC ms-090 [GT]11GA[GT]3GG[GT]4AT[GT]1 197 F:FAM-TCTTGCCTTTGCCACACTAA R:TGGGAAATGTGAGTTTACTTAAACAGT ms-301A [AC]8AT[AC]3TC[AC]2 138 F:HEX-TTCAGCACTAAATGCAGCAGA R:GCACTTAACATGCGATGAGG ms-402A 1. [CAA]2ATA[CAA]8CAG[CAA]2 205 F:HEX-CCAAATGGGCCACATTATTC 2. [AC]8 R:AGGAAAGTGGATGCATTCGT n this study; repeat motif, allele size range in base pairs, and nucleotide sequence all from Wright et Page 6 of 7 (page number not for citation purposes) BMC Evolutionary Biology 2006, 6:53 http://www.biomedcentral.com/1471-2148/6/53 Presgraves DC, Severance E, Wilkinson GS: Sex chromosome meiotic drive in stalk-eyed flies. Genetics 1997, 147:1169-1180. p y 27. Hingle A, Fowler K, Pomiankowski A: Size-dependent mate pref- erence in the stalk-eyed fly Cyrtodiopsis dalmanni. Anim Behav 2001, 61:589-595. 51. Cotton S, Fowler K, Pomiankowski A: Condition dependence of sexual ornament size and variation in the stalk-eyed fly Cyr- todiopsis dalmanni (Diptera: Diopsidae). Evolution 2004, 58:1038-1046. 28. Hingle A, Fowler K, Pomiankowski A: The effect of transient food stress on female mate preference in the stalk-eyed fly Cyrto- diopsis dalmanni. Proc R Soc Lond B 2001, 268:1239-1244. 52. Wright TF, Johns PM, Walters JR, Lerner AP, Swallow JG, Wilkinson GS: Microsatellite variation among divergent populations of stalk-eyed flies, genus Cyrtodiopsis. Genet Res 2004, 84:27-40. 29. Cotton S, Rogers DW, Small J, Pomiankowski A, Fowler K: Varia- tion in preference for a male ornament is positively associ- ated with female eyespan in the stalk-eyed fly Diasemopsis meigenii. Proc R Soc Lond B 2006, 273:1287-1292. y g y p 53. Holehouse KA, Hammond RL, Bourke AFG: Non-lethal sampling of DNA from bumble bees for conservation genetics. Insect Soc 2003, 50:277-285. g 30. Burkhardt D, de la Motte I: How stalk-eyed flies eye stalk-eyed flies: observations and measurements of the eyes of Cyrtodi- opsis whitei (Diopsidae, Diptera). J Comp Physiol A 1983, 151:407-421. 54. Sokal RR, Rohlf FJ: Biometry 3rd edition. New York: Freeman; 1995. 55. Zar JH: Biostatistical analysis 3rd edition. London: Prentice-Hall; 1996. 31. 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https://openalex.org/W2883631829	https://innovation-entrepreneurship.springeropen.com/counter/pdf/10.1186/s13731-018-0085-4	English	null	Enterprise innovation in developing countries: an evidence from Ethiopia	Journal of innovation and entrepreneurship	2,018	cc-by	11,298	RESEARCH Open Access 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. Enterprise innovation in developing countries: an evidence from Ethiopia Megersa Debela Daksa1* , Molla Alemayehu Yismaw1, Sisay Diriba Lemessa1 and Shemelis Ke * Correspondence: mdebela4@gmail.com 1Department of Economics, Haramaya University, Dire Dawa, Ethiopia Full list of author information is available at the end of the article Journal of Innovation and Entrepreneurship Journal of Innovation and Entrepreneurship Daksa et al. Journal of Innovation and Entrepreneurship (2018) 7:6 https://doi.org/10.1186/s13731-018-0085-4 Abstract Enterprise innovation has gained the interest of development policymakers and scholars as the bases for the industrial development. This study comprehensively analyzes the drivers of enterprise innovation in developing countries. The study uses survey data to analyze the determinants of enterprise innovation in Ethiopia using a multivariate probit (MVP) model. For this study, enterprises were grouped into four categories: all-sized, large-sized, medium-sized, and micro- and small-sized enterprises. It appears that engagement in R & D, on-the-job training, and website ownership significantly determine enterprise innovation. This study, unlike previous studies, comprehensively analyzes drivers of innovation by considering enterprises in different sizes and all at the same time. This helps identify factors most relevant for enterprise innovation at all stage which help policymakers get focused on strategy development. Based on the findings, further emphasis on engagement in R & D would help enterprises to become innovative for all categories of enterprises. Furthermore, strengthening the available formal training and diversifying type of the training that is related to skills, knowledge, and techniques that help achieve the long-term objective of the enterprises are worth considering. Enterprises also need to subscribe to different sites that help learn more and access information. Keywords: Enterprise, Innovation, Determinants, Multivariate probit, Ethiopia * Correspondence: mdebela4@gmail.com 1Department of Economics, Haramaya University, Dire Dawa, Ethiopia Full list of author information is available at the end of the article Background Innovation is an engine for economic growth of any economy (Abderrezzak et al. 2016; Abdu and Jibir 2017; African Union Commission (AUC) 2014; Mahendra et al. 2015; NEPAD Planning and Coordinating Agency (NPCA) 2014; van Uden et al. 2016). This is because innovation commitment by a country and/or a firm is often conceptual- ized as one of the important determinants of enterprise-level productivity gains and country-level economic growth (Abdu and Jibir 2017). Enterprises are playing a crucial role in contributing to economic growth of Ethiopia by supporting science, technology, and innovation activities through research, technology transfer, and diffusion for policy formulation framework (The Federal Democratic Republic of Ethiopia (FDRE) 2010). Innovation is very important for enterprises themselves in increasing competitiveness, creating a value, determining the long-term survival, and raising productivity (Anderson and Potočnik 2014; Beyene et al. 2016; Nam et al. 2017). The ability of a country to sustain rapid economic growth, in the long run, also depends on the effectiveness with which its institutions and policies support the knowledge generation, technological Daksa et al. Journal of Innovation and Entrepreneurship (2018) 7:6 Page 2 of 19 Page 2 of 19 transformation, and innovativeness of its enterprises (Ethiopian Science and Agency Technology (ESTA) 2006). Governments and donors in the developing countries have shown increasing interest in promoting enterprise innovations and entrepreneurship to encourage enterprises. This is due to the potential role enterprise innovation play in the enterprise development for the industrial and economic development. Enterprises create job opportunities and income for the youth and poor in a developing country. The impression is that innovation is im- portant for enterprises to become and remain competitive, to move to higher return activ- ities, and to grow and graduate to a larger enterprise status, hence creating new employment and income opportunities. However, the effectiveness of such interventions by understanding the role of innovation in the growth and development of the economy depends on determining factors influencing innovation (Abdu and Jibir 2017). Most business enterprises in developing countries like Ethiopia are small- and medium-sized and face various challenges including lack of processed technological information, inadequate training capabilities at technical and vocational education training (TVET) centers, lack of access to financial and other resources and absence of consultancy support (FDRE 2010), poor infrastructural base, and unfavorable government policies which weaken their innovation activities (Abdu and Jibir 2017; Adebowale et al. Background 2014; Choi and Lim 2017; Dotun 2015; Egbetokun et al. 2016). It is in- teresting to observe that despite all the difficulties, a large share of firms can still innovate in the African context (Egbetokun et al. 2016; Abdu and Jibir 2017). The greatest challenge to understanding the role of innovation in the growth and de- velopment of the economy has been lacking meaningful data to determine the factors influencing innovation. Moreover, there has been a development of new data sources like the Enterprise Data Survey (EDS) collected by the World Bank which have spurred many empirical studies, in the developed countries, on the determinants of a firm’s innovation. Adebowale et al. (2014) argue that some ideas and concepts which have emerged in the innovation systems community have been derived from specific experi- ences in rich countries and cannot be universal templates. Perhaps the conclusion to be drawn from these studies may be misleading, inconclusive, and difficult to generalize to enterprises in developing countries. Empirical studies on determinants of innovation by small firms in Africa are relatively scarce (Abdu and Jibir 2017a; Adebowale et al. 2014). Studies conducted on enterprise innovation so far suffer from several limitations. First, they focus only on product and process innovation determinants. The conclusions and policy recommendations derived from these studies cannot be generalized to other innovation types. This is due to the fact that what fosters innovation in process innovations may inhibit/not affect organizational innovations at all. For instance, Stojčić and Hashi (2014) found that cost factors affect product innovations but do not affect process innovations. The study fur- ther reveals that firm size fosters new process innovations while it hinders new product innovations. The implication is that some determinants of innovations are specific to the type of innovation the enterprises engaged in. Second, enterprises are almost not homogeneous in size, capability, background, and sector types. Under this circum- stance, it is impossible to expect the same factors determining innovation of enterprise (Gebreeyesus 2011). This study reveals that large-sized firms and firms in the manufac- turing sector are more likely to engage in innovative activities. Similarly, Hashi and Daksa et al. Journal of Innovation and Entrepreneurship (2018) 7:6 Page 3 of 19 Page 3 of 19 Stojcic (2013) noted that under different circumstances, firm size could positively/nega- tively determine innovation. This proves that the “one-size-fits-all” principle does not work. Background Third, most of the studies of the enterprise innovation are conducted in the de- veloped countries which may challenge generalization to the developing countries. As developing countries deviate from the developed countries in institutional structures and development infrastructures, it needs due emphasis. This is because the business environment in which enterprises practice may mask the effect of the different factors on the innovation of the enterprise. There is no comprehensive empirical evidence on determinants of enterprise innovation in developing countries including Ethiopia. The existing few studies in Af- rica examined the determinants of innovative activity and attributes of innovation (Gebreeyesus 2011). Given the above research gap, this paper contributes to the narrow literature on innovations of enterprises in Ethiopia in the following ways. First, it ana- lyzes not only the determinants of product and/or process innovations but also the de- terminants of four types of innovations (that is, a new product innovation, a new method of production innovation, a new marketing innovation, and a new organizational structure). Distinguishing enterprises into different sizes helps to identify important factors regarding firms’ size. Second, to address the bias that might arise from pooling a heterogeneous group of firms, this study tries to investigate the deter- minants of innovation by classifying enterprises into all-sized, large-sized, medium-sized, and micro- and small-sized enterprises. Third, contrasting to most of the earlier studies, this study covers not only manufacturing but also retail services and non-retail services. The rest of the paper is organized as follows. The next section presents a brief litera- ture review. In the third section, the data and method of data analysis is presented. Re- sults and discussions are discussed in the “Results and Discussion” section. Lastly, the conclusions and policy implications are discussed. Literature review The Organization for Economic Cooperation and Development (OECD) defines innovation more broadly as the implementation of a new or significantly improved product (that is, a physical good or service), a process, a new marketing method, or a new organizational method in business practices, workplace organization, or external relations (Organizations for Economic Co-operation (OECD) 2010). Enterprise innova- tions can arise at different points in the development process, including conception, R & D, transfer of the technology to the production organization, production, and marketplace usage (Atkinson 2013). A wide range of factors affects innovation process, including firm size and age, re- search and development (R & D) efforts, the quality or skill level of managers/em- ployees, employee participation and motivation, managerial practices and inter-departmental cooperation and knowledge exchange, factors related to the firms’ network and its interactions with outside organizations, and factors specific to the in- dustry (Egbetokun et al. 2016). External market target, capacity building, facilitative support to enterprises, and en- trepreneur’s characteristics determine the innovation ability of enterprise. Enterprises’ Daksa et al. Journal of Innovation and Entrepreneurship (2018) 7:6 Page 4 of 19 Page 4 of 19 characteristics such as size of the enterprises (Hadhri et al. 2016; De Mel et al. 2009; Stojčić and Hashi 2014; Zemplinerová and Hromádková 2012) and enter- prise’s maturity (Zakic et al. 2008) determine innovation of the enterprises. By im- plication, larger and mature enterprises are more innovative than the smaller and less mature enterprises. Studies show that enterprises’ external market target and strategic relation for- mation determine enterprises’ innovation. Foreign market access for the enter- prises would help enjoy the large market size for their goods and services and help earn foreign currency which will have a multiplier effect on their activities. Strategic relation behavior of the enterprises would help them with whom to make collaboration in international and national entities. This would help enter- prises advance their business. Enterprises that use foreign inputs and that have collaboration with foreign are interrelated (Avermaete et al. 2004). Foreign market orientation of enterprises also determines enterprise innovation (De Mel et al. 2009; Stojčić and Hashi 2014; Zakic et al. 2008; Zemplinerová and Hromádková 2012). This shows that firms that are foreign market-oriented have experience and strategic relation with foreign sectors and are more innovative than their counterparts. The enterprise’s capacity level related to investment in human capital of the en- terprises determines the enterprise’s innovation. Literature review 2009), the age of the entrepreneurs, and the gender of the entrepreneurs (Gebreeyesus 2011) explain firms’ innovativeness. Here, the higher the educational level, the younger, and the more the male owners, the more the firms are innovative. Factors exogenous to the enterprises are also found to be important determinants of the enterprises. These factors are less controllable by the enterprises by themselves. En- terprises that are more active in using available external resources and supports are more likely to be innovative. Few studies showed that facilitative supports such as gov- ernment support, availability of patent and copyright (Dotun 2015), better institutional quality at the local, access to finance (Mahendra et al. 2015), and the use of external sources of information (Avermaete et al. 2004) determine firms’ innovation. These studies’ focus contended that external support to the firms determines the firms’ innovation. There are factors which positively and negatively affect enterprise innovations. For instances, foreign ownership of the enterprises (Zemplinerová and Hromádková 2012) and competition (De Mel et al. 2009) negatively affect an enterprise’s innovation. Even, there are also factors that do not affect the firms’ innovative ac- tivities. Characteristics of the entrepreneur (Avermaete et al. 2004), regional net- works, and close customer relations (Romijn and Albaladejo 2002) do not determine enterprises’ innovation. Other studies have emphasized on the importance of the innovation for survival in a volatile environment (Johnson et al. 1997). Some studies that have dealt with enter- prises’ innovation even did not conduct their study by unraveling firms into different respective sizes. Distinguishing enterprises in terms of their size help to identify more relevant factors affecting enterprise innovation. Factor that is more important for a small enterprise may not be important for the large or medium enterprise and vice versa. Identifying factors important in all cases is also worth dealing as it helps policy- makers to get focused in devising enterprise innovation and industrial development strategies. The literature that deals with the characteristics of enterprise innovation activ- ities and connects innovation and other enterprise activities are concerned with the context and content of innovation processes. The focus of the literature, in this case, is whether enterprise innovation activities are related to the existence of R & D activities. The R & D activity is an indispensable part of enterprise innovation activities. Literature review Investment in human capital af- fects the ability, skills, and knowledge of the workforce of the enterprises. These investments affect innovation of the enterprise. Several studies proliferated this issue. For instance, van Uden et al. (2014) analyzed the impact of human capital innovation in developing countries (Kenya, Tanzania, and Uganda) using data from the Enterprise Surveys of the World Bank and found that human capital spurs innovation. Mahendra et al. (2015) also argued that human capital affects innovation abilities of enterprises in Indonesia. Mahendra et al. (2015) further showed that different combinations of human capital affect innovative output de- pending on the context in which these combinations are implemented (manufac- turing or service sector). Moreover, Audretsch et al. (2016) added that academic-based human capital encourages innovativeness of enterprises while business-based human capital does not play a role. The firms’ extent of investment in the R & D, skills of the firms’ workforce, the firms’ investment in know-how (Avermaete et al. 2004; Dotun 2015; Raymond and St-Pierre 2010; Romijn and Albaladejo 2002), and the use of known technology transfer mechanisms (Hadhri et al. 2016) determine the enterprises’ propensity to innovate. This shows that the capacity of the enterprises explains their innovative ability. Empirical evidence shows that the most important factor of innovation is R & D activity though findings are mixed. El Elj and El Elj (2012) argued that the value of the R & D activity is related to the core competencies of the firm and to its efficient innovative processes in Tunisia. However, Aralica et al. (2008) found that continuous engagement in R & D and R & D cooperation has turned out to be insignificant in relation to the share of sales of innovative products in Croatia. Some argue in low- and medium-technology industries, creativity, not technological knowledge, is the driver of innovation, because in those industries, Daksa et al. Journal of Innovation and Entrepreneurship (2018) 7:6 Page 5 of 19 Page 5 of 19 innovation is based on the general knowledge stock of the firm and the creativity to transform such a stock, instead of scientific research (Goedhuys et al. 2014; Santamaría et al. 2009). Studies also witness that owner’s and entrepreneur’s specific characteristics determine enterprise innovation. Owner’s characteristics such as the educational background of the owner, prior experience of owner-manager (Avermaete et al. 2004), owner’s ability personality traits (De Mel et al. Literature review A significant amount of innovation and improvements origi- nates from design improvements like “learning by doing” and “learning by using” (Arrow 1962; Mowery and Rosenberg 1989), and such informal efforts are em- bodied in people and organizations (Teece 1986a, 1986b). These literatures stress an importance of the experience of the enterprises that emanates from the on-the-job training. Daksa et al. Journal of Innovation and Entrepreneurship (2018) 7:6 Daksa et al. Journal of Innovation and Entrepreneurship (2018) 7:6 Page 6 of 19 Other literatures point out the link between innovation and enterprise-level determi- nants of innovation characteristics such as firm size (Aralica et al. 2008; Mahendra et al. 2015). Following the work by Schumpeter (1942), there has been a wide-ranging de- bate on the differences and complementary qualities of small and large firms in the face of innovation and technological change. As per Schumpeter (1942), large firms have ad- vantages in comparison with small ones when taking part in innovation activities and, what is more, these advantages increase according to firm size. In addition, size emerges as a primary internal force driving technological innovation (Alsharkas 2014) and its relevance is motivated by several intertwined arguments. This hypothesis has been reviewed in various empirical studies without any definite conclusion being reached that there is a positive relationship between the propensity to innovate and firm size for Sri Lanka (De Mel et al. 2009), for Lebanese (Hadhri et al. 2016), for Nigeria (Moohammad et al. 2014), and for Ethiopia (Gebreeyesus 2011). On the other hand, some scholars (Martínez-ros and Labeaga 2002); Plehn-Dujowich 2009) argue that firm’s size and innovation abilities are inversely related because they are more dy- namic in the decision to innovate. Some studies found innovation to be negatively re- lated to firm size for Croatia (Aralica et al. 2008). Some of the authors found an inverted-U relationship between firm size and R & D intensity, i.e., the ratio of R & D expenditure or personnel to size, or between firm size and the ratio of patents to size (Koouba, Karim et al. 2010).Others found a positive relationship up to a certain thresh- old and no significant effect for larger firms. The inconclusive results regarding the effect of firm size on innovative capacity of the firms justify the inclusion of many control variables to get robust results (Hadhri et al. 2016). For instance, a systematic review by Becheikh et al. Literature review (2006) shows there are about 40 determinants concerning the characteristics of innovating firms. According to Becheikh et al. (2006), these driving forces of innovation are categorized into internal determinants of innovation and contextual determinants of innovation. Internal deter- minants of innovation include firms’ general characteristics (age of the firm, ownership structure, past performance), firms’ global strategies (export/internalization, external/in- ternal growth), firms’ structure (formalization, centralization, and interaction), manage- ment team (leadership variables and manager-related variables), and functional assets and strategies (R & D, human resources, finance, etc.). Contextual determinants of innovation are firms’ industry-related variables (sector, demand growth, industry con- centration), firm’s regional variables (geographic location and proximity advantage), networking, knowledge/technology acquisition, government and public policies, and surrounding culture. The impact of these internal and contextual determinants of firm’s innovation activ- ities have been studied in developing countries showing varying, inconclusive, and contradictory results (Becheikh et al. 2006; Hadhri et al. 2016). Result and discussion Descriptive statistics results Descriptive statistics results Description of variables used in the study and their descriptive statistics are presented in Table 1. The descriptive result shows that, during the last 3 years, 40% of enterprises introduced a new product innovation; 34% of them introduced a new method of Page 7 of 19 Page 7 of 19 Daksa et al. Journal of Innovation and Entrepreneurship (2018) 7:6 Table 1 Summary of descriptive statistics results Variable Variable description and measurement Obs Mean Std. dev. Min Max h1 (new product innovation) Dummy variable that takes the value one (1) if the enterprise has introduced new product, 0 otherwise. 1487 0.40 0.49 0 1 h3 (new method of production innovation) Dummy variable that takes the value one (1) if the enterprise has introduced new method of production, 0 otherwise. 1488 0.34 0.47 0 1 h5 (new organizational structure innovation) Dummy variable that takes the value one (1) if the enterprise has introduced new organizational structure, 0 otherwise. 1485 0.30 0.46 0 1 h6 (new marketing methods innovation) Dummy variable that takes the value one (1) if the enterprise has introduced new marketing methods, 0 otherwise. 1488 0.34 0.47 0 1 a6b (size) It is a categorical variable that takes the values 0, 1, 2, and 3 if the enterprise is micro, small, medium, and large respectively. It measures the size of the enterprise. 1492 1.71 0.82 0 3 a3b (location of the enterprise) It is a dummy variable that takes the value one (1) if the enterprise is in the capital city, 0 otherwise. 1492 0.62 0.49 0 1 Fage (age) It is the age of the enterprise measured in years. 1476 13.67 12.02 0 89 Fagesqr (age square of the enterprise) It is the age square of the enterprise 1476 331.21 753.31 0 7921 b7 (experience of top manager) It is the years of experience of the top manager measured in years. 1466 14.02 9.95 0 60 b7a (gender of top manager) It is a dummy variable that takes the value one (1) if female, otherwise 0. 1491 0.10 0.30 0 1 c22b (website ownership) It a dummy variable that takes value one (1)1 if enterprise owns website, 0 otherwise. 1485 0.38 0.49 0 1 d3c (share of direct export) It is a continuous variable that measures enterprises’ share of export measured in percentage. Source: Own computation, 2017 production innovation; 30% of them introduced a new organizational structure innovation; and 34% of them introduced new marketing methods. From the descriptive result, it is showed that 1.74% enterprises were micro-enterprises, 46.38% of them were small enterprises, 30.63% of them were medium enterprises, and 21.25% of them were large enterprises. The result also showed that majority of the enterprises (62%) is in the capital city of the country. Descriptive statistics results 1484 4.95 19.10 0 100 l9b (education) It is a continuous variable that measures the percentage of full-time permanent workers who completed secondary school. 1457 68.03 29.73 0 100 l10 (formal training) It is a dummy variable that takes the value one (1) if there is availability of formal training programs for permanent full-time employees, otherwise 0. 1486 0.22 0.41 0 1 l1 (permanent workers) It is a continuous variable that measures the number of permanent full-time workers in the enterprise. 1467 96.31 361.73 1 7600 h7 (formal R & D) It is a dummy variable that takes the value one (1) if the enterprise has spent on formal R & D activities that last 3 years, otherwise 0. 1488 0.14 0.34 0 1 Source: Own computation, 2017 production innovation; 30% of them introduced a new organizational structure innovation; and 34% of them introduced new marketing methods. From the descriptive result, it is showed that 1.74% enterprises were micro-enterprises, 46.38% of them were small enterprises, 30.63% of them were medium enterprises, and 21.25% of them were large enterprises. The result also showed that majority of the enterprises (62%) is in the capital city of the country. Daksa et al. Journal of Innovation and Entrepreneurship (2018) 7:6 Daksa et al. Journal of Innovation and Entrepreneurship (2018) 7:6 Page 8 of 19 Page 8 of 19 About 10% enterprises had a female top manager. There were 38% enterprises that had and own a website. During the last 3 years, about 22% of the enterprises conducted formal training programs for their permanent full-time employees, while only 14% of them spend on formal R & D activities. The average age of the enterprises is 14 years. The mean top manager’s experience is 14 years. On average enterprises, the share of the direct export is about 5%. Out of the full-time permanent workers of the enterprises, 68% of them have above secondary school education level. Table 1 presents the minimum, maximum, and standard deviations. p ; p Source: Own computation 2017 (from World Bank data) Econometric analysis Tables 2 and 3 present the estimated effects of the multivariate probit model on factors affecting enterprises’ innovation (new product innovation, a new method of production Table 2 Multivariate probit results of parameter estimates Variable All-sized Large-sized H1 H3 H5 H6 H1 H3 H5 H6 a6b 0.161 0.216 0.215 0.062 (2.97) (4.04) (3.81)** −1.08 a3b 0.155 0.103 −0.02 0.025 0.153 0.125 0.232 0.194 (2.02)* −1.33 −0.24 −0.3 −0.85 −0.67 −1.21 −1 Fage −0.004 −0.004 −0.002 0.004 −0.004 −0.012 −0.002 0.007 −0.44 −0.43 −0.21 −0.43 −0.24 −0.76 −0.15 −0.4 Fagesqr 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 −0.63 −0.78 −0.08 −0.03 −0.34 −1.04 −0.31 −0.13 b7 0.007 −0.001 −0.003 −0.009 0.003 −0.004 −0.009 0.002 −1.8 −0.28 −0.72 (2.22)* −0.34 −0.5 −1.07 −0.25 b7a 0.086 0.109 0.014 −0.09 −0.438 −0.126 −0.327 −0.452 −0.74 −0.96 −0.11 −0.69 −1.35 −0.4 −0.96 −1.3 c22b 0.283 0.455 0.498 0.561 0.333 0.115 0.248 0.175 (3.44) (5.41) (5.71) (6.43) −1.94 −0.66 −1.35 −0.91 d3c −0.001 0 −0.001 0 0 0.001 −0.004 −0.003 −0.72 −0.08 −0.28 −0.1 −0.07 −0.29 −1.09 −0.78 l9b 0.004 0.003 0.005 0.003 0.001 0.001 0.004 0.003 (3.41)** (2.36)* (3.22)** (2.46)* −0.24 −0.41 −1.2 −0.85 l10 0.475 0.426 0.645 0.499 0.388 0.453 0.537 0.674 (5.20) (4.57) (6.97) (5.24) (2.35)* (2.67) (3.17) (3.85) l1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 −0.49 −1.53 −1.26 −1.27 −0.85 −1.38 −0.82 −1.05 h7 0.574 0.5 0.731 1.164 0.336 0.482 0.953 1.458 (5.13) (4.51) (6.59) (9.59)** −1.8 (2.46)* (5.00) (6.48) _cons −1.408 −1.468 −1.615 −1.21 −0.484 −0.327 −1.004 −1.212 (7.22) (7.37) (7.45) (5.54) −1.09 −0.75 (2.16)* (2.40)* N 1376 280 p < 0 05; p < 0 01 Page 9 of 19 Daksa et al. Econometric analysis Journal of Innovation and Entrepreneurship (2018) 7:6 Page 9 of 19 Table 3 Multivariate probit innovation determinants result (continued) Variable Medium-sized Micro- and small-sized H1 H3 H5 H6 H1 H3 H5 H6 a6b – – – – – – – – – – – – – – – – a3b 0.194 0.099 0.055 0.139 0.147 0.149 −0.147 −0.062 −1.35 −0.7 −0.36 −0.92 −1.3 −1.28 −1.11 −0.49 Fage −0.014 0.015 0.013 0.01 −0.001 −0.029 −0.029 −0.007 −0.69 −0.75 −0.75 −0.48 −0.05 −1.46 −1.64 −0.38 fagesqr 0.0 0.0 0.0 0.0 0.0 0.0 0.001 0.0 −0.31 −0.86 −1.55 −0.51 −0.64 −1.08 −1.66 −0.46 b7 0.006 −0.001 0.009 −0.002 0.01 0.009 −0.004 −0.017 −0.83 −0.15 −1.15 −0.28 −1.64 −1.23 −0.56 (2.22) b7a 0.156 0.102 0.01 0.12 0.156 0.325 0.12 −0.119 −0.81 −0.47 −0.05 −0.52 −0.97 (2.10)* −0.63 −0.62 c22b 0.264 0.113 0.364 0.463 0.313 1.027 0.792 0.828 −1.93 −0.81 (2.49)* (3.20)** (2.36)* (7.55) (5.57) (6.02) d3c 0.001 −0.002 −0.005 −0.003 −0.005 0 0.004 0.003 −0.19 −0.68 −1.45 −0.95 −1.51 −0.12 −1.26 −0.85 l9b 0.002 0.003 0.004 0 0.007 0.003 0.005 0.005 −0.88 −1.24 −1.71 −0.07 (3.71) −1.66 (2.36)* (2.24)* l10 0.329 0.276 0.452 0.251 0.675 0.494 0.813 0.564 (2.20)* −1.82 (2.90) −1.64 (3.80) (2.61) (4.41) (3.10) l1 0.002 0.002 0.008 0.003 −0.008 0.015 0.007 0.003 −0.79 −0.61 (3.00) −1.16 −0.79 −1.39 −0.61 −0.27 h7 0.709 0.626 0.781 1.164 0.866 0.76 0.654 1.242 (3.91) (3.50) (4.21) (6.28) (3.68) (3.15) (2.87) (5.18) _cons −0.934 −1.068 −1.655 −1.214 −1.49 −1.562 −1.266 −1.028 (2.71) (2.92) (4.47) (3.16) (4.99) (5.14) (3.71) (2.93) N 425 671 p < 0.05; *p < 0.01 S O t ti 2017 Table 3 Multivariate probit innovation determinants result (continued) innovation, a new marketing method innovation, and a new organizational structure innovation) based on two scenarios (regardless of enterprises’ size and based on their size). Analyzing determinants of enterprises’ innovation endeavors by segregating enter- prises into different size helps to identify important size-dependent factors that affect enterprises’ innovation. It helps to uncover factors which determine enterprises’ innovation abilities regardless of the enterprises’ size. In what follows, we present and discuss the determinants of enterprise innovation. Then, we conclude and recommend. + indicates variables are significant in determining the innovation Innovation in all-sized enterprises The multivariate probit regression result shows that website ownership, the percentage of full-time permanent workers who completed secondary school, the availability of for- mal training programs for permanent full-time employees, and engagement of the Daksa et al. Journal of Innovation and Entrepreneurship (2018) 7:6 Page 10 of 19 Page 10 of 19 enterprises in R & D activities significantly affect the four enterprises’ innovations irre- spective of the enterprises’ size (see Table 4 for a summary of the main results). The implication of this finding is that enterprises, regardless of their size, that have access to information, have more educated permanent full-time workers, have a regular on-the-job training program for the workers, and conduct research and development are more innovative than their counterparts. Table 4 Main results of the finding Variables Website ownership: 1 yes, 0 otherwise Percentage of full-time permanent workers who completed secondary school Formal training programs for its permanent, full-time employees: 1 yes, 0 otherwise Spending on formal research and development activities last 3 years: 1 yes, 0 otherwise All-sized New product innovation (H1) + + + + New method of production innovation (H3) + + + + New organizational structure innovation (H5) + + + + New marketing method innovation (H6) + + + + Large-size New product innovation (H1) + + New method of production innovation (H3) + + New organizational structure innovation (H5) + + New marketing method innovation (H6) + + Medium- sized New product innovation (H1) + + New method of production innovation (H3) + + New organizational structure innovation (H5) + + + New marketing method innovation (H6) + + + Micro- and small-sized New product innovation (H1) + + + + New method of production innovation (H3) + + + + New organizational structure innovation (H5) + + + + New marketing method innovation (H6) + + + + + indicates variables are significant in determining the innovation Page 11 of 19 Daksa et al. Journal of Innovation and Entrepreneurship (2018) 7:6 Daksa et al. Journal of Innovation and Entrepreneurship (2018) 7:6 Page 11 of 19 Enterprises which have their own website are more likely innovative than those do not have a website. Social networking sites like website provide information about indi- viduals and their networks which enables enterprises to create online social communi- ties shared by external stakeholders. Innovation in all-sized enterprises A website helps enterprises interact with external factors such as customers and public institutions. This helps enterprise get, transfer, and assimilate external knowledge within the enterprise and then generate innovation. Moreover, according to the triple helix theory, the success of innovation endeavors de- pends on what integration and cooperative interaction develop between the academia, the private sector, and the government which is shaped by the social networking sites. Our finding is in line with that of Scuotto et al. (2016), Martins (2016), Guo et al. (2016), and Del Giudice et al. (2016). Having a website may help enterprises to use all possible available resources in the world via the Internet. These resources may be re- lated to new technologies (production), knowledge, and techniques helpful in upgrad- ing the method of production, management of resources, marketing of the products, and so on. They may also use the Internet to identify areas of more demanded products they focus on. Enterprises may conduct an assessment of their product, a method of production, and management through an online survey using their website. Thus, web- site ownership may determine enterprise innovation through the provision of important information, resources, and online survey services. Tables 2 and 3 show that different aspects of human capital (general level of school- ing and formal on-the-job training) ignite enterprises’, regardless of size, innovation of all types. Enterprises investing in formal training programs for its permanent and full-time employees are more likely innovative than otherwise because it is a worker with knowledge and skill who can generate new knowledge and ability to absorb new knowledge created by other enterprise’s employees. Another component of human cap- ital which is a driving force for innovation in this study is a level of schooling attained by the permanent employees. The result shows that the percentage of employees of the enterprises who completed secondary school increases the enterprises’ chance of inno- vativeness. This is because a high number of workers who completed secondary school generates a high level of knowledge and techniques and induces enterprises to develop innovative new practices. The employee of the enterprises with a high school education level may learn from each other, and this may have spillover effects. The spillover ef- fects of this education even may spread to the enterprise’s employee with a lower level of education. Innovation in all-sized enterprises In this way, even employees with a lower level of education may gain ex- perience and this would stimulate the whole activities of the enterprise. The enterprise’s employee with more than a high school education may also have different technical education and experience. Thus, schooling and on-the-job training are an enabling fac- tor in profitable innovation which suggests that investments in skills help expand the group of firms in the economy that have the potential to innovate. This finding is cor- roborated by Abdu and Jibir (2017), D’Este et al. (2014), Dostie (2014, 2018), Sun et al. (2017), van Uden et al. (2014), and van Uden et al. (2016). The result reveals that an enterprise’s propensity to introduce innovation is higher when it spends on R & D. Involvement in research and development would help the enterprise search new things, to adopt, to develop, and to use them to achieve the en- terprise’s objectives. As research and development are concerned with searching new mechanisms that solve problems, enterprises also use research and development to Daksa et al. Journal of Innovation and Entrepreneurship (2018) 7:6 Page 12 of 19 Page 12 of 19 advance to their predetermined goals. Research and development may help enterprises to use the available internal and external resources. Research and development added to the on-the-job training would enhance the absorption capacity and stock of know- ledge of the enterprises that would induce innovation of the enterprises. The findings of Rehman (2016) conducted in India and those of Abdu and Jibir (2017) in Nigeria corroborate this finding. The study support that R & D has a positive impact on the product and process innovation. Another study also shows that enterprises that re- ceived a grant for research and development increase the probability that a firm intro- duces new goods and services to the world (Jaffe and Le 2015). A study by Yuan et al. (2014) shows that R & D investment intensity positively determines the firm’s innovation though the relationship is weak. However, this study showed that the effects of R & D on process innovation and any product innovation are much weaker. The top manager’s experience in years determines a new method of marketing innovation. A longer time the manager stays in the enterprises enriches the experience of the manager in every aspect of the enterprises. Innovation in all-sized enterprises It might also provide an opportunity for the manager to deal with the innovation of the enterprise. The manager of the en- terprise knows the areas that need improvement, and probably, it is the top manager that is exactly keen for the accomplishment of the strategic objective of the enterprise. In this case, the longer the stay of the top manager in the enterprise, the more experi- enced is the manager about the enterprise. It is argued that managers are likely to have better insights into future business opportunities, threats, niche markets, products, technologies, and market development; in this case, top managerial experience is ex- pected to be positively related to innovative activity and its performance. Managerial experience enhances both the propensity to innovate and the innovative firm perform- ance, as measured by the share of sales accounted for by new products (Balsmeier and Czarnitzki 2014). Thus, the experience of the top manager would help peruse marking innovation that helps achieve the objective. However, a study by Yuan et al. (2014) indi- cated that the top management team’s tenure and firm innovation are negatively related. An enterprise’s size determines innovation in all cases except the new market innovation. The size of the firm goes with the capital and human capital. The higher the size of the enterprises, the more they can afford training, R & D, and education and the more the enterprises are innovative. A larger enterprise can amortize fixed costs over a broader base and will, therefore, be more innovative than smaller firms. More- over, due to their broad base of resources and capabilities, large enterprises are more likely innovative as compared to small ones. The assertion that the size of the enter- prise positively affects the innovativeness of the enterprise is also supported by van Uden et al. (2014), van Uden et al. (2016), Leyden et al. (2014), Chowhan (2016), and Mahendra et al. (2015). Location of the enterprise significantly determines new product innovation. The fact that enterprises that are located in the capital city of the country are more innovative than enterprises located outside the capital city can be explained by the compounding effect of the city and the localization (urbanization) economies of the enterprises. The compounding effect of the city is related to the government emphasis on all sectors in the city including the enterprise development. Innovation in large-sized enterprises For the large-sized enterprise, the MVP regression shows that only the availability of formal training programs for permanent full-time employees and the engagement in formal R & D activities significantly determine the four types of enterprise innovations. This finding suggests that enterprises that emphasize on the on-the-job training of the employee and research and development are more innovative than others. This finding convinces that enterprise innovation whether it is a new product or a new process or new management or new market innovation, human capital accumulation through training, research, and development is indispensable. In this study, it is also indicated that in the all-sized enterprises, training and R & D enhance the innovation of the enterprises. Innovation in medium-sized enterprises Regarding medium-sized enterprise innovation, the MVP regression shows that avail- ability of formal training programs for permanent full-time employees and engagement in formal R & D activities determine new product innovation. Engagement in R & D activities determine a new method of the production innovation. The new organizational structure innovation is determined by website ownership, the availability of formal training programs for permanent full-time employees, the number of per- manent full-time workers, and the engagement in formal R & D activities. Here, per- manent full-time worker increases determines the new organizational structure innovation. The explanation for this is that with an increased number of the full-time permanent workers, diverse ideas and experiences would interact that adds to the en- terprise innovativeness. Website ownership and engagement in R & D determine a new method of marketing innovation. In the medium-sized enterprises, the only variable that affects the four enterprise innovations is an engagement in formal R & D. Innovation in all-sized enterprises The localization (urbanization) econ- omies’ effect is related to that enterprise densely populated in the city which may easily Daksa et al. Journal of Innovation and Entrepreneurship (2018) 7:6 Page 13 of 19 Page 13 of 19 learn from each other either in the formal or informal or in both ways. This may thus help enterprises located in the capital city to be more innovative than others. The asser- tion here is that in the capital cities’ information, the capital (human and physical) eas- ily and freely moves from one enterprise to another. This finding is corroborated by the case of Silicon Valley which is well known for being a learning region and where a successful innovation system has been implemented (Doloreux 2003). Porter and Stern (2001) also argue that location matters for innovation; particularly, most attractive loca- tions enhance the environment for innovation. Innovation in micro- and small-sized enterprises For the micro- and small size, the regression result shows that new product innovation is determined by website ownership, a percentage of full-time permanent workers who completed secondary school, the availability of formal training programs for permanent full-time employees, and engagement in formal R & D activities. The new method of production innovation is determined by the sex of the top manager, the website owner- ship, and the availability of formal training programs for permanent full-time em- ployees and engagement in R & D activities. Here, micro- and small enterprises, which have a female as a top manager, are more innovative in a new method of production Daksa et al. Journal of Innovation and Entrepreneurship (2018) 7:6 Page 14 of 19 Page 14 of 19 innovation. Some empirical studies also contend that female representation in top man- agement improves firm performance that focuses on innovation (Dezsö and Ross 2012). In contrast, in hiring more female managers, companies can be more innovative, but having a top female at the top position negatively influences the innovation if the number of female is lower in the top management team (Lyngsie and Foss 2017). The new organizational structure innovation is determined by website ownership, a percentage of full-time permanent workers who completed secondary school, availabil- ity of formal training programs for permanent full-time employees, and engagement in R & D activities. The new method of marketing innovation is determined by years of experience of a top manager, website ownership, a percentage of full-time permanent workers who completed secondary school, availability of formal training programs for permanent full-time employees, and engagement in R & D activities. The top manager’s experience determines the new method of marketing innovation in micro- and small enterprises. This is explained that as the top manager works more in the sector, he/she will be experienced in dealing with the selling of product and services. For the micro- and small enterprise, the regression results showed that website own- ership, availability of formal training programs for permanent full-time employees, and engagement in R & D activities affect the four enterprises innovations. Conclusions This study comprehensively examined the main determinants of an enterprise’s innovation in Ethiopia using a secondary data collected by World Bank. To achieve the objective, the study MVP model was used. This study categorized the enterprises into four groups, unlike other studies which focus on either enterprise of a specific size or enterprises regardless of size. Our findings show that in all-sized enterprises, website ownership, a percentage of full-time permanent workers whose education is above the secondary school, availability of on-the-job training, and engagement in R & D activ- ities are factors that affect enterprises’ innovations. The MVP regression result indi- cated that for the large-sized enterprises, only the availability of formal training programs for permanent full-time employees and the engagement in R & D activities determine the four enterprise innovations. For the medium-sized enterprises, the re- gression result shows that engagement in R & D fosters the four innovations. In the case of micro- and small enterprises, the variables that affect the four enterprise inno- vations are website ownership, the availability of formal training programs for perman- ent full-time employees, and engagement in R & D activities which encourage four of the innovations for micro- and small enterprises. The finding of the study has strong theoretical implications. First, the finding that school- ing and training and R & D drive innovativeness in performance of the enterprise goes with several empirical findings. For instance, schooling and training are important sources of innovation (Abdu and Jibir 2017; D’Este et al. 2014; Dostie 2014, 2018; van Uden et al. 2014; van Uden et al. 2016). Further, R and D contributes to the innovation (Abdu and Jibir 2017; Jaffe and Le 2015; Yuan et al. 2014). And this goes back to replicate Becker’s (1964) notion that maintaining humans possess human capital (skills, knowledge, ability) that can be improved and can impact how people act and affect the business entity. Second, the finding that shows website ownership drives an innovation of enterprise replicates the works of Scuotto et al. (2016), Martins (2016), Guo et al. (2016), Del Daksa et al. Journal of Innovation and Entrepreneurship (2018) 7:6 Page 15 of 19 Page 15 of 19 Giudice et al. (2016), and Bresciani and Ferraris (2016) which contend that social net- working sites, global knowledge, and enterprise embeddedness contribute to the innovation performances of enterprises. Conclusions And this further goes in line with the phenomenon reflected by Schumpeter (1942) that creative destruction produces prod- uct and process innovation and knowledge-intensive entrepreneurship (that can be ob- tained with the help of information through a website) for entrepreneurs that strive to cope with uncertainty generate changes or creative destructions. The finding of the study also has strong policy implications. It suggests that de- velopment partners, policymakers, and enterprises should emphasize on R & D activities, regular on-the-job training, education, and development of a website (information access via the Internet). Specifically, the following policy recommen- dations help an enterprise enhance their innovation performance: first, conduct- ing on-the-job training on a regular basis to upgrade employee’s schooling, skill, and efficiency; second, developing and expanding enterprise’s website for acquir- ing reliable information; and third, activating new and strengthening the existing R & D activities which are salient strategies that can promote enterprises’ innova- tions and achieve their objectives. Conducting on-the-job training on a regular basis to upgrade employee’s skill and ef- ficiency would boost the capacity of the employee of the enterprises. This can be con- ducted based on the identified areas on which employee needs training. In this case, careful human power planning that considers the needs of the enterprises and em- ployees is vital. Training that can be pursued can be specific to the enterprise innova- tions or general. Indeed, it should also be conducted in a regular, sustainable, and variety of manner that ensures the sustainability of the enterprise operation. Website ownership of the enterprises is indispensable to get information worldwide in this globalization era. Here, an enterprise needs to develop their own website for gaining reliable information that boasts their innovation. Only, developing website does not suffice for promotion of the enterprise innovation; the enterprise also needs to sub- scribe to the international institutions that encourage their betterment. The R & D activities can be strengthened by allocating a reasonable amount of budget for R & D, by encouraging their workers to conduct R & D, and by making some linkages with institutions that have ample experience in R & D activities. Con- cerned bodies may incentivize their worker to conduct R & D that result in important enterprise innovations that would have a long-lasting impact on the productivity and profitability of the enterprises. Conclusions Finally, this study is limited to the Ethiopian enterprises and difficult to generalize to all developing countries. The study also used all enterprises. Therefore, future researchers that emphasize on the enterprises’ innovation better consider different countries in the develop- ing countries. Future researchers may also study innovation performances of enterprises based on the sector type, for instance, manufacturing enterprises and trade enterprises. Data source and analytical methods This study used the 2015 Ethiopia Enterprise Surveys (ES) data collected by the World Bank (World Bank 2016) from June 2015 to February 2016. The ES is a panel data Daksa et al. Journal of Innovation and Entrepreneurship (2018) 7:6 Page 16 of 19 Page 16 of 19 which are an ongoing World Bank project in collecting both objective data based on enterprises’ experiences and enterprises’ perception of the environment in which they operate. The sample for the 2015 Ethiopia’s enterprise survey was selected using strati- fied sampling, following the standard methodology. Three levels of stratification were used in the country: industry, establishment size, and region. Industry stratification was designed in the way that follows: the universe was stratified into four manu- facturing industries (food and beverages), textile and garments including leather, non-metallic mineral products, and other manufacturing and three service sectors (transportation, retail) and other services. Size stratification was defined as follows: small (5 to 19 employees), medium (20 to 99 employees), and large (more than 99 employees). Regional stratification for the 2015 Ethiopia ES was done across six geographic regions: Addis Ababa and Dire Dawa City administrations and Amhara, Oromia, SNNPR, and Tigray regional states. For this study, the data were pooled together. Competing interests p g The authors declare that they have no competing interests. Stating the multivariate probit model Journal of Innovation and Entrepreneurship (2018) 7:6 Page 17 of 19 Page 17 of 19 Y ij ¼ X 0 ijBj þ εij ð1Þ ð1Þ Y ij ¼ X 0 ijBj þ εij where Yij (j = 1,...,m) represents the enterprise innovation (in our case, m = 4) taken up by the ith entereprise (i = 1,..., n), Xij is a 1 × k vector of observed variables that are ex- pected to correlate with the enterprises innovation, Bj is a k × 1 vector of unknown pa- rameters (to be estimated), and εij is the random error term. In this specification, each Yj is a binary variable, and thus, Eq. (1) is a system of m equations (m = 4, in this case) to be estimated: Y 1 ¼ α1 þ XB1 þ ε1 Y 2 ¼ α2 þ XB2 þ ε2 Y 3 ¼ α3 þ XB3 þ ε3 Y 4 ¼ α4 þ XB4 þ ε4 8 > > < > > : ð2Þ Y 1 ¼ α1 þ XB1 þ ε1 Y 2 ¼ α2 þ XB2 þ ε2 Y 3 ¼ α3 þ XB3 þ ε3 Y 4 ¼ α4 þ XB4 þ ε4 8 > > < > > : ð2Þ ð2Þ with Y 1; Y 2; Y 3; Y 4 as a set of four latent variables underlying each of the enterprise’s innovation such that Y j ¼ 1 if Y j > 0; 0 otherwise. Additional file Additional file 1: Supporting Material. (DTA 1925 kb) Availability of data and materials Additional file Additional file 1: Supporting Material. (DTA 1925 kb) Stating the multivariate probit model Behavioral response models with more than two possible outcomes are either multinomial or multivariate. Multinomial models are suitable when respondents can choose only one outcome among the set of mutually exclusive and collect- ively exhaustive choices. However, in this study, the innovation variables are not mutually exclusive, considering the possibility of the simultaneous involvement of innovation types and the potential correlations between them. Specifically, we examine factors related to different innovations with the following enterprise in- novations: new product innovation, new method of production innovation, new organizational structure innovation, and new organizational innovation. The first innovation-dependent variable, new product innovation (h1), takes the value 1 if the enterprise has introduced new or significantly improved products or services during the last 3 years, otherwise 0. The second innovation-dependent variable, new method of production innovation (h3), takes the value 1 if the enterprise has introduced any new or significantly improved methods of manufacturing products or offering services during the last 3 years, otherwise 0. The third innovation-dependent variable, new organizational structure innovation (h5), takes the value 1 if the enterprise has introduced any new or significantly improved organizational structures or management practices during the last 3 years, other- wise 0. The fourth innovation-dependent variable, new marketing method innovation (h6), takes the value 1 if the enterprise has introduced new or signifi- cantly improved marketing methods during the last 3 years, otherwise 0. We apply the multivariate probit (MVP) to estimate the jointly dependent variables that exploit a system of simultaneous equations. It is a special case of seemingly unrelated regression (SUR) when the dependent variable is of categorical type. In circumstances where cross-equation error terms are corre- lated and explanatory variables are same across equations, the MVP model can generate more efficient parameter estimates than single-equation estimation approaches. The description of a regression model is given as: Daksa et al. Additional file 1: Supporting Material. (DTA 1925 kb) Additional file 1: Supporting Material. (DTA 1925 kb) Authors’ contributions Authors contributions All authors have contributed substantially to the development of the manuscript. All authors read and approved the final manuscript. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. A th d t il Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Availability of data and materials y See additional supporting files (Additional file 1). Author details 1 1Department of Economics, Haramaya University, Dire Dawa, Ethiopia. 2Department of Economics, Jigjiga University, Jigjiga, Ethiopia. References Doloreux, D. (2003). Regional innovation systems in the periphery: the case of the Beauce in Québec (Canada). 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https://openalex.org/W4387528592	https://pubs.rsc.org/en/content/articlepdf/2023/sc/d3sc03991a	English	null	Systematic exploration of accessible topologies of cage molecules <i>via</i> minimalistic models	Chemical science	2,023	cc-by	12,147	Open Access Article. Published on 11 October 2023. Downloaded on 10/24/2024 5:44:46 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Cite this: Chem. Sci., 2023, 14, 12506 Cite this: Chem. Sci., 2023, 14, 12506 Andrew Tarzia, a Emma H. Wolpert, b Kim E. Jelfs b and Giovanni M. Pavan ac Andrew Tarzia, a Emma H. Wolpert, b Kim E. Jelfs b and Giovanni M. Pavan ac All publication charges for this article have been paid for by the Royal Society of Chemistry All publication charges for this article have been paid for by the Royal Society of Chemistry Cages are macrocyclic structures with an intrinsic internal cavity that support applications in separations, sensing and catalysis. These materials can be synthesised via self-assembly of organic or metal–organic building blocks. Their bottom-up synthesis and the diversity in building block chemistry allows for ﬁne- tuning of their shape and properties towards a target property. However, it is not straightforward to predict the outcome of self-assembly, and, thus, the structures that are practically accessible during synthesis. Indeed, such a prediction becomes more diﬃcult as problems related to the ﬂexibility of the building blocks or increased combinatorics lead to a higher level of complexity and increased computational costs. Molecular models, and their coarse-graining into simpliﬁed representations, may be very useful to this end. Here, we develop a minimalistic toy model of cage-like molecules to explore the stable space of diﬀerent cage topologies based on a few fundamental geometric building block parameters. Our results capture, despite the simpliﬁcations of the model, known geometrical design rules in synthetic cage molecules and uncover the role of building block coordination number and ﬂexibility on the stability of cage topologies. This leads to a large-scale and systematic exploration of design principles, generating data that we expect could be analysed through expandable approaches towards the rational design of self-assembled porous architectures. Received 31st July 2023 Accepted 11th October 2023 DOI: 10.1039/d3sc03991a rsc.li/chemical-science Received 31st July 2023 Accepted 11th October 2023 spatial arrangement of atoms inside and outside cage compounds. aDepartment of Applied Science and Technology, Politecnico di Torino, Corso Duca degli Abruzzi 24, 10129 Torino, Italy. E-mail: andrew.tarzia@polito.it; giovanni. pavan@polito.it bDepartment of Chemistry, Molecular Sciences Research Hub, Imperial College London, White City Campus, Wood Lane, London, W12 0BZ, UK cDepartment of Innovative Technologies, University of Applied Sciences and Arts of Southern Switzerland, Polo Universitario Lugano, Campus Est, Via la Santa 1, 6962 Lugano-Viganello, Switzerland † Electronic supplementary information (ESI) available: One PDF le with all referenced supporting information. See DOI: https://doi.org/10.1039/d3sc03991a Chemical Science View Article Online View Journal \| View Issue © 2023 The Author(s). Published by the Royal Society of Chemistry aDepartment of Applied Science and Technology, Politecnico di Torino, Corso Duca degli Abruzzi 24, 10129 Torino, Italy. E-mail: andrew.tarzia@polito.it; giovanni. pavan@polito.it aDepartment of Applied Science and Technology, Politecnico di Torino, Corso Duca degli Abruzzi 24, 10129 Torino, Italy. E-mail: andrew.tarzia@polito.it; giovanni. pavan@polito.it Open Access Article. Published on 11 October 2023. Downloaded on 10/24/2024 5:44:46 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. p For more eﬃcient modelling, coarse-grained (CG) or mini- malistic models simplify and approximate molecular repre- sentations to low-resolution models. By nature, their low resolution provides access to low-cost simulations, allowing larger systems to be modelled for longer times, or in this case, allowing for vast numbers of systems to be modelled. In particular, minimalistic models (or “toy models”) allow us to zoom out to very low resolutions and systematically generate large amounts of qualitative data to learn from, eventually translating that into atomistic models or experimental designs.29,30 With complete control over the model parameters, toy models allow for extrapolation beyond known chemical space. For example, Martin et al. show in a series of papers how minimal models that capture the structural features of metal– organic frameworks can aid in interpreting the limits of mate- rials optimisation.31–33 Similarly, Wolpert et al. used CG models of cages, treating them as hard-polyhedra, to map the phase space of their packing behaviour as a function of simple inter- actions.34 Evans et al. use a minimal model to explore placement and conguration eﬀects on molecular motors in metal–organic frameworks.35 Using high-resolution models, Pesce et al. studied crowding-reactivity relationships in host-guest binding by articially modifying model parameters.36 Using stk (https://github.com/lukasturcani/stk),37 we built linear, tritopic and tetratopic building blocks made up of three, four and ve beads, respectively (Fig. 1(a)), where the beads have varying force eld parameters (see below). We then place those building blocks on cage topologies9 in stk and perform a series of optimisation steps to attempt to get a single “lowest energy” conformer for analysis. The beads in a given system dene the input parameters (target bond lengths and angles; bead property ranges are dened in Section S2†). By building and optimising a cage, which is dened by a topology and set of building blocks with specic beads (hence, specic input parameters), we are eﬀectively testing the matching of those parameters to a topological constraint. High energy structures imply that some bond, angle or torsion term is far from their input targets. Here, we apply a bottom-up computational approach to build 1000s of minimal cage models using stk37 and OpenMM.38 This work does not represent a rigorous coarse-graining based on some experimental or higher resolution data but focuses on the role of a few building block parameters on the geometric stability and eventual cage structure of diﬀerent topologies. Open Access Article. Published on 11 October 2023. Downloaded on 10/24/2024 5:44:46 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. We outline how this work could feed into future computational and experimental cage screening and decision-making. In partic- ular, we use this model to eﬃciently explore parameter space and study the eﬀect of distinct features, such as exibility, which are diﬃcult to isolate in atomistic models. We design the model and associated soware towards automated rational design and we have made the outcomes of these predictions freely available online in an easily accessible manner for use by experimental researchers. The force eld used for each model is based on bonding, angular, torsional and excluded volume terms, and has a potential energy form E = Ebond + Eangle + Etorsion + Eexcl.vol., (1) (1) where the functional forms of each term are Ebond ¼ 1 2kbondðr r0Þ2 (2) Eangle ¼ 1 2kangleðq q0Þ2 (3) (2) Etorsion = ktorsion(1 + cos(p4 −40)) (4) Eexcl:vol: ¼ ﬃﬃﬃﬃﬃﬃﬃ 3i3j p si þ sj 2r 12 : (5) (4) (5) 1 Introduction 1 The successful synthesis of a specic cage is determined by whether the building blocks self-sort into the chemist's desired product (generally this is a single species, but could be a tar- geted mixture). The eventual cage structure has two related but distinct structural characteristics: topology and geometry. Cage topology strictly includes information about the connectivity of building blocks in the cage; throughout, we use the denition introduced by Santolini et al.9 This terminology is commonly used in the POC literature but is translatable to any cage-like molecule. The cage geometry describes the coordinates of atoms or building blocks in the cage structure, which has been closely linked to regular shapes or polyhedra while rigid building blocks are used.10 Indeed, under these conditions, building block geometry and stoichiometry can predict topo- logical outcomes, which infer geometrical properties. But researchers are targeting more complex structures11,12 (towards improved tunability) through exible, unsymmetrical, or mixed components.13–16 Therefore, the degrees of freedom to be considered will only grow, making the use of existing design rules for cages less viable. Porous cage-like molecules are oen formed through the bottom-up self-assembly of building blocks with varying connectivities and geometries, which transfers into the prop- erties of the cage. The broad term “cage” applies to macrocyclic structures containing an intrinsic void1 widely studied for various types of applications, e.g. catalysis,2 sensing,3 separa- tions,4,5 and as porous liquids.6 Their bottom-up self-assembly aﬀords a vast pool of building blocks, allowing for the tunability of their properties. Cages can be entirely organic (porous organic cages, POCs)7 or contain metals (metal–organic cages, MOCs).8 Through rational selection from this vast chemical space and control of the self-assembly outcome and cage structure, researchers can achieve ne control over the Computational chemistry can help to simplify the relation- ship between building blocks and self-sorting outcomes by predicting or rationalising the structure and energetics of diﬀerent potential topologies, showing, for example, whether © 2023 The Author(s). Published by the Royal Society of Chemistry 12506 \| Chem. Sci., 2023, 14, 12506–12517 Chemical Science View Article Online Chemical Science View Article Online Chemical Science View Article Online Edge Article Edge Article a particular combination of building blocks and topology are accessible.17–21 Structure generation and evaluation of the rela- tive stability of diﬀerent topologies can help drive or rationalise experimental eﬀorts.16,22–26 However, these calculations are costly or too approximate (oen neglecting solvent/ion eﬀects and exibility), and the thermodynamics of each step is not always enough of the picture (e.g., when kinetic traps are present). When introducing asymmetry, for example, combi- natorial explosion quickly leads to an intractable number of isomers, topologies or congurations that must be studied to consider self-sorting outcomes.23,27,28 Therefore, new approaches to tackling these prediction and rationalisation problems are needed. the key features of their constituent building blocks. This approach is informed by the design rules used by experimental chemists, which tend to be based on placing rigid building blocks on high-symmetry geometries.39 Our model evaluates the role of diﬀerent building block angles in cage stability (of a series of dened topologies; Table S1†). We then use the relative cage stability of diﬀerent topologies to approximate self- sorting outcomes. We focus on the role of internal building block angles, in particular, we evaluate the eﬀect of changing the ditopic ligand bite-angle, which has been a key variable in designing MOC topologies,40 and pyramid angles of tritopic or tetratopic building blocks (including the planar case). The model's design is modular and general (not parameterised to any specic chemical system), so it can be applied to unknown systems. Fig. 1 summarises the minimalistic model and Fig. 1(a) shows the ligand model schematics, where the labels match the symbols in Table S2.† For comparison to previous cage design methods, we dene the target bite angle of a ditopic ligand based on the two internal bac angles, as qbite = 2(q0 −90), where q0 is the target bac angle on both sides. However, this only applies when the baab torsion is near 0°. Open Access Article. Published on 11 October 2023. Downloaded on 10/24/2024 5:44:46 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. 2 Computational methods (b) Model construction and optimisation workﬂow built on stk, OpenMM, resulting in (c) the lowest energy conformer for a given input with a structure and properties. The two structures highlight the visual diﬀerence between low and high-energy structures. (d) Schematic of diﬀerent self-sorting outcomes (unstable, mixed and selective) approximated in this work, where small circles correspond to diﬀerent topologies, and (e) the mapping of those outcomes to a discrete, accessible topology map based on two general model parameters, A and B. The topology map shows points in phase space as circles, which are coloured by the topologies that are stable in those regions. (in kJ mol−1) and si is the size of bead i (in Å). Eexcl.vol. is a force term dened such that there is a penalty for bead overlap. When beads are connected by two or less bonds, the Eexcl.vol. term is turned oﬀusing the “bondCutoﬀ” parameter. Table S2† denes the bead classes used and the ranges of their parameters. For bonds, r0 is determined through Lorentz–Berthelot mixing rules, e.g., r0 ¼ ri þ rj 2 , where ri and rj are the equilibrium bond lengths assigned to bead types i and j, respectively. We have selected parameters such that the bond, angle and torsion terms are on the same scale as all-atom molecular force elds (when present) with kbond = 1 × 105 kJ molnm−2, kangle = 1 × 102 kJ molrad−2, ktorsion = 50 kJ mol−1. The rigidity in our system derives from the limited internal degrees of freedom in the cage building blocks. In this initial version of our model, we have used the same 3i = 10 kJ mol−1 and si = 1 Å for all beads. This model mimics a “good solvent” case, where solute–solvent interactions are energetically favourable. Therefore, this model assumes that cage collapse is a function of the geometric constraints of the building blocks, not inter-building block interactions or the hydrophobic eﬀect. Section S2† contains details about handling the angles in tetratopic building blocks and how we dene alchemical torsions. simulation with soened bond and angle potential terms and optimisations of cage models aer beads have been shied away from the cage centroid (full details in Section S3†). These processes are designed to help exploration of conformational space. 2 Computational methods r is the bond length (or distance between two beads), r0 is the equilibrium bond length, q is the angle, q0 is the equilibrium angle, 4 is the torsion, 40 is the equilibrium phase oﬀset, p = 1, 3i is the strength of nonbonded interaction for bead i In this work, we dene a minimalistic model to evaluate the accessible topologies in cage-like molecules, towards an understanding of their self-sorting behaviour, as a function of © 2023 The Author(s). Published by the Royal Society of Chemistry Chem. Sci., 2023, 14, 12506–12517 \| 12507 Chemical Science Chemical Science Fig. 1 Outline of the minimalistic cage model. (a) Input to the model, including deﬁning bond lengths and internal angles in ditopic, tritopic an tetratopic building blocks, and topology graphs. Bead types are provided in the ﬁgure and described in Table S2;† semicircles represe connections between building blocks. The main parameters of interest are highlighted: the internal ditopic angle (bac) and the two angl deﬁning the tritopic (bnb) and tetratopic (bmb) building blocks. (b) Model construction and optimisation workﬂow built on stk, OpenMM, resultin in (c) the lowest energy conformer for a given input with a structure and properties. The two structures highlight the visual diﬀerence betwee low and high-energy structures. (d) Schematic of diﬀerent self-sorting outcomes (unstable, mixed and selective) approximated in this wo where small circles correspond to diﬀerent topologies, and (e) the mapping of those outcomes to a discrete, accessible topology map based o two general model parameters, A and B. The topology map shows points in phase space as circles, which are coloured by the topologies that a stable in those regions. Chemical Science Edge Artic emical Science Edge Article Open Access Article. Published on 11 October 2023. Downloaded on 10/24/2024 5:44:46 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 1 Outline of the minimalistic cage model. (a) Input to the model, including deﬁning bond lengths and internal angles in ditopic, tritopic and tetratopic building blocks, and topology graphs. Bead types are provided in the ﬁgure and described in Table S2;† semicircles represent connections between building blocks. The main parameters of interest are highlighted: the internal ditopic angle (bac) and the two angles deﬁning the tritopic (bnb) and tetratopic (bmb) building blocks. 2 Computational methods Each step uses OpenMM (inspired by other applications of OpenMM with CG models41,42) to perform local energy mini- misation or molecular dynamics.38 This approach is similar to those we have applied on all-atomistic cage models.9,25 The optimisation sequence neglects the role of temperature in the accessibility of diﬀerent conformers and searches for the lowest internal energy model. We expect the geometrical stability we compute in this paper and self-assembly outcome to depend on temperature, which we aim to consider in future models. Throughout, we directly compare the energy per number of building blocks in a cage (Eb) as eﬀective formation energies. The energy scale dened by the force eld terms above is alchemical, and so we determine cage stability based on the data our model produces such that we can extract useful distinctions. The force eld is modular, which we take advan- tage of herein, e.g., to explore the role of a particular type of exibility by modifying the torsional term in the ligand back- bone (by design, this is the only torsional term present). By default, we explore the most constrained case with the torsion restriction “on” (i.e., preorganised building blocks), where the two ditopic binding sites face the same direction, but in Section S3.3,† we explore how topology accessibility changes without this constraint. We have applied a multi-step optimisation sequence to attempt to nd the lowest energy conformer of each cage model. The sequence is made up of seven steps including a constrained geometry optimisation (constraints applied to bonds not formed during cage construction), a molecular dynamics 12508 \| Chem. Sci., 2023, 14, 12506–12517 © 2023 The Author(s). Published by the Royal Society of Chemistry Chemical Science View Article Online Chemical Science View Article Online Edge Article complementarity)43,44 and huge PdxL2x cages.40 Fig. 2(a) shows that our model produces this behaviour, with the expected topology being stable at the appropriate bite-angle. Fig. 2(b) and (c) show geometrical agreement between stable structures with square-planar tetratopic building blocks in topologies Tet6Di12 (target bite angle of 90°) and Tet12Di24 (target bite angle of 120°) with experimental crystal structures.45,46 This also highlights the chemical generality of our model, where agreement is achieved for square-planar Pd(II) systems and the geometrically equiva- lent Cu paddle-wheel in (b). 3 Results and discussion 3 (a) Relationship between target tritopic angle, target ditopic internal angle and energy for Tri4Di6 topology; the colour map is Eb. White squares show high-energy points. (b) Example low energy structures along the blue line in (b) with varying ditopic and tritopic angles. Green are the a beads, orange are the n beads, black are the b beads, and grey are the c beads. (c) Overlap of a porous organic cage (CCDC code: FOXLAG47) and a metal–organic cage (CCDC code: TIXFIP50) with two Tri4Di6 models from our phase space with tritopic angles of 120° and ditopic angles of 125° and 135°, respectively. In atomistic models, grey are carbon, blue are nitrogen, cyan are palla- dium, hydrogens and solvent are not shown for clarity. 3.1 Eﬀect of building block angles on topology stability Green are the a beads, cyan are the m beads, black are the b beads, and grey are the c beads. (b) Comparison of minimal model of Tet6Di12 with target bite angle of 90° with a crystal structure (CCDC: SUPPID).45 (c) Comparison of minimal model of Tet12Di24 with target bite angle of 120° with a crystal structure (CCDC: BIMXIF).46 The minimal models are minimum energy points in (a) with the same colour as the line at the bottom. In atomistic models, grey are carbon, blue are nitrogen, red are oxygen, cyan are palladium, hydrogens and solvent are not shown for clarity. Fig. 2 (a) Relationship between ditopic bite-angle and topology preference for square-planar tetratopic building blocks with torsion restrictions. The lowest energy structure for each selected topology is shown; horizontal bars are colour-coordinated to the points in the plot. Green are the a beads, cyan are the m beads, black are the b beads, and grey are the c beads. (b) Comparison of minimal model of Tet6Di12 with target bite angle of 90° with a crystal structure (CCDC: SUPPID).45 (c) Comparison of minimal model of Tet12Di24 with target bite angle of 120° with a crystal structure (CCDC: BIMXIF).46 The minimal models are minimum energy points in (a) with the same colour as the line at the bottom. In atomistic models, grey are carbon, blue are nitrogen, red are oxygen, cyan are palladium, hydrogens and solvent are not shown for clarity. Fig. 2 (a) Relationship between ditopic bite-angle and topology preference for square-planar tetratopic building blocks with torsion restrictions. The lowest energy structure for each selected topology is shown; horizontal bars are colour-coordinated to the points in the plot. Green are the a beads, cyan are the m beads, black are the b beads, and grey are the c beads. (b) Comparison of minimal model of Tet6Di12 with target bite angle of 90° with a crystal structure (CCDC: SUPPID).45 (c) Comparison of minimal model of Tet12Di24 with target bite angle of 120° with a crystal structure (CCDC: BIMXIF).46 The minimal models are minimum energy points in (a) with the same colour as the line at the bottom. In atomistic models, grey are carbon, blue are nitrogen, red are oxygen, cyan are palladium, hydrogens and solvent are not shown for clarity. Fig. 3.1 Eﬀect of building block angles on topology stability Over the last twenty years, the relationship between self-sorting outcome and ligand internal angles in square-planar Pd(II)- based systems has driven the design of impressive cage systems, including heteroleptic cages (through shape outcome and ligand internal angles in square-planar Pd(II)- based systems has driven the design of impressive cage systems, including heteroleptic cages (through shape Fig. 2 (a) Relationship between ditopic bite-angle and topology preference for square-planar tetratopic building blocks with torsion restrictions. The lowest energy structure for each selected topology is shown; horizontal bars are colour-coordinated to the points in the plot. Green are the a beads, cyan are the m beads, black are the b beads, and grey are the c beads. (b) Comparison of minimal model of Tet6Di12 with target bite angle of 90° with a crystal structure (CCDC: SUPPID).45 (c) Comparison of minimal model of Tet12Di24 with target bite angle of 120° with a crystal structure (CCDC: BIMXIF).46 The minimal models are minimum energy points in (a) with the same colour as the line at the bottom. In atomistic models, grey are carbon, blue are nitrogen, red are oxygen, cyan are palladium, hydrogens and solvent are not shown for clarity. Fig. 3 (a) Rela internal angle White squares structures alon angles. Green b beads, and g (CCDC code: TIXFIP50) with angles of 120° atomistic mod dium, hydroge Fig. 2 (a) Relationship between ditopic bite-angle and topology preference for square-planar tetratopic building blocks with torsion restrictions. The lowest energy structure for each selected topology is shown; horizontal bars are colour-coordinated to the points in the plot. Green are the a beads, cyan are the m beads, black are the b beads, and grey are the c beads. (b) Comparison of minimal model of Tet6Di12 with target bite angle of 90° with a crystal structure (CCDC: SUPPID).45 (c) Comparison of minimal model of Tet12Di24 with target bite angle of 120° with a crystal structure (CCDC: BIMXIF).46 The minimal models are minimum energy points in (a) with the same colour as the line at the bottom. In atomistic models, grey are carbon, blue are nitrogen, red are oxygen, cyan are palladium, hydrogens and solvent are not shown for clarity. Fig. 2 (a) Relationship between ditopic bite-angle and topology preference for square-planar tetratopic building blocks with torsion restrictions. The lowest energy structure for each selected topology is shown; horizontal bars are colour-coordinated to the points in the plot. 3 Results and discussion We generated 2890 cages in our cage space from a pool of 287 distinct building block combinations and 13 topologies, with and without torsion restriction. We explored the eﬀect of cage topology, building block coordination number, building block exibility and target internal angles of the two building blocks on topology accessibility relationships and approximations to self-sorting behaviour (Fig. 1). Note that the cage space is dis- cretised over the target internal angle values, which are shown in all plots. We implemented an optimisation sequence, described in Section S3,† that is reasonably robust, but does result in some instabilities in the phase space (Section S5†). However, we show that the optimised cage models have struc- tural parameters that match the input force eld parameters and that most of the strain exists in the angle terms (Section S4†). Open Access Article. Published on 11 October 2023. Downloaded on 10/24/2024 5:44:46 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Similarly, we show that our model produces structures akin to those expected for the Tri4Di6 topology based on tetrahedral MOCs and POCs.47–49 Fig. 3(a) summarises the relationship between tritopic and ditopic building block angles and cage stability for the Tri4Di6 topology. The data shows that the stable regions shitowards 180° in internal ditopic angle as the 3.1 Eﬀect of building block angles on topology stability Over the last twenty years, the relationship between self-sorting outcome and ligand internal angles in square-planar Pd(II)- based systems has driven the design of impressive cage systems, including heteroleptic cages (through shape Fig. 2 (a) Relationship between ditopic bite-angle and topology preference for square-planar tetratopic building blocks with torsion restrictions. The lowest energy structure for each selected topology is shown; horizontal bars are colour-coordinated to the points in the plot. Green are the a beads, cyan are the m beads, black are the b beads, and grey are the c beads. (b) Comparison of minimal model of Tet6Di12 with target bite angle of 90° with a crystal structure (CCDC: SUPPID).45 (c) Comparison of minimal model of Tet12Di24 with target bite angle of 120° with a crystal structure (CCDC: BIMXIF).46 The minimal models are minimum energy points in (a) with the same colour as the line at the bottom. In atomistic models, grey are carbon, blue are nitrogen, red are oxygen, cyan are palladium, hydrogens and solvent are not shown for clarity. Fig. 3.1 Eﬀect of building block angles on topology stability 3 (a) Relationship between target tritopic angle, target ditopic internal angle and energy for Tri4Di6 topology; the colour map is Eb. White squares show high-energy points. (b) Example low energy structures along the blue line in (b) with varying ditopic and tritopic angles. Green are the a beads, orange are the n beads, black are the b beads, and grey are the c beads. (c) Overlap of a porous organic cage (CCDC code: FOXLAG47) and a metal–organic cage (CCDC code: TIXFIP50) with two Tri4Di6 models from our phase space with tritopic angles of 120° and ditopic angles of 125° and 135°, respectively. In atomistic models, grey are carbon, blue are nitrogen, cyan are palla- dium, hydrogens and solvent are not shown for clarity. Chem. Sci., 2023, 14, 12506–12517 \| 12509 © 2023 The Author(s). Published by the Royal Society of Chemistry Edge Article View Article Online Edge Article View Article Online Chemical Science tritopic building block becomes less planar, exemplied by structures in Fig. 3(b). This outcome, again, is consistent with the geometry of a tetrahedron. We show in Fig. 3(c) the struc- tural similarity between two models from our phase space (diﬀering by input ditopic angle) to known experimental structures. The two low-energy models have internal ditopic angles approximately matched to the atomistic building blocks of the experimental structures. What is less obvious about the data in Fig. 3 is why there are stable points at low internal angles for smaller tritopic angles (structures 1–3 in Fig. 3(b)). We posit that these structures are geometrically stable in the framework of this model, but may be diﬃcult to realise using real chemical structures. However, this highlights new geometries that we could target in future design studies based on our models. interesting question about the degree of deviation that is possible from existing design rules, and if we can control for or predict that leniency. An interesting case is the Tet4 2Di8 topology, termed a double- walled tetrahedron.28,54–56 Using a consistent energy scale, there are no stable structures in the whole angle space (see details in Fig. S33†), and further analysis shows that there is a systematic strain in the structure, which we propose to be due to the required twist in the ditopic building blocks bridging some of the tetratopic sites (this can be seen in known experimental structures of Pd(II) cages in this topology). 3.2 Mapping accessible topologies and their selectivity Next, we distil a large cage space into what we term “accessible topology maps”. These gures map building block parameters to their calculated accessibility for all studied cage topologies. The outcomes of self-assembly are governed by experimental conditions, thermodynamics (i.e., free energy) and kinetics, which our model does not consider. However, the accessible topology maps, based on internal cage energies, are an approximation to this problem based on what topologies are permitted (or not) for given building block parameters. Under this model, if only a single species is stable at a given point in phase space, we assume that self-sorting will occur and produce only that species. Fig. 1(d) shows the diﬀerent accessibility outcomes (all unstable, mixed or selected), and how that can be used to map cage space, where A and B in this work are the target tritopic/tetratopic pyramid angles and target internal ditopic angles. Given the geometrical nature of our approach, it is prom- ising that our models agree with literature examples, e.g., from ref. 53 and their ndings of stable angle-topology combinations in metal–organic polyhedra (they performed a detailed review of the angles in building block pairs and their eventual topology). In particular, we nd overlapping stable regions for the Tri4Di6, Tet6Di12, Tri8Di12, Tet6Tri8, and Tet12Di24 topologies (all topol- ogies shown in Fig. S1†). We note that for Tet12Di24, they also found another stable structure, but the formation of this structure involves a decrease in symmetry of the angles around the tetratopic building blocks, which is not studied in our model. Therefore, we would not expect to nd that stable conguration. Similarly, we support the ndings in ref. 10 for our studied topologies (Tri2Di3, Tet2Di4, Tri4Di6, Tet6Di12, Tri8Di12, Tet6Tri8). In ref. 10, they point out how concerted rotations within building blocks play a signicant role, which our model will not currently capture due to its simplicity. However, the granularity of our approach begins to highlight how tuning the force eld may better explain transitions between angles in our space. Additionally, there is an To compute accessible topology maps, we must dene an energy per building block (Eb) value below which a structure is deemed accessible. Because our force eld does not correspond to any specic experimentally relevant system, we chose this parameter based on the threshold that provided the maximum degree of self-sorting (Fig. 4(a)); the selected value is 0.3 kJ mol−1. Open Access Article. Published on 11 October 2023. Downloaded on 10/24/2024 5:44:46 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 11 October 2023. Downloaded on 10/24/2024 5:44:46 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. We have explored the relationship between internal building block angles and cage internal energy for all topologies in this work (Section S6 and S7†). The number of stable regions (or wells) and the smoothness in these angle maps tends to increase as the degrees of freedom increase (i.e., for larger topologies). Although, some of the larger topologies do not have this eﬀect. Also, some topologies show two stable wells for a given pyramid angle, for example, at tritopic angle 110° in Fig. 3(a), that deviate from the single well for the planar tritopic or tetratopic case. Visualising structures in these types of surfaces, we see structures with inverted tetratopic or tritopic building blocks as in Fig. 3(b) 1–3. For other topologies, we see that there are low energy structures up until some critical internal ditopic angle that leads to instability (Fig. S16 and S20†). This shows that there are topological eﬀects determining the energy surface associated with these two types of internal angles. The observed topological dependence is non-trivial and may be related to some inherent exibility. For example, studies have shown that the Tri4 2Di6 topology results in exible cages36 and here the topology shows a at energy surface, while Tri4Di6 has the double-well behaviour and tends to result in more rigid cage structures.51 However, studies on POCs show that there is exibility in the window apertures of Tri4Di6 cages.52 © 2023 The Author(s). Published by the Royal Society of Chemistry 3.1 Eﬀect of building block angles on topology stability Therefore, the torsion constraint at 0° leads to strained structures, although the cage models look reasonable visually. While we are not aiming to develop an exhaustive model in this manuscript, this type of outcome poses interesting questions for our approach. Can we nd the building block features that lead to well-dened stable regions in the angle space of a specic topology? Are the exi- bility in the bonds and angles, or the torsion restriction important factors? The requirement of non-zero torsions is present for Tet8Di16 also. Young et al. highlight cases of torsion restrictions with diﬀerent target angles,10 and how they result in cages with symmetric structures. This issue is also exemplied for the Tri6Di9 trigonal prism structure, which, by denition, requires a tritopic angle of 60° and 90° for an ideal trigonal prism structure. Hence, increased exibility in the tritopic angle term would likely modify the accessible cage space for this topology. 3.3 Preorganisation eﬀect on topology accessibility So far, we have enforced preorganisation on the ditopic building blocks, where the binding beads are forced to face the same direction with a restricted torsion along the ligand backbone (see Section S2† for more information). This is one way in which these building blocks could be preorganised, where the rigid, well-dened binding site orientation facilitates self-assembly processes and allows the application of predictive geometrical rules. Chemically speaking, this corresponds to rigid building blocks with few rotatable bonds, resulting in a constant relative orientation of the reactive bonding atoms. Increased exibility of the building blocks or the cages makes computational structure prediction more challenging for atomistic simulations/calculations due to the increased degrees of freedom and the likelihood of multiple low-energy states to consider. Importantly, small deviations from the ideal geome- tries of rigid building blocks can lead to changes in self- assembly outcomes.10,57 Therefore, we aim to use our toy model to explore the impact of building block preorganisation (at least in one form) by switching oﬀthis torsion restriction (Fig. 5(a)). This will inherently lead to more exible cages, which we assume will always increase the accessibility of building block and topology combinations under the conditions of this model. Fig. 4 (a) Percentage of selected topologies (i.e., only one topology is stable for a given building block combination) as a function of the threshold Eb for accessibility for cages formed from ditopic and either tritopic (3C) or tetratopic (4C) building blocks, with and without restricted torsions, described further in Section S3.3.† Accessible topology maps for cages formed with torsion restrictions, ditopic building blocks and (b) tritopic or (c) tetratopic building blocks. The colour map for each sub-ﬁgure is shown in the corresponding legend. Regarding selection, Fig. 4(a) shows that the percentage of selected cage structures decreases signicantly when pre- organisation is switched oﬀ. A geometrical eﬀect of removing this torsion restriction on the cage structures is shown in Fig. 5(a), where helicity occurs without torsion restrictions, which is akin to recent work in Pd2L4 cages.58 Further, we see that there would be a mixed eﬀect of turning oﬀtorsion restrictions on pore size; many unrestricted cages are collapsed, but the same seems to be true for unstable restricted cages with torsion restrictions. 3.2 Mapping accessible topologies and their selectivity This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. © 2023 The Author(s). Published by the Royal Society of Chemistry 3.2 Mapping accessible topologies and their selectivity Note that this maximum occurs for 4-connected structures (4C), while the maximum is at lower Eb for 3-con- nected structures (3C). This model focuses on geometrical stability, ignoring many other factors (e.g., dispersion interac- tions, sterics, computation methodology, cage properties and © 2023 The Author(s). Published by the Royal Society of Chemistry 12510 \| Chem. Sci., 2023, 14, 12506–12517 Chemical Science View Article Online Chemical Science View Article Online Edge Article his article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Edge Article a single species forms experimentally (see discussion of Tri4Di6 and Tri4 2Di6 above). We suggest that cage topologies could be selected based on such data to avoid issues with sensitivity to experimental conditions. We also show that the self-sorting propensity depends on the pyramidal angles of the tritopic or tetratopic building block, where planar building blocks lead to more selected isomers at higher energy thresholds (Fig. S42†). Focusing on the smallest, stable topology, Fig. S44 and S45† show regions of stability for most topologies, where the largest topologies tend to be favoured at larger internal angles. Therefore, it is possible to design for a particular cage topology if desired. solvent) that ultimately aﬀect the a threshold in any computational attem i.e. 0.3 kJ mol−1 is entirely model dep Fig. 4 clearly shows diﬀerent d topologies with 3-connected and 4-co This is summarised clearly in Fig. 4(a selected systems is higher for 4-con nected cages (under the rigid regime up to higher energy thresholds. It is enumerated topologies, as in the wor this behaviour.18 Fig. S43† shows tha cages is higher for the 4-connected t nected topologies. This outcome, fro suggests that 3-connected topologies tritopic building blocks, will suﬀer during self-assembly than the 4-conne there are many examples where our m Fig. 4 (a) Percentage of selected topolog stable for a given building block combi threshold Eb for accessibility for cages fo tritopic (3C) or tetratopic (4C) building restricted torsions, described further i topology maps for cages formed with building blocks and (b) tritopic or (c) tet colour map for each sub-ﬁgure is shown © 2023 The Author(s). Published by the R Open Access Article. Published on 11 October 2023. Downloaded on 10/24/2024 5:44:46 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 11 October 2023. Downloaded on 10/24/2024 5:44:46 AM. 3.4 Relationship between building blocks, shape and topology The shape of cage molecules determines the properties of their intrinsic pores and inuences their packing behaviour.34,49 As dened,9,10 the topology of a cage molecule does not necessarily provide information on the eventual cage geometry. Here, we systematically mapped the relationship between topology, shape and building block parameters. We calculate a series of shape measures for a given cage structure, which are the devi- ation of that cage from ideal shapes, using the SHAPE v2.1 soware.61 We calculate the shape only for some topologies (shapes with vertex numbers 3 to 8 to avoid the cost of 12-vertex calculations), and we do so on the centroids of either the tritopic/tetratopic building blocks or the ditopic building blocks in the cage structure. For example, for the Tri4Di6 topology, we calculate the shapes based on the positions of the four tritopic building blocks (compared to four-vertex shapes, e.g., square and tetrahedron) and based on the positions of the six ditopic building blocks (compared to six-vertex shapes, e.g., octahe- dron). Therefore, some topologies will have two shape measures (more detail in Section S11†). Table S3† shows the ideal shape of each topology, where the naming convention states the geom- etry and the number of vertices in the shape deviation measure. Importantly, these deviation measures are unitless and allow quantitative comparison between diﬀerent shapes. Fig. 5 (a) Schematic of the preorganisation modiﬁed in this work, where the baab torsion (4; Newmann projection is shown) is either restricted (top; rigid) or not restricted (bottom; ﬂexible). Representa- tions of Tet2Di4 structures and the torsion force ﬁeld term in both cases are shown. (b) The eﬀect of this restriction on the proportion of accessible structures for each topology. The diﬀerence in the blue and white bins represents the eﬀect of ﬂexibility. shis from the smallest to the largest topology as the internal angles increase with unstable regions in the top-right-hand corner. However, this is not the case when preorganisation is on. Further, diﬀerent topologies are preferred (as the smallest stable structure) with and without torsion restrictions for a given pair of building block pairs. There are regions that were unstable or favoured larger topologies, that now favour smaller topologies when preorganisation is not present. As expected for such a minimal model, the eﬀect of preorganisation is negli- gible at higher internal angles, where the torsion has a smaller eﬀect. 3.3 Preorganisation eﬀect on topology accessibility Regardless, simple models such as this can begin to uncover where and how to introduce exibility in the most useful and robust way, avoiding the pitfalls of challenging synthetic outcomes and optimising materials properties.59,60 Open Access Article. Published on 11 October 2023. Downloaded on 10/24/2024 5:44:46 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. 3.3 Preorganisation eﬀect on topology accessibility Shape persistence or porosity is not directly related to exibility, although the average pore size52 should be considered for exible cages, rather than a static image, which provides a less clear image of cage porosity. solvent) that ultimately aﬀect the appropriate choice of this threshold in any computational attempt to predict self-sorting; i.e. 0.3 kJ mol−1 is entirely model dependent. Fig. 4 clearly shows diﬀerent degrees of self-sorting for topologies with 3-connected and 4-connected building blocks. This is summarised clearly in Fig. 4(a), where the percentage of selected systems is higher for 4-connected cages than 3-con- nected cages (under the rigid regime) and remains signicant up to higher energy thresholds. It is possible that including all enumerated topologies, as in the work by Poole et al. may alter this behaviour.18 Fig. S43† shows that the number of unstable cages is higher for the 4-connected topologies than the 3-con- nected topologies. This outcome, from our very simple model, suggests that 3-connected topologies, at least with symmetric tritopic building blocks, will suﬀer from more competition during self-assembly than the 4-connected topologies. However, there are many examples where our model predicts mixing, but For small ditopic internal angles, removing the torsion restriction has a strong impact, resulting in many low energy cages in the angle map (Fig. S37†). This results in more mixing in the accessible topology maps (Section S10†) than in the restricted case. Focusing on the smallest accessible topology at each point, the topology maps show that the preferred topology © 2023 The Author(s). Published by the Royal Society of Chemistry Chem. Sci., 2023, 14, 12506–12517 \| 12511 Edge Article View Article Online Edge Article View Article Online Chemical Science Fig. 5 (a) Schematic of the preorganisation modiﬁed in this work, where the baab torsion (4; Newmann projection is shown) is either restricted (top; rigid) or not restricted (bottom; ﬂexible). Representa- tions of Tet2Di4 structures and the torsion force ﬁeld term in both cases are shown. (b) The eﬀect of this restriction on the proportion of accessible structures for each topology. The diﬀerence in the blue and white bins represents the eﬀect of ﬂexibility. Chemical Science ensed under a Creative Commons Attribution 3.0 Unported Licence. eﬀectiveness of geometry-based design principles, and our data shows that it increases the competition between diﬀerent cage topologies. However, it is not immediately clear whether these results are false positives due to the model's simplicity. © 2023 The Author(s). Published by the Royal Society of Chemistry 3.4 Relationship between building blocks, shape and topology 7 Map of (a) OC-6 shape and (b) CU-8 shape for the tetratopic and tritopic building blocks in the Tet6Tri8 topology. The colour map shows the deviation from the ideal shapes. Black squares highlight stable cage structures. (c) Low energy structure examples. (d) Approximate pore radius of structures 1–5. Orange are the n beads, cyan are the m beads, and black are the b beads. Chemical Science View Article Online becomes less robust for certain topologies or larger topologies. This may be a bias from the design of the problem. Our interest here is how available low-energy structures with controllable shapes are. For example, the shape maps for Tet6Di12 and Tri8Di12 tell very diﬀerent stories about the shape diversity over their stable structures. For the application of binding/ separating isotropic guests, an ideal (symmetric) shape is useful. However, precisely controlled binding environments are oen asymmetric, and controlling the cage and pore's shape (and dynamics) is critical to designing enzyme-like hosts. Therefore, understanding the relationship between cage inputs to eventual shape is crucial. size, which suggests this topology may be ripe for controlled adaptability, where small changes in building block preference (through external stimuli, for example) could result in large h i  i Fig. 6 Distribution of the deviation from the ideal shapes for (a) Tri6Di9 (shape measure: TPR-6), (b) Tet6Di12 (shape measure: OC-6), (c) Tri8Di12 (shape measure: CU-8) and (d) Tet8Di16 (shape measure: SAPR-8) with torsion-restrictions for tritopic or tetratopic building blocks. Blue distributions are all cages, and orange is for stable cages. This data is on a log scale due to the high proportion of cages near zero. Fig. 7 Map of (a) OC-6 shape and (b) CU-8 shape for the tetratopic and tritopic building blocks in the Tet6Tri8 topology. The colour map shows the deviation from the ideal shapes. Black squares highlight stable cage structures. (c) Low energy structure examples. (d) Approximate pore radius of structures 1–5. Orange are the n beads, cyan are the m beads, and black are the b beads. Edge Article Chemical Science View Article Online Edge Article Edge Article Fig. 6 Distribution of the deviation from the ideal shapes for (a) Tri6Di9 (shape measure: TPR-6), (b) Tet6Di12 (shape measure: OC-6), (c) Tri8Di12 (shape measure: CU-8) and (d) Tet8Di16 (shape measure: SAPR-8) with torsion-restrictions for tritopic or tetratopic building blocks. Blue distributions are all cages, and orange is for stable cages. 3.4 Relationship between building blocks, shape and topology Analysing the data, we see that there are nearly no deviations from shape values of zero for small topologies, indicating a symmetry in the ditopic building block positions regardless of the molecular changes seen in Fig. 5(a). The exibility of larger topologies also allows for the stability of structures far from their ideal shape. However, we see that deviations from the ideal shape do not necessarily become more common for larger topologies. For example, the distribution of Tet6Di12 shapes in this data set are thinner than Tri6Di9 (Fig. 6 also shows the comparison of Tri8Di12 and Tet8Di16). For the exible Tri4 2Di6,36 we see a signicant deviation in the tritopic and ditopic building block shapes for stable structures. And removing torsion restrictions allows for these structures to be low energy, which is the case for other topologies (including the more rigid, Tri4Di6). However, this result could suggest the generation of nonsensical “stable” cages with unrealistic shapes. For exible systems, the distribution of shapes at equilibrium is likely the more important feature and something we can extract for low cost from this model. Fig. 5(b) summarises the change in the proportion of accessible structures with and without restricted torsions. The diﬀerence between topologies with 3-connected and 4-con- nected building blocks is interesting; the eﬀect of pre- organisation on topology stability is much more variable for 3- connected topologies. However, this could be a feature of the studied topologies, not the connectivity. For example, the Tet4 2Di8 topology ignores this trend (all restricted cages are deemed unstable in our model; see above). The change in accessibility of a given topology due to exibility (the gap between white and blue bars in Fig. 5(b), larger gaps suggest larger eﬀect of exibility) may be a useful indicator of the degree of diﬃculty of the rational design of a certain topology with exible components. Our model does not consider the free energy or varying exibility within cage bonds and angles, all of which would signicantly impact the self-sorting of exible structures. Overall, the role of exibility will diminish the This analysis suggests that, under the design of this model, a stable cage in the rigid regime will likely result in a shape near the expected ideal shape of that topology. And that this result © 2023 The Author(s). Published by the Royal Society of Chemistry 12512 \| Chem. Sci., 2023, 14, 12506–12517 Fig. 3.5 Beyond ditopic building blocks We applied the same protocol as above to the common Tet6Tri8 topology, which switches between two main geometries as a function of the tritopic and tetratopic angle (Fig. 7 and S38†). These data agree with the geometrical analysis performed by Tranchemontagne et al. regarding this topology.53 Their anal- ysis highlights the range of deviations that occur in real molecular structures, due to exibility in bonded interactions, for example. Comparing their analysis to the width of stability shown in our model suggests that we are considering more rigid connections than the experimental systems. Low energy struc- tures for this topology have good shape similarity to octahedral (OC-6) and cubic (CU-8) measures, depending on whether it is measured from the tetratopic or tritopic building blocks, respectively. However, Fig. 7(c) shows that the overall cage geometry changes signicantly; therefore, measuring the shape of cages based on decoupled building block positions may be limited. The distinction between 80° and 90° tetratopic building blocks is stark, with regards to the stable geometries and pore 3.4 Relationship between building blocks, shape and topology This data is on a log scale due to the high proportion of cages near zero. Open Access Article. Published on 11 October 2023. Downloaded on 10/24/2024 5:44:46 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 6 Distribution of the deviation from the ideal shapes for (a) Tri6Di9 (shape measure: TPR-6), (b) Tet6Di12 (shape measure: OC-6), (c) Tri8Di12 (shape measure: CU-8) and (d) Tet8Di16 (shape measure: SAPR-8) with torsion-restrictions for tritopic or tetratopic building blocks. Blue distributions are all cages, and orange is for stable cages. This data is on a log scale due to the high proportion of cages near zero. Fig. 6 Distribution of the deviation from the ideal shapes for (a) Tri6Di9 (shape measure: TPR-6), (b) Tet6Di12 (shape measure: OC-6), (c) Tri8Di12 (shape measure: CU-8) and (d) Tet8Di16 (shape measure: SAPR-8) with torsion-restrictions for tritopic or tetratopic building blocks. Blue distributions are all cages, and orange is for stable cages. This data is on a log scale due to the high proportion of cages near zero. becomes less robust for certain topologies or larger topologies. This may be a bias from the design of the problem. Our interest here is how available low-energy structures with controllable shapes are. For example, the shape maps for Tet6Di12 and Tri8Di12 tell very diﬀerent stories about the shape diversity over their stable structures. For the application of binding/ separating isotropic guests, an ideal (symmetric) shape is useful. However, precisely controlled binding environments are oen asymmetric, and controlling the cage and pore's shape (and dynamics) is critical to designing enzyme-like hosts. Therefore, understanding the relationship between cage inputs to eventual shape is crucial. Fig. 7 Map of (a) OC-6 shape and (b) CU-8 shape for the tetratopic and tritopic building blocks in the Tet6Tri8 topology. The colour map shows the deviation from the ideal shapes. Black squares highlight stable cage structures. (c) Low energy structure examples. (d) Approximate pore radius of structures 1–5. Orange are the n beads, cyan are the m beads, and black are the b beads. size, which suggests this topology may be ripe for controlled adaptability, where small changes in building block preference (through external stimuli, for example) could result in large changes in cage conguration. © 2023 The Author(s). Published by the Royal Society of Chemistry Chemical Science materials, and is open-sourced (https://github.com/ andrewtarzia/CGExplore), which facilitates future work. We expect that our work could be integrated with automatic optimisation64 and enhanced sampling36,51 into a platform for the rational design of cages based on shape, exibility and porosity. et al.63 performed knowledge engineering of experimental data to develop an algorithm for rational MOC design, and eﬃcient atomistic model generation. Their approach is akin to ours, where they build up an understanding of which building blocks will be stable in which topologies based on extracted experi- mental data. Better input models can help avoid costly geometry optimisations at the atomistic level by improving initial guesses and simplifying computational workows. We have limited this work to symmetrical building blocks of a single size, with no control for stereochemistry, no consider- ation of intermolecular interactions between components (which can strongly impact cage formation), and no solvation or ions. In particular, while this work includes topologies of cages formed by multi-dentate building block interactions (e.g., subcomponent self-assembly), this iteration does not include the necessary detail at the metal centres to properly encode the twist and stereochemistry present in these MOCs.65 We do not model the actual self-assembly process, which is likely neces- sary to predict self-sorting outcomes generally, and our approach will likely result in false positives. Additionally, it is expected that many self-sorting outcomes can be overcome by choice of experimental conditions. The eﬀect and degree of exibility in cage structures are likely to depend on the solvent and environment. Further, the nature of kinetic traps and stable intermediates in cage formation is not trivial to understand. Including higher resolution CG models (e.g., the Martini force eld66) may help overcome inconsistencies in the phase space, especially for larger molecules. Additionally, the existing models using the Martini force eld for solvent molecules, for example, will help to introduce the eﬀect of solvent in cage formation. Indeed, to study interesting phenomena such as stability cliﬀs in the cage phase space, we require higher CG resolution. Understanding these sharp changes in stability or properties over a phase space, and whether they are topological eﬀects, could be useful in controlling self-assembly in complex cage structures. Open Access Article. Published on 11 October 2023. Downloaded on 10/24/2024 5:44:46 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Data availability The code for structure generation and analysis can be found at https://github.com/andrewtarzia/CGExplore and on Zenodo at https://doi.org/10.5281/zenodo.8184816. Data for this paper, including structure les and properties, are available at Zenodo at https://doi.org/10.5281/zenodo.8136345. Topology maps and structure images are available at https:// andrewtarzia.github.io/selfsort/. Our model opens up interesting questions about the role of dynamics in controlled self-assembly; the balance between achieving stability and control over the outcome of self- assembly is not trivial. We nd that increased exibility leads to more stable cage topologies, resulting in an increased competition. However, this is an observation of this model and the role of exibility in real systems is multifaceted and in need of detailed study. Low-cost computational tools can be used to approach the prediction and design of less trivial cases involving larger, more exible, or unsymmetrical ligands that are near-intractable to study atomistically on large scales.23,25,28 We propose that this will lead to much more eﬃcient screening and design of cage molecules as, for example, a step in approaches similar to that by Young et al.10 Our soware (based on stk and OpenMM) is extendable to many other cage types and 4 Conclusions In this work, we have introduced an approach to generating and analysing minimal cage-molecule models for their exploration and design. Our minimal model, which contains few parame- ters, oﬀers more eﬃcient access to new insights than traditional techniques by mapping the underlying relationships between cage design parameters. We systematically plot the accessible topology space based on these parameters with and without ditopic ligand preorganisation, where increased exibility tends to result in a drastic increase in stable structures. Importantly, we nd good agreement between our model and existing experimental data available for a series of topologies of porous organic and metal–organic cages, where we found that the re- ported topology is stable in our model for the same building block angles (Section 3.1). And in the cases where our model does not agree with previous work, we can highlight the struc- tural features causing this outcome, highlighting potential new areas for design in cage molecules. Therefore, the minimal model we have introduced, which is extendable to other mole- cules and materials, could provide guidance in design problems and initial property predictions for high-throughput screening; this type of approach has shown promise in metal–organic framework design.31–33,59 12514 \| Chem. Sci., 2023, 14, 12506–12517 3.6 Application to future screening Here, we aim to highlight the utility of this model and the next directions we can take. Immediately, it is clear that our model only provides a geometrical understanding of topology stability, and will not capture the changes in self-assembly outcomes that have been studied experimentally (e.g., how metal identity may change the kinetics).57 In particular, our model, based only on connectivity, is expected to provide multiple solutions approx- imating what could be expected during the self-assembly process. However, there is an insight to gain from these solu- tions, especially if the experimental and model data disagree. Through this oversimplication, we can begin to pinpoint the dominant factors in many diﬀerent self-assembly outcomes. One particularly interesting use of these models, and our previous CG models of 2D covalent organic frameworks,62 is to provide more realistic initial structures for atomistic model construction, through soware like stk. Recently, Kondinski © 2023 The Author(s). Published by the Royal Society of Chemistry Chem. Sci., 2023, 14, 12506–12517 \| 12513 Edge Article View Article Online Edge Article View Article Online Chemical Science Chemical Science The data produced in this work, accessible topology maps and images of self-sorting outcomes are provided through an open interface at https://andrewtarzia.github.io/selfsort/ (we discuss its usage in Section S13†). 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https://openalex.org/W2950643777	http://oops.uni-oldenburg.de/4516/1/2019-049_raap_article_fimmu-10-01383.pdf	English	null	Role of the Pruritic Cytokine IL-31 in Autoimmune Skin Diseases	Frontiers in immunology	2,019	cc-by	5,871	MINI REVIEW MINI REVIEW published: 21 June 2019 doi: 10.3389/ﬁmmu.2019.01383 Role of the Pruritic Cytokine IL-31 in Autoimmune Skin Diseases Bernhard F. Gibbs, Nikolaos Patsinakidis and Ulrike Raap* Division of Experimental Allergy and Immunodermatology, University of Oldenburg, Oldenburg, Germany Many autoimmune skin diseases, such as bullous pemphigoid (BP), psoriasis and certain types of chronic urticaria, are associated with intensive pruritus. While histamine and neuropeptides have previously been ascribed to play a role in itch that accompanies these diseases, recent evidence suggests that the pruritogenic cytokine interleukin (IL)-31 is a major driver of pruritic responses. IL-31 was originally shown to be produced by activated helper T cells, particularly Th2 cells, mast cells, macrophages and dendritic cells. However, more recent evidence demonstrated that eosinophils are a major source of this cytokine too, particularly in bullous pemphigoid. Basophils have also been shown to express the cytokine which, through autocrine action, strongly supports the production of other Th2-type cytokines from these cells. These investigations suggest that the dynamic recruitment of eosinophils and basophils in some autoimmune skin diseases could play an important role in the severity of IL-31-mediated itch. Furthermore, these studies suggest that IL-31, in addition to its pruritic actions, also has potential immunomodulatory roles in terms of supporting Th2-type immunity, which often underpins IgE-associated autoimmune diseases (such as bullous pemphigoid and urticaria) as well as allergies. While the role of IL-31 in psoriasis remains to be clariﬁed, current evidence shows that this cytokine plays a major role in BP, chronic spontaneous urticaria and dermatomyositis. This suggests potential use of IL-31 receptor-blocking therapeutic approaches (e.g., Nemolizumab) for the treatment of IL-31-associated disorders. Reviewed by: Zhi Liu, *Correspondence: Ulrike Raap raap.ulrike@klinikum-oldenburg.de INTRODUCTION Specialty section: This article was submitted to Autoimmune and Autoinﬂammatory Disorders, a section of the journal Frontiers in Immunology Specialty section: This article was submitted to Autoimmune and Autoinﬂammatory Disorders, a section of the journal Frontiers in Immunology Received: 25 March 2019 Accepted: 31 May 2019 Published: 21 June 2019 IL-31 was ﬁrst described by Dillon et al as a T cell-derived cytokine that was predominantly produced by The cells (1). Interestingly, mice which overexpressed IL-31 were observed to develop severe pruritus, alopecia and skin lesions (1). Furthermore, animal models of airway hypersensitivity exhibited increased IL-31 receptor expressions and these expressions, as well as those of IL-31 receptors in the skin, were also veriﬁed in respective human tissues (1). Human and mouse IL-31 share 31% amino acid homology and the genes for this cytokine are, respectively, located on chromosome 12q24.31 and chromosome 5 (1). Received: 25 March 2019 Accepted: 31 May 2019 Published: 21 June 2019 IL-31 is a member of the gp130/IL-6 family of cytokines and is a four helix bundle cytokine that acts as a ligand for the heterodimeric IL-31 receptor A (IL-31RA), a novel type I cytokine receptor, and the oncostatin M receptor (OSMR), the latter of which increases IL-31 binding aﬃnity to IL-31RA. Both of these IL-31 receptors are expressed on a variety of diﬀerent cell types including T cells, dorsal root ganglia (DRG), keratinocytes, dendritic cells, eosinophils, basophils, and macrophages (2–7). The functional activity of IL-31 Keywords: IL-31, itch, autoimmunity, bullous pemphigoid, psoriasis, eosinophils, basophils Edited by: Ralf J. Ludwig, Universität zu Lübeck, Germany Reviewed by: Zhi Liu, University of North Carolina at Chapel Hill, United States Victoria Patricia Werth, University of Pennsylvania, United States Edited by: Ralf J. Ludwig, Universität zu Lübeck, Germany Chronic Spontaneous Urticaria It has long been recognized that autoimmune mechanisms are an important component of CsU [reviewed in (33)]. CsU is deﬁned by the rapid appearance of pruritic wheals lasting <24 h but which may occur repeatedly over a period of at least 6 weeks. The autoimmune association in CsU is either due to IgE mediated autoallergy against thyroid peroxidase or IgG auto-antibodies against the α-subunit of the high-aﬃnity IgE receptor(FcεRI) and, more rarely, against IgE. All of these possible autoimmune mechanisms activate mast cells and basophils, cells which have been centrally implicated with CsU (34–36). Alongside its pruritogenic actions, IL-31 has a proinﬂammatory role too due to the upregulation of proinﬂammatory gene expressions in T cells, including CCL2 and granulocyte colony-stimulating factor (21). Interestingly, CCL2 has recently been implicated in driving basophil traﬃcking in systemic lupus erythematosus (SLE) and in severe allergic reactions (22, 23). IL-31 release by CD4+ T cells is mostly restricted to Th2 cells, although Th1 cells can produce this cytokine in vitro following exposure to IL-4 (21). Epidermal keratinocytes are also a major target for IL-31, where it induces chemokine gene expressions for GRO1α (CXCL1), I-309 (CCL1), MIP-1β (CCL4), TARC (CCL17), MIP-3β (CCL19), MDC (CCL22), and MIP-3 (CCL23), [(1), reviewed in(11)]. Furthermore, Singh et al. showed that IL-31 increased epidermal basal-cell proliferation in mice resulting in thickening of the epidermal skin layer but increased transepidermal water loss (14). This impairment of skin barrier function occurs due to IL-31 decreasing ﬁlaggrin expression in human keratinocytes (24). Interestingly, reduced ﬁlaggrin expressions are strongly While mast cells and basophils are the main sources of histamine, antihistamine treatment in patients with CsU does not fully eliminate itch, suggesting that IL-31 may have some input in pruritic mechanisms associated with CsU. Indeed, we observed increased IL-31 serum levels in CsU patients, although these levels were generally lower than those seen in atopic dermatitis patients (30). These observations have recently been conﬁrmed by Lin et al. who demonstrated signiﬁcantly higher levels of IL- 31 in CsU patients with most severe pruritus intensity compared to milder forms but not with urticarial activity per se (37). This supports the notion that IL-31 contributes to itch rather than other aspects of disease activity. In our own investigations, we subsequently discovered that the main cellular sources of IL-31 in skin lesions of CsU patients are basophils (7). Citation: Gibbs BF, Patsinakidis N and Raap U (2019) Role of the Pruritic Cytokine IL-31 in Autoimmune Skin Diseases. Front. Immunol. 10:1383. doi: 10.3389/ﬁmmu.2019.01383 June 2019 \| Volume 10 \| Article 1383 1 Frontiers in Immunology \| www.frontiersin.org IL-31 in Autoimmune Skin Diseases Gibbs et al. associated with atopic dermatitis and have been shown to enhance Staphylococcus aureus colonization (24, 25). appears to depend on the expression of both the IL-31RA and OSMR since, in basophils, we recently demonstrated that blocking of either receptor type decreases IL-31-induced IL-4 and IL-13 release (7). Increased levels of IL-31 have now been shown in various inﬂammatory skin diseases including prurigo nodularis (12), atopic dermatitis (26–28), contact eczema (29), chronic spontaneous urticaria (CsU) (30), as well as in a subset of patients with mastocytosis (31). In an animal model of atopic dermatitis anti-IL-31 treatment signiﬁcantly reduced scratching in mice (16), underlining the important role of IL-31 in mediating pruritus. This seems to apply to humans too, where IL-31 levels were shown to correlate with disease severity in atopic dermatitis (27, 28). Interestingly, CsU patients respond to omalizumab therapy which was recently shown to be associated with reduced IL-31 serum levels (32). This suggests that this IL-31 may be modulated by IgE-dependent mechanisms and this further highlights the potential of anti-IL-31 as a therapeutic approaches as well as targeting IgE-positive eﬀector cells. Subsequent signaling following receptor complex binding involves the Janus kinase–signal transducer and activator of transcription pathway, the phosphoinositide-3-kinase–Akt pathway as well as the mitogen-activated protein kinase pathway (4, 8–10). IL-31 signaling controls a wide range of biological functions and immunomodulatory eﬀects, such as the release of chemokines, proinﬂammatory cytokines, regulation of cell proliferation and stimulation of DRG sensory neurons which are responsible for induction of itch [reviewed in (11, 12)]. IL-31 IN AUTOIMMUNE SKIN DISEASES In dermatological diseases, IL-31 is of particular interest in terms of itch and inﬂammation as well as impaired skin-barrier function arising from IL-31-induced tissue remodeling (13, 14). The cytokine was not only found to induce severe pruritus in mice, where levels of IL-31 correlated with scratching behavior, but a NC/Nga mouse model of atopic dermatitis demonstrated the potential therapeutic beneﬁt of blocking anti-IL-31 antibodies (1, 15, 16). Before the discovery of IL-31, histamine was widely considered to be the most important pruritic mediator and the relative lack of therapeutic control of itch by H1- antihistamines was ascribed following the discovery of additional H4-receptor input in pruritus [reviewed in (17)]. This is partly supported by a recent successful clinical trial using an H4- receptor antagonist in atopic dermatitis (18), indicating potential future combinational H1- and H4-receptor antagonist/inverse agonist approaches. However, recent data from human clinical trials using nemolizumab (CIM331), a humanized antibody against interleukin-31 receptor A, clearly indicates that IL-31 is an important mediator of itch in humans too (19, 20). IL-31 has thus far been shown to play a prominent role in common inﬂammatory skin diseases, such as atopic dermatitis, particularly in relation to pruritus. However, since pruritus is also a major feature in bullous pemphigoid, chronic spontaneous urticaria (CsU) and other autoimmune diseases there is a potential role for IL-31 in some of these diseases too. Frontiers in Immunology \| www.frontiersin.org Bullous Pemphigoid p g The autoimmune blistering skin disease, bullous pemphigoid (BP), is accompanied by severe pruritus, thus suggesting possible involvement of IL-31. Indeed, Salz et al reported high IL-31 levels in BP blister ﬂuids as well as an association of this cytokine with granulocytes (50). More recently, we also showed that IL- 31 is expressed in the lesional skin of BP patients, particularly in eosinophils from skin tissue and also within blister ﬂuids (51). Moreover, eosinophils were demonstrated to be the major cellular source of IL-31 in BP and isolated peripheral blood eosinophils secreted substantial amounts of IL-31 (51). Similarly to basophils, IL-31 also has chemotactic eﬀects on eosinophils and additionally stimulates these cells to release reactive oxygen species and the chemokine CCL26 (5). FIGURE 1 \| Overview of the main cellular sources and potential roles of IL-31 in autoimmune skin diseases. Red arrows denote direct effects, black indirect effects. Double arrows denote autocrine activation. were responsive to IL-31 stimulation which gave rise to release of the archetypal Th2-type cytokines IL-4 and IL-13 from these cells. Surprisingly, however, IL-31 failed to cause degranulation of basophils, as determined by the expression of the degranulation markers CD63 and CD203c as well as histamine release (7). Since basophils are predominant sources of both IL- 31 and histamine, inhibition of basophil function is of therapeutic interest in controlling itch-related symptoms in CsU. Furthermore, basophils not only signiﬁcantly inﬁltrate the skin in CsU (38) but are also crucial contributors of IL-4 and IL-13 which support IgE class-switching and, in the case of IL-4, also Th2 immunity [reviewed in (39)]. IgE-dependent basophil activation can be therapeutically targeted with the monoclonal antibody omalizumab, which prevents free-circulating IgE from binding to cells such as basophils and also mast cells, leading to the down- regulation of cell-surface FcεRI expressions as well as the degree of IgE sensitization. Since the autoimmune component of CsU is thought to involve autoantibodies against either FcεRI or IgE a reduction of cell-surface IgE and/or FcεRI due to omalizumab presents an important therapeutic strategy. Indeed, omalizumab has been used successfully to treat patients with CsU (40, 41). The fact that IL-31 levels are also reduced following omalizumab treatment further supports the role of basophils in CsU (32). This is underlined by reports of a rapid reduction in vivo of both FcεRI and IgE expressions on basophils due to omalizumab (42). Chronic Spontaneous Urticaria Basophils produced and released IL-31 in response to IgE-dependent stimulation and were even more responsive in terms of secreting the cytokine following incubation with the bacterial peptide N-Formylmethionyl-leucyl-phenylalanine (7). Basophils also expressed both IL-31 and OSMR receptors, and June 2019 \| Volume 10 \| Article 1383 Frontiers in Immunology \| www.frontiersin.org 2 IL-31 in Autoimmune Skin Diseases Gibbs et al. possibly due to a greater turnover of basophils compared to their skin tissue-ﬁxed mast cell counterparts (43). FIGURE 1 \| Overview of the main cellular sources and potential roles of IL-31 in autoimmune skin diseases. Red arrows denote direct effects, black indirect effects. Double arrows denote autocrine activation. In addition to our observations regarding IL-31 inducing Th2- type cytokine release from basophils, we found that the cytokine also facilitates basophil chemotaxis (7). This indicates that IL-31 plays a role in the orchestration and accumulation of basophils in inﬂammatory skin diseases such as CsU. Since basophils are majorly involved in the pathogenesis of CsU, targeting IL- 31 directly (e.g., by nemolizumab) may prevent both IL-31- mediated basophil recruitment as well as the autocrine eﬀects of this cytokine in stimulating the release of IL-4 and IL-13 from basophils. Such approaches using nemolizumab (CIM331) have recently been tested successfully (also in terms of reducing itch) in atopic dermatitis in both humans and other primates (19, 20, 44–46). Similarly, the anti-canine-IL-31 monoclonal antibody, lokivetmab, has been used to treat atopic dermatitis in dogs (47–49). However, the potential clinical beneﬁts of anti-IL- 31 monoclonal antibodies have yet to be reported for CsU (and indeed other autoimmune skin diseases). Frontiers in Immunology \| www.frontiersin.org CONCLUDING REMARKS The published literature to date shows that IL-31 is diﬀerentially associated with autoimmune skin diseases, especially those where pruritus is a major symptom, such as CsU and bullous pemphigoid. Here, recent evidence shows that basophils and eosinophils are major sources of this cytokine, respectively, especially within aﬀected skin tissues. However, the input of other IL-31-producing cells, especially CD4+ T cells, in these autoimmune diseases still needs to be elucidated in terms of IL- 31-mediated immunomodulatory roles. IL-31 possibly plays a role in the traﬃcking of basophils to aﬀected tissue sites because of its known actions in supporting the release CCL2 and CCL26 chemokines. Basophil-derived IL-4, the release of which is also driven by IL-31, may support the development of Th2 cells and subsequent further IL-31 production. The interplay of IL- 31 and various immune cells that are known to play a role in autoimmune skin diseases is summarized in Figure 1. Overall, IL-31 is a crucial driver for pruritus in certain autoimmune skin diseases and the recent development of blocking antibodies oﬀers exciting new therapeutic opportunities to combat itch- related symptoms. Alopecia Areata In addition to its role in pruritic skin diseases, IL-31 was originally also associated with alopecia areata, an autoimmune disease involving inﬂammation of the hair follicles and a loss of immune privilege [reviewed in (70)]. However, although IL-31 and hair loss was described in mice (1) it was not subsequently observed in patients with alopecia areata (60). Autoimmune Connective Tissue Diseases Affecting the Skin g Pruritus is a prominent symptom in several autoimmune connective tissue diseases such as lupus and dermatomyositis, although these diseases can aﬀect multiple organ systems and are often supervised by dermatologists. In lupus erythematosus there is currently only sparse evidence to suggest a role of IL- 31 and, as yet, no reports speciﬁcally dealing with cutaneous forms of this autoimmune disease. Although pruritus does often accompany cutaneous lupus erythematosus it is usually mild in severity and (65), possibly indicating that IL-31 is not a major player in this disease. In systemic lupus erythematosus (SLE) IL-31 serum levels were not shown to be signiﬁcantly increased compared to healthy controls (66). However, in stark contrast, a more recent study reported signiﬁcant increases in IL-31 serum levels in SLE as well as identiﬁcation of IL-31 polymorphisms that were associated with SLE in the Chinese population (67). While the role of IL-31 in SLE is still unclear these IL-31 polymorphisms may nonetheless serve as novel genetic markers of susceptibility to SLE (67). ACKNOWLEDGMENTS Dermatomyositis is another autoimmune disease that involves the skin and is sometimes involves considerable pruritus(68), although the muscles and sometimes the lungs can also be aﬀected. In a recent article, Kim et al showed that itch correlated The authors would like to thank the Deutsche Forschungsgemeinschaft for providing research funding (Translationale Pruritusforschung RA 1026/3-1). AUTHOR CONTRIBUTIONS All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. Bullous Pemphigoid While mast cells may also be targeted by omalizumab, the reductions in FcεRI receptor density resulting from omalizumab therapy are known to occur sooner in basophils than for skin mast cells, It is, however, as yet unclear whether BP patients display elevated serum levels of IL-31, since the currently published literature has reported conﬂicting observations. Our own investigation showed only minor elevations in serum IL-31 levels vs. non-BP controls (51) compared to Salz et al (50), who observed signiﬁcant increases, and Kulczycka-Siennicka et al. (52), who reported reduced levels of the cytokine. Despite these diﬀerent observations regarding IL-31 serum levels, it is clear that eosinophils are one of the major sources for the increased IL-31 levels in BP blister ﬂuids, which often contain a highly enriched accumulation of eosinophils. Because BP patients experience pruritus particularly in lesional skin the increases in IL-31 levels in blister ﬂuids oﬀer a plausible explanation for this localized itch. Basophils have also been reported to be associated with BP (53) but their potential contribution to IL-31 in this disease is not yet known. Eosinophil-derived IL-31 may possibly participate in the traﬃcking of basophils to lesional tissues aﬀected by BP and this may further be supported by IL-31-induced CCL26 release from eosinophils (5). Furthermore, there are several case reports of successful therapy of BP using omalizumab, suggesting potential basophil involvement (54–56). However, Freire et al recently showed that the mast cells and eosinophils make up most of the IgE-expressing cells in BP skin (57) and both cell June 2019 \| Volume 10 \| Article 1383 3 IL-31 in Autoimmune Skin Diseases Gibbs et al. types are widely reported to participate in various autoimmune skin diseases [reviewed in (58, 59)]. It is thus conceivable that the clinical beneﬁt of omalizumab therapy may largely due to a reduction in eosinophil and mast cell function. with increased cutaneous severity where IL-31 and IL-31RA gene expressions in lesional skin were upregulated compared with either non-lesional skin or that from healthy controls (69). IL-31 mRNA expression also positively correlated with itch score and immunoreactivity for IL-31 and IL-31RA was greater in lesional skin. Furthermore, lesional dermatomyositis skin contained signiﬁcantly more IL-31-producing cells, of which CD4+ cells were the most abundant IL-31-expressing cell type (70). Psoriasis It was originally thought that IL-31 does not play a role in psoriasis, based on comparisons of cellular expressions of the cytokine (both at mRNA level and immunoreactivity) together with controls (12, 29, 60). However, more recent investigations have reported that serum IL-31 levels are signiﬁcantly elevated in psoriasis and that chronic itch associated with psoriatic skin is accompanied by increased transcription of IL-31 (61, 62). Narbutt et al further showed that IL-31 serum levels were signiﬁcantly reduced after narrowband UVB phototherapy, coinciding with a substantial reduction in pruritus in these patients (61). This study indicates that itch accompanying psoriasis might be associated with IL-31. Interestingly, skin mast cells have been shown to express elevated IL-31 in psoriatic skin compared to healthy controls (63). In contrast, however, Czarnecka-Operacz et al. failed to observe any correlation between itch severity in psoriasis and IL-31 (64). 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https://openalex.org/W4384826450	https://www.frontiersin.org/articles/10.3389/fnins.2023.1208554/pdf	English	null	Effects of health qigong exercise on upper extremity muscle activity, balance function, and quality of life in stroke patients	Frontiers in neuroscience	2,023	cc-by	8,951	OPEN ACCESS OPEN ACCESS EDITED BY Lingxiao He, Xiamen University, China REVIEWED BY Sanghee Moon, Ithaca College, United States Edgar Hernandez, National University of Colombia, Colombia CORRESPONDENCE Zhonglou Zhang 9081435@qq.com Xiaolei Liu liuxiaolei99@hotmail.com RECEIVED 19 April 2023 ACCEPTED 04 July 2023 PUBLISHED 19 July 2023 CITATION Yang H, Li B, Feng F, Zhang L and Liu X (2023) Effects of health qigong exercise on upper extremity muscle activity, balance function, and quality of life in stroke patients. Front. Neurosci. 17:1208554. doi: 10.3389/fnins.2023.1208554 OPEN ACCESS EDITED BY Lingxiao He, Xiamen University, China REVIEWED BY Sanghee Moon, Ithaca College, United States Edgar Hernandez, National University of Colombia, Colombia OPEN ACCESS EDITED BY Lingxiao He, Xiamen University, China REVIEWED BY Sanghee Moon, Ithaca College, United States Edgar Hernandez, National University of Colombia, Colombia CORRESPONDENCE Zhonglou Zhang 9081435@qq.com Xiaolei Liu liuxiaolei99@hotmail.com RECEIVED 19 April 2023 ACCEPTED 04 July 2023 PUBLISHED 19 July 2023 CITATION Yang H, Li B, Feng F, Zhang L and Liu X (2023) Effects of health qigong exercise on upper extremity muscle activity, balance function, and quality of life in stroke patients. Front. Neurosci. 17:1208554. doi: 10.3389/fnins.2023.1208554 REVIEWED BY Sanghee Moon, Ithaca College, United States Edgar Hernandez, National University of Colombia, Colombia Huixin Yang 1, Baolong Li 2, Lin Feng 2, Zhonglou Zhang 1* and Xiaolei Liu 3* 1 Institute of Nation Traditional Sport, Harbin Sport University, Harbin, China, 2 The Second Affiliated Hospital of Heilongjiang University of Traditional Chinese Medicine, Harbin, China, 3 Chinese Traditional Regimen Exercise Intervention Research Center, Beijing Sport University, Beijing, China Introduction: This study explored the effects of Qigong exercises on upper extremity muscle activity, balance function, and quality of life in stroke patients. Methods: A total of 30 stroke patients were randomly allocated to either control group or Qigong group. In the Qigong group, participants completed an intervention of Qigong Baduanjin over 8 weeks. Data on the electromyographic activities of the biceps brachii muscle, triceps brachii muscle, and muscle coordination were obtained using surface electromyography and the co- contraction ratio (CCR). Data on balance were obtained using the PK254P balance function detection system. Quality of life was measured using the brief version of the World Health Organization Quality of Life scale. COPYRIGHT © 2023 Yang, Li, Feng, Zhang and Liu. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). OPEN ACCESS The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Results: The results for the Qigong group showed a significant difference in CCR of the triceps brachii muscle (p < 0.01). Concerning balance (assessed using the open-eye test), there was a significant decrease (p < 0.05) in Y-axis trajectory deviations and the Y-axis speed in the Qigong group. In the closed- eye test, the peripheral area of the Qigong group was significantly lower than that of the control group (p < 0.05). Significant differences were also observed in physical health (p < 0.05), psychological health (p < 0.01), environment (p < 0.01), and the total scores for quality of life (p < 0.01) in the Qigong group. Discussion: We conclude that Qigong exercises improve the quality of life in stroke patients and have positive effects on the coordination of limb extremities and balance function. Qigong, upper extremity, muscle activity, balance function, quality of life, stroke patients TYPE Original Research PUBLISHED 19 July 2023 DOI 10.3389/fnins.2023.1208554 TYPE Original Research PUBLISHED 19 July 2023 DOI 10.3389/fnins.2023.1208554 Effects of health qigong exercise on upper extremity muscle activity, balance function, and quality of life in stroke patients OPEN ACCESS EDITED BY Lingxiao He, Xiamen University, China REVIEWED BY Sanghee Moon, Ithaca College, United States Edgar Hernandez, National University of Colombia, Colombia CORRESPONDENCE Zhonglou Zhang 9081435@qq.com Xiaolei Liu liuxiaolei99@hotmail.com RECEIVED 19 April 2023 ACCEPTED 04 July 2023 PUBLISHED 19 July 2023 CITATION Yang H, Li B, Feng F, Zhang L and Liu X (2023) Effects of health qigong exercise on upper extremity muscle activity, balance function, and quality of life in stroke patients. Front. Neurosci. 17:1208554. doi: 10.3389/fnins.2023.1208554 1. Introduction According to a bibliometric analysis of the clinical studies on Qigong, the top 15 most commonly studied diseases/conditions are diabetes, chronic obstructive pulmonary disease, hypertension, stroke, cervical spondylosis, lumbar disc herniation, insomnia, knee osteoarthritis, lower back pain, osteoporosis, coronary heart disease, breast cancer, periarthritis of the shoulder, depression, and metabolic syndrome. Thus, considerable research has shed light on the effects of Qigong following a stroke (Zhang et al., 2020). Frontiers in Neuroscience 01 frontiersin.org Yang et al. Yang et al. 10.3389/fnins.2023.1208554 strength and function, and the relationship between upper limb extremity muscle activities and balance function. Stroke is the second leading cause of death (after cancer) globally (Feigin et al., 2017), accounting for 11.8% of all-cause mortality (Valery et al., 2018), and there are roughly 17 million new cases each year (Crichton et al., 2016). Once the sensory or motor conduction in stroke patients is impaired, they may lose muscle strength or control, partly or wholly (Vahlberg et al., 2013, 2017). Such impairments can impede patients’ daily activities and lead to long- term weakness, paresis, or falls (Taylor-Piliae et al., 2014; Pereira et al., 2019). In this study, we evaluated Qigong exercises on stroke patients’ upper extremity muscle activity, balance function, and quality of life, regarding the benefits of Qigong. It showed the data of the stroke patients before and after the experiment to see how Qigong works on the physical health, like the muscles and joints, and mental health, like the quality of life. In a recent study, a 10-day mind–body interactive exercise program, Chan-Chuang qigong, was shown to benefit subacute stroke inpatients and offer useful adjunctive rehabilitative care for stroke inpatients. Among the covariates in this study, neurologic deficit, muscle strength, low-frequency to high-frequency ratio, and anxiety were significantly associated with changes in quality of life (Chen et al., 2019). A longer, 12-week supervised Baduanjin exercise intervention was effective and safe in modulating cerebral hemodynamics, reducing blood pressure, and improving anthropometric parameters and related psychological outcomes in older community-based adults at risk for ischemic stroke. After the 12-week intervention period and additional 12-week follow-up period, the Baduanjin exercise group displayed significant changes in most cerebral hemodynamic parameters compared to the control group: lower systolic blood pressure, diastolic blood pressure, plasma total cholesterol levels, waist circumference, hip circumference, and waist/hip ratio; and improved mood, self- confidence, self-esteem, quality of life, and sleep quality (Zheng et al., 2019). 2.2. Subjects The sample size was ascertained using G-power before the experiment. By setting the effect size to 0.25, power to 0.8 and alpha to 0.05, we can get 29 as the minimum of the total number of the subjects. In addition, considering 15% attrition rate, at least 35 subjects should be recruited at the very beginning. Therefore, a total of 41 patients with a clinical diagnosis of stroke were voluntarily recruited from the Second Affiliated Hospital of the Heilongjiang University of Chinese Medicine. They were in Brunnstrom stages IV–V. The inclusion criteria for patients were as follows: (1) aged less than 75 years; (2) had experienced their first, officially diagnosed stroke; (3) were at the stage of motor recovery (Brunnstrom IV-V stage); (4) experienced the onset of stroke more than 1 week but no more than 3 months ago; (5) were able to walk more than 6 meters freely without any support; (6) had a Mini-Mental State Exam score higher than 24. Patients were excluded if they had (1) a hearing impairment, a neurological impairment, or sensory aphasia that influenced training, or (2) severe comorbidities (e.g., diabetes, heart disease, cancer, brain tumor). A total of 11 patients withdrew from the study, meaning that 30 patients were studied and the requirements of the G-power calculation were met. Research has examined whether Qigong exercises can improve physical and mental health indices such as sleep quality, gait performance, and muscle strength (Wang et al., 2014; Wu et al., 2016; Klich and Milert, 2018; Moreno-Segura et al., 2018). In stroke patients, the stability of the core muscle group is weak, and the trunk and pelvis cannot be stabilized during anti-gravity activities, which may weaken the motor and balance function in patients (Feigin et al., 2014; Valery et al., 2018). As a low-intensity activity, Qigong exercises can increase patients’ muscle flexibility and strength, thus alleviating muscle tension and improving balance, mental health, and quality of life (Feigin et al., 2015; Crichton et al., 2016). Therefore, Qigong exercises have been used to improve balance (Taylor-Piliae et al., 2014; Xu et al., 2017) and endurance (Xu et al., 2017) in the rehabilitation of stroke patients. However, there are few studies about the positive effects of Qigong on stroke patients with moderate impairments of upper limb All the information regarding the intervention was concealed from the subjects and the testers, but not the researchers, ensuring the study was blinded. 2.1. Design This study was based on an 8-week randomized controlled design with two groups (a control group and a Qigong group). The data were collected at the baseline, after 4 weeks and at the end of week 8 (Figure 1). Subjects in the Qigong group practiced Qigong Baduanjin while the control group subjects did not receive or perform any regular physical activity. The study protocol was approved by the Internal Review Board of the Heilongjiang University of Chinese Medicine. All eligible subjects signed an informed consent form before the study. Data from all the subjects admitted to the hospital consecutively were collected with clinical registration (Registry Number: ChiCTR2100048031) and ethical approval (Approval Number:2021-K135) by the Ethics Committee of the Second Affiliated Hospital of the Heilongjiang University of Chinese Medicine. 1. Introduction Meanwhile, regular Baduanjin training has been associated with less loss of cognitive function in patients after stroke. Compared to the control group, Baduanjin training significantly increased Montreal Cognitive Assessment scores for global cognitive function from 16 weeks after the intervention. It also improved scores for immediate recall and short-term delayed recall from 16 weeks after the intervention, and long-term delayed recognition scores at 24 weeks after the intervention in the memory domain. In addition, it shortened the time taken to complete the Trail Making Test-A and Trail Making Test-B tests from 16 weeks after the intervention in the executive functioning domain. These effects were maintained from 4 weeks until after follow-up (i.e., 28 weeks after the intervention). In the mixed linear model analyses, significant main effects or interaction effects for exercise were found for those measures (Zheng et al., 2020). Frontiers in Neuroscience 2.2. Subjects Subjects did not know which group they have been placed in until the experiment finished. As the control group, they 02 frontiersin.org Yang et al. 10.3389/fnins.2023.1208554 FIGURE 1 The flowchart of the experiment. FIGURE 1 The flowchart of the experiment. 2.4. Measurements and procedure were treated with the same Qigong methods after the experiment. The subjects were randomly allocated using random, computer-generated numbers into the Qigong group or the control group such that there were 15 subjects per group. An independent investigator generated the random numbers and was not involved in the intervention and assessment. Subject characteristics such as age, gender, duration of disease, and education were collected via a questionnaire at the beginning of the study. Four instruments were used as measurement tools. The muscle activity of the biceps brachii and triceps brachii was assessed using sEMG (Thought Technology, SA7550, Canada) and the Ag/AgCI electrode (CH55D) with an inter-electrode distance of 2 cm. These data were collected at baseline, after 4 weeks and at the end of week 8. Frontiers in Neuroscience 2.3. Intervention Surface electromyography (sEMG) is an inexpensive, quantitative, non-invasive, and painless method to evaluate neuromuscular function (Ford et al., 2008). In the mid-twentieth century, sEMG was used to recognize neuromuscular patterns, identify the roles of different muscles in functional movements, and determine prognosis following nerve injury (Vahlberg et al., 2013, 2017). In the following decades, sEMG was used to examine neurological factors such as the coordination between limbs, patterns of muscle activation and co-activation, and biofeedback effects. More recently, sEMG has been used to identify and treat gait disorders in stroke survivors (Liu et al., 2011; Pollock et al., 2014; Pereira et al., 2019). The control group did not receive any specific exercise training and these participants were asked to maintain their routine medical or rehabilitative treatment and original physical activity. They were able to avail themselves of the Qigong exercises after the study. The Qigong group completed the Baduanjin exercises 3 days a week for 40 min a day based on their routine medical or rehabilitative treatment at the Heilongjiang University of Chinese Medicine. The Qigong master had a 10-years teaching experience. Each session consisted of 5–10 min warm-up and cool-down routines that focused on stretching, rhythmic breathing, and relaxation followed by 30 min of Qigong practice (including movements and repetitions). All the subjects gathered together to train as part of the same community and were guided by qualified Baduanjin exercise coaches. The whole process for the Qigong group was video-recorded so patients could practice in their own time. The PK254P balance function detection system (Wu and Gong, 2019) is used to test the balance of patients when standing upright with their eyes opened and closed. In this study, the PK254P was used to collect data from two groups, the control group and Qigong group. 03 frontiersin.org Yang et al. Yang et al. 10.3389/fnins.2023.1208554 The indexes included the trajectory circumference (TL), the peripheral area (Area), the X-axis trajectory deviations (X dev.), and the Y-axis trajectory deviations (Y dev.). The indexes indicate the stability of the center of gravity in stroke patients. The X-axis speed (X speed) and the Y-axis speed (Y speed) show the micro-control of the center of gravity. X and Y axes are the speed indices of PK254P. X axis represents the velocity in the left and right direction. Y axis represents the velocity in the anteroposterior direction. 3.1. Characteristics of study participants Of the 30 participants, 15 were in the control group, and 15 were in the Qigong group. Table 1 shows the detailed characteristics of the participants at baseline. At baseline, there were no significant differences between the groups for the continuous variables (age and duration of disease). The chi-squared test for categorical variables (gender, lesion nature, Brunnstrom stage, and education) also showed no significant differences between the groups at baseline. Furthermore, there was no significant difference in the bias of the impairments, especially the postural tone, which is also listed in Table 1. Before testing, each patient sat on the testing chair with their knees bent so that their lower legs were at 90 degrees to their thighs and the floor. Testers shaved the skin on each patient’s upper limbs and swabbed the area with alcohol before attaching the electrodes. Electrodes were attached to the biceps brachii and triceps brachii muscles. Patients were familiar with the function of the electrodes and understood the instructions that followed. The temperature of the test room was kept between 22 and 28°C. During testing, the sEMG signals were collected in five-second segments and the measurement for each position (at elbow extension and elbow flexion) was repeated five times, following the system prompt. The sEMG results of the biceps brachii and triceps brachii muscles in the intermediate three activities were recorded, as per the method used by Zhu et al. (2014). 3.2.2. CCR of biceps brachii and triceps brachiif As shown in Table 2, main effects of time were found for the CCR of triceps brachii (p < 0.01), in which subjects’ scores significantly decreased from baseline to week 8, regardless of group allocation. After week 8, the CCR of triceps brachii in the Qigong group changed by 0.07 ± 0.07 (p = 0.008), which indicated a significant improvement compared with pre-test. Using the Pk254P balance function detection system, the TL, Area, X dev., Y dev., X speed, and Y were selected as the speed indexes in each group. The smaller these indexes, the better the individual’s balance ability. TABLE 1 Baseline characteristics of two groups (n = 30). Variable Control group (n = 15) Qigong group (n = 15) p-value Age (year) 54.02 ± 38.41 52.85 ± 32.85 0.88 Length of disease (day) 57.57 ± 60.34 52.57 ± 56.12 0.77 Gender Male 10 11 0.28 Female 5 4 0.91 Lesions nature Cerebral infarction 11 10 0.83 Encephalorrhagia 4 5 0.72 Brunnstrom stage IV 4 7 0.58 V 11 9 0.58 Postural tone High 4 3 0.99 Low 11 12 The WHOQOL-BREF was used to assess patients’ quality of life and included an assessment of each participant’s physical, mental, and social states, and their environment before the intervention, then 4 and 8 weeks after the intervention. All assessments were completed by the same tester who had been trained for the work and did not know of the difference between the two groups. TABLE 1 Baseline characteristics of two groups (n = 30). 3.2.1. sEMG results of biceps brachii muscle and triceps brachii muscleif The co-contraction ratio (CCR) represented the ratio of normalized agonistic muscle activity to normalized antagonistic muscle activity during the intervention. A decreasing CCR value signified a decrease in antagonist co-activation, higher coordination, and smoother movement (Lim et al., 2016). CCR was calculated according to the formula reported by Hammond et al. (1988) as follows: As shown in Table 2, there was a significant main effect of time for the biceps brachii (elbow extension). No significant main or interaction effects were found for biceps brachii (elbow flexion) and triceps brachii (elbow extension and elbow flexion). Frontiers in Neuroscience 2.3. Intervention In addition, the World Health Organization Quality of Life-Brief version (WHOQOL-BREF) scale was used to test each patient’s quality of life. Comparison between groups was performed using a mix-model repeated ANOVA (group × time) while comparisons within each group were conducted using paired t-tests. 2.5. Statistical analysis The software SPSS 17.0 (SPSS Inc., Chicago, United States) was used for statistical analysis, and a two-sided p < 0.05 was set to determine statistical significance. Measurement data are expressed as means and standard deviations (SD). The Kolmogorov–Smirnov test was used to test the normality of distributions and the chi-squared test was used to compare the categorical variables for the two groups at baseline. The Kruskal-Wallis test and one-way analysis of variance (ANOVA) were used to analyze the categorical variables and continuous variables at baseline and across groups, respectively. 04 frontiersin.org Yang et al. 10.3389/fnins.2023.1208554 TABLE 2 Descriptive statistics of all outcome variables across groups at baseline and post-intervention. TABLE 2 Descriptive statistics of all outcome variables across groups at baseline and post-intervention. TABLE 2 Descriptive statistics of all outcome variables across groups at baseline and post-intervention. Qigong group Control group Group Time Interaction effect V0 V1 V2 V0 V1 V2 F/P F/P F/P Biceps brachii (elbow extension) 41.13 ± 13.48 36.08 ± 15.05 29.99 ± 11.29 42.39 ± 17.96 37.39 ± 15.87 30.64 ± 9.78 0.156/0.697 3.327/0.047 0.003/0.996 Triceps brachii (elbow flexion) 93.45 ± 25.56 96.17 ± 23.29 97.08 ± 21.54 91.87 ± 30.49 98.31 ± 32.72 100.08 ± 23.67 0.062/0.806 0.275/0.747 0.042/0.951 CCR of triceps brachii 0.30 ± 0.06 0.27 ± 0.07 0.23 ± 0.06* 0.31 ± 0.07 0.28 ± 0.06 0.24 ± 0.05* 0.341/0.565 7.570/0.002 0.038/0.961 Triceps brachii (elbow extension) 31.80 ± 9.74 30.75 ± 10.09 28.12 ± 10.56 29.15 ± 13.47 28.08 ± 9.69 27.64 ± 12.35 1.065/0.313 0.269/0.756 0.063/0.933 Biceps brachii (elbow flexion) 95.12 ± 26.33 100.64 ± 25.50 109.02 ± 31.79 92.99 ± 26.59 98.94 ± 26.48 102.58 ± 26.18 0.288/0.597 1.048/0.356 0.052/0.941 CCR of biceps brachii 0.26 ± 0.06 0.23 ± 0.06 0.20 ± 0.04 0.24 ± 0.11 0.22 ± 0.07 0.21 ± 0.07 0.068/0.797 1.869/0.176 0.144/0.816 V0, V1, V2 indicated pre-test, after 4-week intervention, after 8-week intervention, respectively. Represented for p < 0.05; represented for p < 0.01. TABLE 3 Comparison of static balance indexes in the open-eye of biped (X ± S). TABLE 3 Comparison of static balance indexes in the open-eye of biped (X ± S). Qigong group Control group Group effect Time effect Interaction effect V0 V1 V2 V0 V1 V2 F/P F/P F/P X dev. 3.3. Effects of Qigong exercises on balance function A main effect of time was also found for psychological health. In the Qigong group, the scores increased significantly at week 8 (p < 0.01). At the same time, the control group showed a significant increase (p < 0.05) at week 8.if As shown in Table 3, in the open-eye test, a main effect of time was found for Y dev. (p < 0.05), reflecting improvements in both the Qigong group and the control group after 8 weeks. In the Qigong group, the scores of the Y speed and Area significantly decreased (p < 0.05 and p < 0.01, respectively) after the 8-week intervention. No significant main or interaction effects were found for social relationship scores. However, for the environment scores, a main effect of group (p < 0.05) was found, demonstrating a significant difference between the Qigong group and the control group. The scores for the Qigong group increased continually over the study and showed a significant improvement at week 8 (p < 0.01). As shown in Table 4, in the closed-eye test, except for the scores of Area, in which the Qigong group showed a significant decrease, no significant improvement was found. 2.5. Statistical analysis (mm) 7.00 ± 1.76 6.87 ± 2.36 6.09 ± 1.83 7.48 ± 2.23 6.95 ± 1.94 6.62 ± 2.18 0.803/0.375 2.417/0.100 0.181/0.817 Y dev. (mm) 7.74 ± 1.74 7.52 ± 2.15 6.57 ± 1.95 7.67 ± 2.20 7.24 ± 1.84 6.43 ± 1.69* 0.230/0.634 4.616/0.013 0.035/0.966 X speed (mm/s) 11.09 ± 2.45 10.61 ± 2.84 10.30 ± 1.55 11.43 ± 2.82 10.43 ± 3.25 10.33 ± 1.62 0.024/0.876 1.619/0.208 0.114/0.859 Y speed (mm/s) 11.87 ± 3.07 11.65 ± 2.21 10.35 ± 1.82* 11.57 ± 3.22 11.33 ± 2.71 10.57 ± 1.78 0.062/0.805 3.971/0.028 0.208/0.782 TL (mm) 524.83 ± 170.49 504.43 ± 172.53 429.26 ± 139.87 529.95 ± 189.99 521.81 ± 199.70 510.43 ± 129.54 1.327/0.256 1.418/0.248 0.658/0.513 Area (mm2) 759.65 ± 202.45 686.48 ± 243.47 607.00 ± 133.55** 747.19 ± 227.74 636.62 ± 266.38 612.24 ± 128.13* 0.363/0.550 4.884/0.014 0.182/0.801 V0, V1, V2 indicated pre-test, after 4-week intervention, after 8-week intervention, respectively. , V0 compared with V2, P < 0.05, P < 0.01. TABLE 3 Comparison of static balance indexes in the open-eye of biped (X ± S). TABLE 3 Comparison of static balance indexes in the open-eye of biped (X ± S). Frontiers in Neuroscience 3.3.1. Effects of Qigong exercises on quality of lifehif The results showed a significant main effect of time (p < 0.001) for the total WHOQOL-BREF score (Table 5). In the Qigong group, significant differences were found at week 8 (p < 0.01) as compared with baseline. Previous studies have suggested that training can improve the balance of the human body and thus enhance balance control (Horak et al., 1997). Studies also found that 4 weeks of intensive practice of Tai Chi improved standing balance in healthy seniors and 12 weeks of short- form Tai Chi produced specific standing balance improvements in people with a chronic stroke that outlasted training for 6 weeks (Au- Yeung et al., 2009). One session of Qigong exercise improved energy and balance between the upper and lower halves of the body (Lin et al., 2018), For physical health, there was a significant main effect of time (p < 0.05) but no main effect of group or interaction. The score of the Qigong group had significantly decreased at week 8 (p < 0.05) and there was a significant difference between the pre-test and post-test (p < 0.05). 05 frontiersin.org Yang et al. 10.3389/fnins.2023.1208554 10.3389/fnins.2023.1208554 flexion movements. For example, the elbows are bent and risen in the first movement (Figure 3); stretched and extended sideways in the second movement (Figure 2). Moreover, the palms are pushed up and pressed down alternately with the elbows bent in the third movement (Figures 4, 5). Three muscle movements are mainly used in this process, including eccentric contraction, concentric contraction, and isokinetic contraction (Ge et al., 2004) with the coordination of agonistic muscles and antagonistic muscles (Christou et al., 2003). Stroke patients usually have high CCR accompanied by central nervous system damage, which directly leads to abnormal changes in coordinated muscle contraction (Desrochers et al., 2016). Qigong exercises require slow and even, coherent and gentle, loose and tight combination, breathing and consciousness together. Patients can perceive the internal and external changes of the limbs, and thus stimulate and improve the central nervous system. In this way, Qigong exercises may promote the coordination and exertion of agonistic and antagonistic muscles in stroke patients.h and the addition of Baduanjin to conventional cardiac rehabilitation exercise further improved exercise endurance, the reserve of heart function, and quality of life (Yu et al., 2018). Another study suggested that Chan-Chuang Qigong, a mind–body interactive exercise, is an appropriate and desirable healthcare intervention for subacute stroke inpatients. 3.3.1. Effects of Qigong exercises on quality of lifehif The Chan-Chuang Qigong intervention was found to improve the physical and mental aspects of quality of life when considering the severity of stroke impairment, muscle strength, sympathovagal balance, anxiety, and depression, supporting the view that Chan-Chuang Qigong is a simple and safe adjunctive approach for stroke inpatients when practiced for at least 10 days (Chen et al., 2019). After an 8-week intervention, data relating to upper extremity muscles were collected for sEMG in the current study. The findings showed that patients who practiced the Qigong exercise experienced a significant decrease in CCR of upper limb muscles compared with the control group. This suggested that Qigong exercise positively reduced muscle spasms in stroke patients. In Qigong exercises, the core movements of the upper limbs are “Chen Jian Zhui Zhou (Drop Shoulders and Sink Elbows).” It can be demonstrated vividly in Figure 2. The movement in Figure 2 is “Zuo You Kai Gong Si She Diao (Posing as an Archer Shooting Both Left- and Right-Handed),” which requires the subjects to lower down the shoulders and elbows when they are shooting. In Starting movement, two hands become a ball posture, require sink the shoulders and drop the elbows, and keep a hollow space under the armpits. Both hands do upward and downward can minimize shoulder shrugs, keep shoulders and elbows relaxed, and to some extent, loosen the joints and the surrounding ligaments (Gao et al., 2014). In the action of Figure 3, the shoulder muscles force downward to prevent the shoulder from lifting, then drop the hands while relaxing the shoulders, elbows and chest. Therefore, with the joint tendons relaxed and tensed, when maintaining upper limb movement, the patients only need to overcome their own gravity without using additional force. Since too much exertion of antagonistic muscles would result in spasms in stroke patients (Becker et al., 2019), it is highly probable that Qigong exercises can relieve the exertion of antagonist muscles by making patients relax their upper limbs as much as possible. The transformations, which smooth out the gaps between the movements, are primarily Horse-stance (Figure 6) and Standing- stance based. It requires the exercisers to bend their knees slightly, take a step sideways to either the left or the right with single foot, and withdraw the step back. The gravity center of the body shifts from either foot to the place between the feet. 3.3.1. Effects of Qigong exercises on quality of lifehif In the lower limb movement transformation, the lower limb muscle strength is exercised, and the balance ability and stability of the lower limbs are improved. In the open-eye balance test, the Qigong group performed significantly better after 8 weeks, especially in Y dev., Y speed index, and Area index, which indicated that Qigong exercise improved the balance of the stroke patients. However, the Qigong group did not differ from the control group in the closed-eye test. It can be seen that in the absence of sensory assistance, the Qigong group had no improvement in performance in the left, right, front or back directions. Unlike in the opened-eye test, the maintenance of static posture depends more on attention than dynamic posture change. This may also be the main reason why there is no significant change in the center of gravity micro-control index in stroke patients without conscious assistance. The decrease of muscle strength in stroke patients is one of the main causes of motor function damage, including balance. The center of gravity is one of the most important factors affecting balance. The big toe and the first metatarsal bone also play a major role in adjusting bodily posture (Meyer et al., 2004). Qigong exercise involves movements in six directions, front and back, left and right, up and down. The gravity center of the body can be either in the up, down, left and right directions. The basic technical movements of Qigong require In Qigong exercises, agonist muscle are properly exerted and antagonist muscles are kept at a corresponding degree of relaxation, which is conducive to unblocking the meridians, slipping the joints and strengthening the muscles and bones. The upper limbs are flexed at the elbows, which not only ensures smooth movement of the skeletal muscles and joints, but also maintains stability and improves muscle strength. In the qigong movements, the upper limbs all have elbow TABLE 4 Comparison of static balance indexes in closed-eye of biped (X ± S). Qigong group Control group Group effect Time effect Interaction effect V0 V1 V2 V0 V1 V2 F/P F/P F/P X dev. (mm) 8.96 ± 1.72 8.57 ± 2.13 7.78 ± 1.70 9.24 ± 2.10 9.05 ± 1.83 8.95 ± 2.24 2.531/0.119 1.542/0.220 0.565/0.570 Y dev. Frontiers in Neuroscience frontiersin.org 3.3.1. Effects of Qigong exercises on quality of lifehif FIGURE 3 Liang Shou Tuo Tian Li San Jiao (holding the hands high with palms up to regulate the internal organs). Qigong group Control group Group effect Time effect Interaction effect V0 V1 V2 V0 V1 V2 F/P F/P F/P Physical health 13.69 ± 2.25 14.21 ± 2.20 15.18 ± 3.00 13.50 ± 1.97 14.04 ± 1.79 14.82 ± 2.48 0.307/0.583 4.465/0.015 0.025/0.973 Psychological health 12.17 ± 1.65 13.11 ± 2.49 14.87 ± 2.60** 11.97 ± 1.71 12.92 ± 2.06 13.33 ± 2.49* 2.585/0.115 9.776/0.000 1.410/0.250 Social relationships 10.07 ± 3.60 10.96 ± 2.57 12.06 ± 2.97 9.71 ± 3.38 10.41 ± 3.57 10.98 ± 2.79 1.642/0.207 2.725/0.072 0.142/0.868 Environment 13.26 ± 2.15 14.28 ± 2.13 15.02 ± 2.68 12.98 ± 2.16 13.12 ± 2.24 13.36 ± 2.37 6.851/0.012 2.364/0.101 1.003/0.370 Total 54.36 ± 4.80 56.25 ± 4.83 60.78 ± 5.25 54.04 ± 6.43 55.25 ± 5.11 58.24 ± 5.40* 1.586/0.215 13.018/0.000 0.564/0.571 V0, V1, V2 indicated pre-test, after 4-week intervention, after 8-week intervention, respectively. , V0 compared with V2, P < 0.05, P < 0.01. FIGURE 2 Chen Jian Zhui Zhou (drop shoulders and sink elbows) in the movement Zuo You Kai Gong Si She Diao (posing as an archer shooting both left- and right-handed). FIGURE 3 Liang Shou Tuo Tian Li San Jiao (holding the hands high with palms up to regulate the internal organs). FIGURE 2 Chen Jian Zhui Zhou (drop shoulders and sink elbows) in the movement Zuo You Kai Gong Si She Diao (posing as an archer shooting both left- and right-handed). FIGURE 3 Liang Shou Tuo Tian Li San Jiao (holding the hands high with palms up to regulate the internal organs). FIGURE 3 Liang Shou Tuo Tian Li San Jiao (holding the hands high with palms up to regulate the internal organs). FIGURE 2 Chen Jian Zhui Zhou (drop shoulders and sink elbows) in the movement Zuo You Kai Gong Si She Diao (posing as an archer shooting both left- and right-handed). FIGURE 3 Liang Shou Tuo Tian Li San Jiao (holding the hands high with palms up to regulate the internal organs). the control group. Therefore, Qigong exercises effectively improved the balance of the center of gravity in stroke patients. that the rotation of upper body and the transfer of gravity center should be completed during the horse stance and standing stance, especially when the exercisers bend their knees and squat down. Frontiers in Neuroscience frontiersin.org 3.3.1. Effects of Qigong exercises on quality of lifehif (mm) 9.91 ± 2.37 9.61 ± 2.29 8.61 ± 1.88 9.62 ± 2.80 9.48 ± 2.06 8.10 ± 2.05 0.675/0.416 4.827/0.011 0.076/0.924 X speed (mm/s) 15.43 ± 2.69 15.09 ± 2.94 14.57 ± 2.95 15.52 ± 2.84 14.43 ± 3.25 14.43 ± 2.181 0.001/0.978 0.739/0.477 0.422/0.650 Y speed (mm/s) 15.52 ± 2.97 15.30 ± 3.18 14.22 ± 1.93 14.95 ± 3.12 14.52 ± 3.71 14.52 ± 1.778 0.336/0.565 1.324/0.272 0.573/0.562 TL (mm) 669.48 ± 163.17 693.43 ± 229.03 667.91 ± 211.56 736.81 ± 196.08 721.33 ± 221.88 724.24 ± 196.01 1.455/0.234 0.043/0.952 0.135/0.863 Area (mm2) 968.57 ± 294.27 935.3 ± 373.95 771.04 ± 244.46* 935.71 ± 289.66 913.81 ± 326.38 875.19 ± 224.04 0.108/0.744 2.244/0.116 0.703/0.489 V0, V1, V2 indicated pre-test, after 4-week intervention, after 8-week intervention, respectively. V0 compared with V2, P < 0.05, P < 0.01. TABLE 4 Comparison of static balance indexes in closed-eye of biped (X ± S). TABLE 4 Comparison of static balance indexes in closed-eye of biped (X ± S). 06 frontiersin.org Yang et al. 10.3389/fnins.2023.1208554 TABLE 5 WHOQOL-BREF score comparison (X ± S). TABLE 5 WHOQOL-BREF score comparison (X ± S). the control group. Therefore, Qigong exercises effectively improved TABLE 5 WHOQOL-BREF score comparison (X ± S). Qigong group Control group Group effect Time effect Interaction effect V0 V1 V2 V0 V1 V2 F/P F/P F/P Physical health 13.69 ± 2.25 14.21 ± 2.20 15.18 ± 3.00 13.50 ± 1.97 14.04 ± 1.79 14.82 ± 2.48 0.307/0.583 4.465/0.015 0.025/0.973 Psychological health 12.17 ± 1.65 13.11 ± 2.49 14.87 ± 2.60** 11.97 ± 1.71 12.92 ± 2.06 13.33 ± 2.49* 2.585/0.115 9.776/0.000 1.410/0.250 Social relationships 10.07 ± 3.60 10.96 ± 2.57 12.06 ± 2.97 9.71 ± 3.38 10.41 ± 3.57 10.98 ± 2.79 1.642/0.207 2.725/0.072 0.142/0.868 Environment 13.26 ± 2.15 14.28 ± 2.13 15.02 ± 2.68 12.98 ± 2.16 13.12 ± 2.24 13.36 ± 2.37 6.851/0.012 2.364/0.101 1.003/0.370 Total 54.36 ± 4.80 56.25 ± 4.83 60.78 ± 5.25 54.04 ± 6.43 55.25 ± 5.11 58.24 ± 5.40* 1.586/0.215 13.018/0.000 0.564/0.571 V0, V1, V2 indicated pre-test, after 4-week intervention, after 8-week intervention, respectively. , *V0 compared with V2, P < 0.05, P < 0.01. FIGURE 2 Chen Jian Zhui Zhou (drop shoulders and sink elbows) in the movement Zuo You Kai Gong Si She Diao (posing as an archer shooting both left- and right-handed). 3.3.1. Effects of Qigong exercises on quality of lifehif For example, in the fifth movement (Figures 7–9), exercisers should rotate the upper body in a circle and transfer the gravity center from the left to the right, or from the right to the left, when shifting the Horse stance to either one side Horse stance. In the sixth movement (Figure 10), exercisers extend their legs fully first. When lifting the arms above the head, exercisers begin to rise the upper body with the arms, thus the upper body recurves and both the legs and arms can be fully stretched. Quality of life was assessed in terms of physical health, psychological state, social relationships, environment, and a total score. Qigong is a whole-body exercise, which is a mind–body exercise that harmonizes body, breath and mind. When practicing Qigong, the consciousness is dominant and the breathing is coordinated with the movements. Each movement has a breathing method to match with. For example, exercisers inhale when rising the arms and exhale when putting down the arms in the first movement; inhale when turning the hands into the palms and exhale when stretching sideways in the second movement; inhale when rotating the arms outward, exhale when withdrawing the arms back, and hold the breath for a second when keeping the posture still in the fourth movement. Previous studies found that during Qigong practice, the maximum pressure on the sole of the foot is mainly concentrated on the first metatarsal bone and the big toe, while in normal walking it is more concentrated on the second, third, fourth, and fifth areas (Mao et al., 2006). The results in the present study showed that the Y dev. and Y speed index of the Qigong group was significantly lower than that of The results indicated that the score for physical health in the Qigong group improved significantly while psychological health also improved significantly by the end of the 8 weeks, similar to the results 07 Yang et al. 10.3389/fnins.2023.1208554 FIGURE 6 Horse stance. FIGURE 6 Horse stance. FIGURE 4 The right palm is pushed up and the left palm is pressed down. FIGURE 6 Horse stance. FIGURE 4 FIGURE 4 The right palm is pushed up and the left palm is pressed down. and quality of life (Ying et al., 2019). Frontiers in Neuroscience frontiersin.org 3.3.1. Effects of Qigong exercises on quality of lifehif Furthermore, paced breathing synchronized with rhythmic muscle contraction leads to more resilient activation of the parasympathetic response than either alternating contractions or breathing alone, which may help explain the benefits of mind–body disciplines (Chin and Kales, 2019). The number of Qigong movements is small, and the route is not complicated, easy to learn. Practitioners can adjust the intensity and difficulty of the movements according to their own physical conditions. Before the experiment, most of the stroke patients were afraid of taking up Qigong exercise due to their limitations in physical activity. However, after being exposed to Qigong exercises, they were able to complete the basic movements, which may explain why the Qigong group had higher scores for their psychological state than the control group after 8 weeks and why as the study progressed, the psychological state of stroke patients improved. FIGURE 5 The left palm is pushed up and the right palm is pressed down. In our study, the control group showed some improvements but were less marked than the Qigong group in the same amount of time. Although there was no significance between the groups, the Qigong group performed better than the control group after 8 weeks, which has been shown in some of the data comparisons except for the significant ones. For example, Qigong group has made greater progress in the TL of static balance in the open-eye of biped in Table 3, the Y speed of static balance in the closed-eye of biped in Table 4, the Social relationships of WHOQOL-Bref in Table 5.i Regarding the insignificant results between groups, we have also revised our experimental design and put it down to three reasons. First, an 8-week intervention was not enough for more significant effects. Second, the results was not that noticeable with low frequency of Qigong exercise, 3 times a week in this experiment. Third, the time spent on Qigong exercise each time should be longer. We would like The left palm is pushed up and the right palm is pressed down. of earlier studies. In a 6-month Baduanjin exercise program for breast cancer survivors, there were significant improvements in heart rate variability, shoulder range of motion on the affected side, depression, of earlier studies. Frontiers in Neuroscience Feigin, V. L., Forouzanfar, M. H., Krishnamurthi, R., Mensah, G. A., Connor, M., Bennett, D. A., et al. (2014). Global and regional burden of stroke during 1990-2010: findings from the global burden of disease study 2010. Lancet 383, 245–255. doi: 10.1016/S0140-6736(13)61953-4 Data availability statement All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors. Ethics statement to observe the effect of Qigong exercise on stroke patients and obtain the optimal scheme by increasing the length, frequency and time in the future design and experiment. The studies involving human participants were reviewed and approved by the Ethics Committee of the Second Affiliated Hospital of Heilongjiang University of Chinese Medicine. The patients/ participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article. 3.3.1. Effects of Qigong exercises on quality of lifehif In a 6-month Baduanjin exercise program for breast cancer survivors, there were significant improvements in heart rate variability, shoulder range of motion on the affected side, depression, 08 frontiersin.org Yang et al. 10.3389/fnins.2023.1208554 FIGURE 9 The movement of gravity center to either the up and the down from Yao Tou Bai Wei Qu Xin Huo (swinging the head and lowering the body to relieve stress). FIGURE 7 The movement of gravity center to either the left or the right from Yao Tou Bai Wei Qu Xin Huo (swinging the head and lowering the body to relieve stress). FIGURE 9 FIGURE 7 The movement of gravity center to either the left or the right from Yao Tou Bai Wei Qu Xin Huo (swinging the head and lowering the body to relieve stress). FIGURE 7 The movement of gravity center to either the left or the right from Yao Tou Bai Wei Qu Xin Huo (swinging the head and lowering the body to relieve stress). FIGURE 9 The movement of gravity center to either the up and the down from Yao Tou Bai Wei Qu Xin Huo (swinging the head and lowering the body to relieve stress). FIGURE 9 The movement of gravity center to either the up and the down from Yao Tou Bai Wei Qu Xin Huo (swinging the head and lowering the body to relieve stress). FIGURE 10 Liang Shou Pan Zu Gu Shen Yao (moving the hands down the back and legs and touching the feet to strengthen the kidneys). FIGURE 8 The movement of gravity center to either the front and the back from Yao Tou Bai Wei Qu Xin Huo (swinging the head and lowering the body to relieve stress). FIGURE 8 The movement of gravity center to either the front and the back from Yao Tou Bai Wei Qu Xin Huo (swinging the head and lowering the body to relieve stress). FIGURE 10 Liang Shou Pan Zu Gu Shen Yao (moving the hands down the back and legs and touching the feet to strengthen the kidneys). FIGURE 10 Liang Shou Pan Zu Gu Shen Yao (moving the hands down the back and legs and touching the feet to strengthen the kidneys). 09 Frontiers in Neuroscience frontiersin.org Yang et al. 10.3389/fnins.2023.1208554 Crichton, S. L., Bray, B. D., McKevitt, C., Rudd, A. G., and Wolfe, C. D. A. (2016). Patient outcomes up to 15 years after stroke: survival, disability, quality of life, cognition and mental health. J. Neurol. Neurosurg. Psychiatry 87, 1091–1098. doi: 10.1136/jnnp-2016-313361 Christou, E. A., Yang, Y., and Rosengren, K. S. (2003). Rapid Communication. Taiji Training Improves Knee Extensor Strength and Force Control in Older Adults. The Journals of Gerontology: Series A 58, M763–M766. doi: 10.1093/gerona/58.8.M763 Funding After 8 weeks of intervention, Qigong exercises can effectively improve the upper extremity muscle activity, balance, and quality of life in stroke patients. Qigong exercise not only improves balance but the coordination and exertion of antagonistic and active muscles in stroke patients, and quality of life. The psychological state of the Qigong group was also significantly improved in this study but given the small sample size, further studies are needed to provide stronger evidence with larger samples. In addition, multivariate analyses may be applied to understanding the relationship between hypertension, diabetes, obesity, hyperlipidemia, non-sudden stroke, internal capsule involvement, pontine morphology, and other factors, and the motor dysfunction associated with stroke. This research was funded by the Natural Science Foundation of Heilongjiang Province (No. LH2020H009). Author contributions HY, BL, ZZ, and XL: conceptualization. HY, BL, LF, and XL: methodology. HY and LF: software. HY, ZZ, and XL: validation. HY and XL: formal analysis, writing—original draft preparation, and writing—review and editing. HY, BL, and LF: investigation and data curation. HY, ZZ, BL, and LF: resources. XL: supervision. HY: project administration and funding acquisition. All authors have read and agreed to the published version of the manuscript and made substantial contributions to conception and design. Desrochers, P., Kairy, D., Pan, S., Corriveau, H., Tousignant, M., and Elè Ne Corriveau, H. (2016). Tai chi for upper limb rehabilitation in stroke patients: the patient’s perspective. Disabil. Rehabil. 39, 1313–1319. doi: 10.1080/09638288.2016.1194900 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. Frontiers in Neuroscience 5. Limitations There are some limitations in this study that should be acknowledged. 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https://openalex.org/W4231422362	https://periodicos.ufpb.br/ojs2/index.php/tpa/article/download/38857/20813	English	null	Practices and Knowledges	Teoria e Prática em Administração	2,018	cc-by	10,484	Practices and Knowledges Silvia Gherardi University of Trento - Italy silvia.gherardi@unitn.it Teoria e Prática em Administração, volume 8, número 2 (special issue), ano 2018 Practices and Knowledges Gherardi p. 33-59 DOI: http://dx.doi.org/10.21714/2238-104X2018v8i2S-38857 Submission: Mar/05/18 –Second version: Mai/13/18 –Acceptance: Mai/15/18 Teoria e Prática em Administração, volume 8, número 2 (special issue), ano 2018 Practices and Knowledges p. 33 59 DOI: http://dx.doi.org/10.21714/2238-104X2018v8i2S-38857 Submission: Mar/05/18 –Second version: Mai/13/18 –Acceptance: Mai/15/18 Abstract The present article retraces the way in which, over the years, I have developed a post- humanistic approach to social practice on the basis of an Actor-Network sensibility and the way in which practice theory appeared in the literature on learning and knowing in organizations. From this background, I propose an epistemology of practice grounded on relationality, multiplicity, and transformation, and I approach practice as an empirical phenomenon from the perspective of knowing as an activity situated in working and organizing. From this point of view the central interest in practice theories becomes practice as a collective and knowledgeable doing. Keywords: practice, knowledge, post-humanism, embodiment 33 33 Teoria e Prática em Administração, volume 8, número 2 (special issue), ano 2018 Practices and Knowledges Gherardi p. 33-59 DOI: http://dx.doi.org/10.21714/2238-104X2018v8i2S-38857 Submission: Mar/05/18 –Second version: Mai/13/18 –Acceptance: Mai/15/18 In a previous issue of Teoria e Prática em Administração (7/1, 2017) Theodore Schatzki wrote about the relationship of practices and people, offering a way of thinking about practices and people according to which these two phenomena are equally real while mutually dependent and co-responsible for social life. In the present article, I wish to explore a complementary view on practices offering to think about their relationship with knowledge. We have a common point of departure in assuming that practices have to do with activities, and we acknowledge that many practice scholars are concerned with what people do (e.g., Shove et al., 2012). Nevertheless, I argue that doing is not enough for defining a practice and that the concept of practice is more useful for empirical research when it is conceived as a ‘knowledgeable doing’. In the present essay I explore the relationship between knowledge and practice, knowledgeable practices, knowing in practice, and knowledge as a situated activity. I trace a tradition of sociological thought in practice theories that derives from the studies of scientific knowledge, that challenges the conventional understanding of ‘social’ as human-centered, and that operates a re-turn to practice within learning and knowing in organization. I anticipate that my understanding of practice is grounded in an actor-network approach, i.e. ‘in a disparate family of material-semiotic tools, sensibilities, and methods of analysis that treat everything in the social and natural worlds as a continuously generated effect of the webs of relations within which they are located’ (Law, 2009: 141). Abstract Such sensibility is important, within the panorama of contemporary practice theories, because it offers a theoretical conception of the social that does not separate activities, thought, feelings, matter, discourses and their collective cultural-historical forms. Without a theoretical conception of the social, one cannot analyze activities in situ and report on how knowledge always undergoes construction and transformation in use. In this essay, I shall contextualize (in the first section) my approach to practice theories within the broader debate in the philosophy of social theories that discusses the changing status of knowledge and moves towards a definition of knowledge as an activity situated in practice. The precursors of the empirical study of knowing in situ were the so- called laboratory studies and, in section 2, I present their contributions to the study of knowledge practices. From those studies we have learnt how the boundaries between ‘scientific’ and mundane knowledge practices can be blurred, and similarly the boundaries between humans and nonhumans. Therefore, we can move (in section 3) to a formulation of a post-humanist practice theory that joins other post-epistemologies in the project of de- 34 34 Teoria e Prática em Administração, volume 8, número 2 (special issue), ano 2018 Practices and Knowledges Gherardi p. 33-59 DOI: http://dx.doi.org/10.21714/2238-104X2018v8i2S-38857 Submission: Mar/05/18 –Second version: Mai/13/18 –Acceptance: Mai/15/18 Teoria e Prática em Administração, volume 8, número 2 (special issue), ano 2018 Practices and Knowledges Gherardi p. 33-59 DOI: http://dx.doi.org/10.21714/2238-104X2018v8i2S-38857 Submission: Mar/05/18 –Second version: Mai/13/18 –Acceptance: Mai/15/18 centering the human subject as the main source of action and moving from a formulation of practice theory as ‘humans and their practices’ to a vision of practice as the entanglement of humans, materialities, discourses, knowledges and any other relevant element in the situated activities. A posthumanist conception of practice enables a different conception of the ‘social’ in theories of social practice, since the social is not the ‘other’ of the ‘natural’. The social is generated by material practices – as expressed in the term sociomateriality - and an empirical study of social practices focuses on how the social is done and holds together. Abstract Since my main interest is on practices as an empirical phenomenon and how to elaborate a methodological framework for studying practices in organizational settings, in the section 4, 5, 6, I illustrate its main focus: the sensorial and elusive knowledges embedded in knowing in practice; realities as enacted in practices; how all the practice elements achieve agency in their being connected and how practices are woven in a texture of practices. Philosophy of social sciences and the status of knowledge What is knowledge and which one is the ‘correct’ methodology for approaching it is an open question that I need to sketch brieflyi in order to position knowing as an activity situated in practice. It is necessary to start with the discredit of positivism in the 1960s and the loss of credibility of its main assumptions: scientism (scientific method as the only valuable source of knowledge); naturalism (the unity of method across the social and the natural sciences); a regularity notion of causality (the regular association of x and y is both necessary and sufficient to talk about causality); an assumption that explanation entails prediction (and vice versa); a rejection of explanations in terms of mental or subjective states (like intentions or motives), a predilection for quantification and sophisticated statistical analysis, and finally a sharp distinction between facts and values (Baert, 2005). Nevertheless, we have to keep in mind that those positivistic assumptions were strongly associated with the establishment of sociology as a scientific discipline preoccupied with the nature of the scientific method and the distinctiveness of the sociological research. Later, in the 1980s, sociologists became more suspicious of holistic theories such as structuralism, functionalism, and system theory (Sciortino, 2009). A renewed interest in meaning, language and critique arose in the late twentieth- century social sciences (Baert & Dominguez Rubio, 2009) when naturalist philosophy of social science was challenged by three intellectual strands: hermeneutics, Wittgensteinian 35 Teoria e Prática em Administração, volume 8, número 2 (special issue), ano 2018 Practices and Knowledges Gherardi p. 33-59 DOI: http://dx.doi.org/10.21714/2238-104X2018v8i2S-38857 Submission: Mar/05/18 –Second version: Mai/13/18 –Acceptance: Mai/15/18 philosophy, and critical theory. I mention only the first two in an attempt to highlight the move from knowledge conceived within positivism to knowledge conceived as an empirical phenomenon. Gadamer’s (1975) hermeneutics, in contrast with Dilthey, Rickert, and Weber who conceived of historical context, tradition, and prejudice as external factors that bias understanding and rationality, considered those factors as the very elements that make understanding possible. Each specific historical context discloses a horizon of understanding, and the hermeneutical task of the social sciences is to achieve a “fusion of horizons” whereby the interpreters and interpreted enter a hermeneutical dialogue. The liberation of meaning from logics brought into focus the relations between meaning, practices and language. Philosophy of social sciences and the status of knowledge Moreover, the interest in meaning-making lead to the rediscovery of the phenomenological tradition, and in particular of Alfred Schutz’s work (1962, 1964) which focused on the “common-sense world.” Different from scientific rationality, common- sense rationality operates within a taken-for-granted world where people suspend disbelief. Schutz’s phenomenological sociology and Berger and Luckmann’s (1966) constructionist research institutionalized the assumption that the categories, through which people interpret social reality, help as well to create the world (Weinberg 2008). Social constructionism has contributed to the empirical investigations of what counts as genuine knowledge and why. Therefore, knowledge becomes an observable and researchable phenomenon rather than a merely imagined normative ideal. Also for Wittgenstein (1968), meaning is irreducible to any rule-following method but it derives from the use of language within what he called a language-game, and to give an account of the meaning of an utterance we need to describe how the utterance is used within a specific language-game. The agreement reached by using a language-game, is not simply a convergence in opinions but an agreement reached by sharing a specific form of life. Social constructionism also referred to Wittgenstein to argue against the possibility of establishing universal and objective knowledge claims. Schutz’s work, together with Wittgenstein’s, influenced Garfinkel’s (1967) ethnomethodological studies of the micro-mechanisms of social order. Both hermeneutics and ethnomethodology influenced Giddens’ (1984, 1993) structuration theory, which explores the various ways in which people’s sense-making practices contribute to the making of social order. 36 Another important and powerful critique to foundational philosophy of social sciences comes from the so-called pragmatist turn that rediscovered the early pragmatism by Dewey and neo-pragmatism (Rorty 1980, 1982). The Pragmatist school is skeptical of scientific knowledge as representing the inner nature of the external world: knowledge should no longer be seen as mirroring or representing the world “as it really is”. Pragmatists argue instead for the primacy of the agent’s point of view and knowledge acquisition is seen as active, as one of the tools that people have to cope with the world, using as resources (and not as limitations) the conceptual framework, language, and cultural setting in which they are situated. With this critique of foundationalism comes a rejection of any philosophical attempt to capture the scientific method. The boundaries between scientific knowledge and ordinary knowledge become blurred. Knowledge practices: the laboratory and everyday life In the 1970s, the social construction of scientific facts and scientific knowledge became studied as a field of social practices like any other. The ethnographic methodologies were used in laboratory studies (Callon 1986; Clarke & Fujimura 1992; Collins 1985; Gieryn 1999; Knorr-Cetina 1981; Latour and Woolgar 1986; Lynch, 1993; Pickering, 1995; Rheinberger 1997; Traweek 1988) resulting in rich descriptions of the mundane practices related to science, scientists, technologies and innovations. Knowledge practices were looked at as paying attention to the unique relations between things that are brought together in laboratories’ activities, following the ethnomethodological principle that science is what scientists do. In The Manufacture of Knowledge (1981) Karin Knorr Cetina, who was studying a food lab in Berkeley, observed how scientific facts are constructed within the context of social life and are fabricated by social consensus and experimenters’ expectation-based tinkering. Laboratories’ practices were described as an opportunity-directed to networks of scientists connected through resource relationships (either materials and tools) and the raw material of ideas. In her later book, Epistemic Cultures: How the Sciences Make Knowledge (1999), where high-energy physics and molecular biology labs were studied as knowledge cultures, Knorr Cetina examined the way the machineries of knowledge construction are themselves constructed. Objects of knowledge are always in the process 37 Teoria e Prática em Administração, volume 8, número 2 (special issue), ano 2018 Practices and Knowledges Gherardi p. 33-59 DOI: http://dx.doi.org/10.21714/2238-104X2018v8i2S-38857 Submission: Mar/05/18 –Second version: Mai/13/18 –Acceptance: Mai/15/18 of being materially defined, they continually acquire new properties and change the ones they have. In this sense, she presents an ontology of the object of knowledge based on an open-ended becoming rather than a fixed being. From Knorr Cetina (2001) I shall borrow the term ‘epistemic practice’ to refer to the kind of practice that has knowledge as the object of inquiry, while by the term ‘knowing-in-practice’, I refer to a knowledgeable doing in accomplishing a practice. Knowing as a situated activity can be studied either in the context of a situated activity (a practicing) or as the object of practitioners’ reflection on a practice, after practicing. Similarly, we can observe talking in practice and talking on practice. Knowledge practices: the laboratory and everyday life With the progressive institutionalization of Science and Technology Studies (STS) the elusive boundaries between science and non-science, scientific and non-scientific knowledge were taken-for-granted (Roosth & Silbey, 2009) and we can say that the ‘manufacture of knowledge’ is work done within laboratories’ practices in a way not dissimilar from the way it is done in any other working practice. Moreover, in both cases the knowledgeable doing of the expert practitioners are open to the knowledgeable practices of non-experts. We saw it in the case of museums (Star & Griesemer, 1989) where lay persons participate to the production of natural sciences and gay activists contributed to the understanding of AIDS treatments (Epstein, 1996). The so-called ‘daughters of DES’ expanded the knowledge about the long-term consequences of estrogen and became political activists (Bell, 2009) and ordinary patients contribute to medical knowledge and tools’ developments. Laboratories studies and science and technology studies made visible how the concept of practice connects ‘knowing’ with ‘doing’. It conveys the image of materiality, of fabrication, of handiwork, of the craftsman’s skill in the medieval bottega d’arte. From the Latin verb facere, Knorr-Cetina (1981) uses the term ‘facticity’ and Bruno Latour (1987) the ‘fabrication’ of scientific facts and technical artefacts. Knowledge consequently is fabricated by situated practices of knowledge production and reproduction, using the technologies of representation and mobilization employed by scientists. The term ‘knowing-in-practice’ (Gherardi, 2001; Orlikowski, 2002) sanctions the passage from the noun to the verb, suggesting how knowing is an enactment and an accomplishment, rather than a thing or a static property. What is known constitutes itself in knowledgeable doing, in purposeful activities, and it is ‘situated in practice’ (Suchman, 2007). Knowing-in-practice only becomes meaningful in relation to a distinct social practice. Due to its embeddedness in 38 social practice, knowing is necessarily in permanent flux, and it entails a procedural understanding of the ability to act of all the practice elements once connected and reconnected. In other words, knowledge emerges from the context of its production and is anchored by (and in) material supports in that context. To convey a preliminary idea of the theoretical and methodological framework in which working practices may be analyzed as knowing-in-practice, I summon its characteristics in the following way:  A pragmatic stance. Practical knowledge is directed to doing, to making decisions in situations, to solving problems, to maintaining and reproducing a texture of practices;  A specific temporality. Knowledge practices: the laboratory and everyday life Practical knowledge emerges from the situation and from situated activities;  An anchoring in materiality. Practical knowledge uses fragments of knowledge embedded in knowledgeable bodies, objects and technology, and in the material world that interacts with humans and interrogates them;  An anchoring in discursive practices. Practical knowledge uses the discursive mobilization of cues for action and their positions within a narrative scheme that gives sense to what occurs in communication;  A historical-cultural anchoring. Practical knowledge is also anchored by what has happened in the past and has been learned from experience and in experience. If we consider the setting in which practices are accomplished, we have to include within it, its institutional context.  A historical-cultural anchoring. Practical knowledge is also anchored by what has happened in the past and has been learned from experience and in experience. If we consider the setting in which practices are accomplished, we have to include within it, its institutional context. We can observe how practice is here conceived as a location, in which practice elements are contained and are relationally linked the one to the other. Nevertheless, once we recognize that knowledge is an activity and an activity situated in working practices, we can move on and wonder whether materiality has agency and which effect is produced in knowing practices once agency is attributed to both human and nonhumans working together. Humans and nonhumans working together: a posthumanist practice theory For the moment, I keep the term nonhuman to acknowledge that for a long time within a practice theory—inspired by an actor-network sensibility—the nonhuman was used 39 Teoria e Prática em Administração, volume 8, número 2 (special issue), ano 2018 Practices and Knowledges Gherardi p. 33-59 DOI: http://dx.doi.org/10.21714/2238-104X2018v8i2S-38857 Submission: Mar/05/18 –Second version: Mai/13/18 –Acceptance: Mai/15/18 as the ‘other’ of human beings including objects, tools, technologies and any other materiality hanging in a practice. p. 33-59 DOI: http://dx.doi.org/10.21714/2238-104X2018v8i2S-38857 Submission: Mar/05/18 –Second version: Mai/13/18 –Acceptance: Mai/15/18 as the ‘other’ of human beings including objects, tools, technologies and any other materiality hanging in a practice. A concern with materials of different kinds, with language, discourses, with humans, and with their precarious relations was the foundation for conceiving a practice as a knowledgeable mode of ordering heterogeneous materials into a provisional and productive assembly. Within a project of establishing a material-semiotic relationality in which all the practice elements define and shape each other, humans are not privileged over materials as the main (and the only) source of action. The demarcations between ‘social’ and ‘natural’, ‘nature’ and ‘culture’, ‘structure’ and ‘agency’, ‘humans’ and ‘nonhumans’ are the effect of epistemic practices, and ‘material agency’ is temporally emergent in relation to practice (Pickering 1995). Human and nonhuman, meaning and materiality, big and small, macro and micro, social and technical, these are just some of the dualisms undone by the relational epistemology of practice. Therefore, an epistemology of practice is not limited to operating a connection across dualisms, rather it is a proposal to see how all the demarcations are effects of epistemic practices. People are relational effects in the same way that objects are and within a situated practice, humans, discourses, and materials achieve agency in their being entangled. In this sense, we can use the concept of sociomaterial practices (Orlikowski, 2007). The purpose of these concepts is to emphasize that ‘materiality is integral to organizing, positing that the social and the material are constitutively entangled (italics in the original) in everyday life’ (Orlikowski, 2007, pp. 1437). The term refers to the fact that within a practice meaning and matter, the social and the technological are inseparable and one cannot be defined without reference to the other. A position of constitutive entanglement privileges neither humans nor technologies, neither knowing nor doing; it does not even link them in a form of mutual interdependence (as in two-way interactions). Humans and nonhumans working together: a posthumanist practice theory It was from Wanda Orlikowski’s and Susan Scott’s (2008) work that terms such as entanglement, sociomateriality, intra-action, taken from Karen Barad’s (2003; 2007) work, were translated into organization studies. The humanist practice theory was criticized and posthumanism based on the relationalism between the social and the technical joined other families of posthumanist epistemology (Braidotti, 2013). Some examples of posthumanist epistemology are the feminist new materialism (Alaimo & Hekman, 2008), the affect 40 Teoria e Prática em Administração, volume 8, número 2 (special issue), ano 2018 Practices and Knowledges Gherardi p. 33-59 DOI: http://dx.doi.org/10.21714/2238-104X2018v8i2S-38857 Submission: Mar/05/18 –Second version: Mai/13/18 –Acceptance: Mai/15/18 theories (Clough, 2007), the animal studies (Wolfe, 2010) among others (Taylor, 2016). The aim of a posthuman sociology is to identify and map distributed agency. A posthumanist conception of practice enables a different conception of the ‘social’ in theories of social practice since the social is not the ‘other’ of the ‘natural’. The social is generated by material practices, and an empirical study of social practices focuses on how the social is done and holds together. The concept of sociomaterial practices implies not only that the social and the material are co-constituted, but also that nature and culture are entangled. It has a methodological corollary that entails studying how, within a practice, bodies (humans and more-than-humans), matter, and discourses are expressions of the same sociomaterial world. The term ‘embodiment’ expresses how the nature/culture division is blurred in the materiality of bodies encountering a material-semiotic environment. When we study working practices empirically, we should focus on how practical knowledge is embodied and how practitioners rely on sensible knowledge (Strati, 2007) in order to take a practice forward (Gherardi, 2012; 2017). The centrality of bodies – and sensible knowing - in approaching practices is self-evident, not only because humans ‘are’ bodies (Merleau-Ponty, 1945) but also because bodies stand in between the dualism of persons and things (Esposito, 2014). Nevertheless, the body has been overlooked even when humans are considered the carriers of practices. Therefore, to the definition of practice as an array of ‘doings’ and ‘sayings’ (Schatzki, 2001), I prefer to enlarge the focus to (knowledgeable) ‘seeing, saying, and doing’ (Gherardi, 2006), where seeing is taken as the bodily activity representative of all sensible knowing. Humans and nonhumans working together: a posthumanist practice theory Our Western culture is mainly visual and for this reason with ‘seeing’ I locate within practice all the other bodily knowing, like hearing, tasting, touching in order to stress how activities and discourses are grounded in an embodied and pre-verbal presence and that in becoming a practitioner one learns knowledgeable bodily competences that are practice-specific. Embodied, embedded, and other elusive knowledges Embodiment is a concept present in practice theories, and Reich and Hager (2014) consider it one of the six threads of the literature on practice (the others are: knowing-in- practice; sociomateriality of practices; relationality; historical and social shaping of practices; emergent nature of practices). In fact, it is now widely accepted within the social sciences that selfhood is not only social, but also materially embodied. 41 Teoria e Prática em Administração, volume 8, número 2 (special issue), ano 2018 Practices and Knowledges Gherardi p. 33-59 DOI: http://dx.doi.org/10.21714/2238-104X2018v8i2S-38857 Submission: Mar/05/18 –Second version: Mai/13/18 –Acceptance: Mai/15/18 Teoria e Prática em Administração, volume 8, número 2 (special issue), ano 2018 Practices and Knowledges Gherardi p. 33-59 DOI: http://dx.doi.org/10.21714/2238-104X2018v8i2S-38857 Submission: Mar/05/18 –Second version: Mai/13/18 –Acceptance: Mai/15/18 The idea that knowledge is embedded in situated practices is widespread, and I like to recall how the turn to practice within the literature on learning and knowing rediscovered the concept of practice as a way out from the two dominant conceptions of learning, either cognitivist or reified. Practice theories entered the debate on organizational learning and knowing inserting a distance from an individualistic conception of knowledge as residing in the mind and supporting the claims of a social learning theory viewing learning as legitimate participation in the practices of a community (see Gherardi, 2009). At the same time, the turn to practice inserted a distance from the commodification of knowledge, that in the years 2000 dominated the literature on knowledge management, conceiving knowledge a commodity as any other (Gherardi, 2000). The focus on practice theory, within this debate, was devoted to how knowledge was kept inside the practices of a community, how it was transferred to the new members, and how it was changed by being in use. This debate is initially in debt with the formulation of learning as peripheral legitimate participation within a community of practice, but later the concept was turned upside down and the focus was devoted to the practices that generate a community in their accomplishment (Gherardi, 2009). The concern was on practices as sites of knowing (Nicolini, 2011); on knowledge that was tacit, sticky, non-translated into words, corporeal, haptic and generally aesthetic (Strati, 1999, 2007). Within a sociology of learning: ‘‘Knowledge is not what resides in a person’s head or in a book or in data banks. Embodied, embedded, and other elusive knowledges To know is to be capable of participating with the requisite competence in the complex web of relationships among people and activities. On this definition it follows that learning is always a practical accomplishment. Its goal is to discover what to do; when and how to do it, using specific routines and artefacts; and how to give, finally, a reasonable account of why it was done. Learning, in short, takes place among and through other people” (Gherardi et al., 1998: 274). Embodiment and embodied knowledge have been among the main reasons for the turn to practice around the year 2000, leaving behind the classic practice theories of the first generation such as Deweyan pragmatism or activity theory (Miettinen et al. 2009). Thus, organizational and working practices have been considered as the locus of knowledge 42 Teoria e Prática em Administração, volume 8, número 2 (special issue), ano 2018 Practices and Knowledges Gherardi p. 33-59 DOI: http://dx.doi.org/10.21714/2238-104X2018v8i2S-38857 Submission: Mar/05/18 –Second version: Mai/13/18 –Acceptance: Mai/15/18 production, circulation and transformation Tacit knowing, sensible knowledge, and the knowing body become the main elements for approaching practices as the containers of knowing subjects and known objects. From this perspective embodied knowledge and embodied knowing have been studied as competence, mainly as individual competence but also as a learned collective one. Embodied knowledge, as a type of knowledge where the body knows how to act, leads to a skillful performance that emerges from and through reciprocal relations encompassing the body-in-the-world and the world-in-the-body (Dall’Alba et al., 2018). The body, the gendered body, and embodied knowing are highly relevant for practice- based studies (Yakhlef, 2010). And organizational aesthetics has greatly contributed to directing attention to knowing through the hands, the ears, the nose and all the human senses involved in working practices. The knowledge that is kept, distributed, fragmented, used and transmitted while practicing is embodied, sensory and atmospheric. For an empirical study of practices, the problem becomes how to articulate in words embodied experiences (tacit, aesthetic, embodied), i.e. those ‘elusive knowledges’ (Toraldo, et al. 2016) that are learnt but kept silent within a working practice. The term ‘elusive knowledges’ refers to ‘those forms of knowledge that escape literal representation through discourse including alphanumeric symbols’ (Toraldo, et al. 2016: 3). Nevertheless, they may be made articulable by the use of video- based methods. Embodied, embedded, and other elusive knowledges In fact, the authors value video’s ability, in conjunction with interviews or ethnography, to explore the interactions of humans with material settings that reveal facets of nonverbal communication. The authors suggest that video-based methods facilitate access to embodied practical knowledge not because they capture the objective reality of practical knowing but because they promote cross-modal translations (visual/textual) productive of new knowledge that can prompt reflexivity on knowing-in-practice. Knowing-in-practice: realities are enacted in practices Focusing on practices rather than on persons or structures has an implicit methodological corollary: a practice can be seen as the locus of knowledgeable doing, learning and organizing (as we have proposed in the previous sections), at the same time a practice can be seen as way of knowledgeable doing (as in what follows). The second definition implies to consider a practice as a mode of ordering sociomaterial relations and 43 Teoria e Prática em Administração, volume 8, número 2 (special issue), ano 2018 Practices and Knowledges Gherardi p. 33-59 DOI: http://dx.doi.org/10.21714/2238-104X2018v8i2S-38857 Submission: Mar/05/18 –Second version: Mai/13/18 –Acceptance: Mai/15/18 thus inquiry into how practices generate (an unstable) order, and how ordering becomes a relational and performative effect of practices. p. 33-59 DOI: http://dx.doi.org/10.21714/2238-104X2018v8i2S-38857 Submission: Mar/05/18 –Second version: Mai/13/18 –Acceptance: Mai/15/18 thus inquiry into how practices generate (an unstable) order, and how ordering becomes a relational and performative effect of practices. To give a simple example of how different ‘objects’ are the product of different practices (this is a way of expressing the abstract term of empirical ontologies), I shall narrate an exemplar story that has been told several times in the literature on Science and Technology Studies (Joks & Law, 2017; Law & Joks, 2017; Law & Lien, 2013; Law & Singleton, 2013). In my narrative I relay mainly on Law and Lien (2013: 365-369), and my plotline is developed around the question: What a farmed Atlantic salmon is if it is treated as an effect of relational practices? The story comes from an extended ethnography of farming practices, whose focus is on salmon–human enactments in which salmon become slippery and elusive, and farming practices enact separations between humans and salmon. The texture of farming practices enacts what a salmon is, since they define the qualities of both salmon and humans. Imagine that we observe a practitioner fishing dead salmon out of the water, a fish that is dead or alive, or injured. If it is dead it is also something to be put in a bucket and dumped in a tank filled with formic acid and other dead fish. In this case a salmon (precisely a dead salmon) is an object that needs to be sifted out and removed. Imagine now that we open a scientific book searching for a definition of salmon, and there we find a reference to Linnaean systematics, and the physical characteristics of the salmon. Knowing-in-practice: realities are enacted in practices Another salmon is enacted through scientific categories and another set of relations are described where the salmon is located into a web of links that include a taxonomic system, particular genetic attributes, and a specific lifecycle, geographical range and feeding characteristics. An Atlantic salmon is here a scientific object that is done in the context of specific scientific practices, and it is different from the salmon being done by the practitioner at the farm. If we imagine moving in the farm, we observe the practice of vaccinating young salmon or parr that are pumped up through a pipe, arrive in batches in a gush of water, and drop into a container filled with water and anaesthetics. Once they go limp, they are lifted in a metal basket and decanted onto a stainless-steel work surface behind a rapidly moving conveyer belt. What is the salmon here? Another set of relations are established between: i) the practitioner’s hands that reach out, lift the fish, and drop them onto the conveyer belt, ii) the embodied knowledge in the hands that have learnt how to do the sorting, iii) the red or green light of the machine processing the fish (in the right or wrong way), and iv) the wet, 44 Teoria e Prática em Administração, volume 8, número 2 (special issue), ano 2018 Practices and Knowledges Gherardi p. 33-59 DOI: http://dx.doi.org/10.21714/2238-104X2018v8i2S-38857 Submission: Mar/05/18 –Second version: Mai/13/18 –Acceptance: Mai/15/18 Teoria e Prática em Administração, volume 8, número 2 (special issue), ano 2018 Practices and Knowledges Gherardi p. 33-59 DOI: http://dx.doi.org/10.21714/2238-104X2018v8i2S-38857 Submission: Mar/05/18 –Second version: Mai/13/18 –Acceptance: Mai/15/18 busy and noisy vaccination cabin surrounded by pipes and cables, and filled with buckets, tables and machines. Here we see how the ‘the salmon passive’, or perhaps ‘the salmon not passive enough’ is enacted in a web of distributed agency, where salmon passivity and human or vaccination-machine agency are being done relationally, and moment-by- moment, through continual effort, work, and redoing, and this knowledgeable doing is more or less precarious. The object of this practice is fluid – a salmon may change in form between practices - and it is done within a choreography that should be sustained since there is no ‘salmon’ outside the practices that enact it. What we have observed within a practice (either cleaning the water tank or vaccinating the parr) can be observed in the texture of ordering practices forming the farming. Knowing-in-practice: realities are enacted in practices 33-59 DOI: http://dx.doi.org/10.21714/2238-104X2018v8i2S-38857 Submission: Mar/05/18 –Second version: Mai/13/18 –Acceptance: Mai/15/18 Teoria e Prática em Administração, volume 8, número 2 (special issue), ano 2018 Practices and Knowledges Gherardi p. 33-59 DOI: http://dx.doi.org/10.21714/2238-104X2018v8i2S-38857 Submission: Mar/05/18 –Second version: Mai/13/18 –Acceptance: Mai/15/18 She argues, in material-semiotic mode, that each practice generates its own material reality and not that there are different perspectives on a single disease. Mol coined the label praxiography, which unfortunately did not have a wide following, for denoting it as ‘a story about practices’. Praxiography is a method to ‘stubbornly take notice of the techniques that make things visible, audible, tangible, knowable’ (Mol, 2002: 23). A similar concern is expressed by the term ‘ethnography of the object’ (Bruni, 2005) that, in following the trajectory of a clinical health record in a hospital, incorporates Latour’ (1987) methodological injunction to ‘follow the actor’ and translate it in respect to the agency of the material actants. Knowing-in-practice: realities are enacted in practices Other practices measure the ‘right’ salmon, separating it from the rejected ones, the losers who grow too slowly and follow a different trajectory that I do not exemplify here. The point is that in a relational world, control and ordering are impossible without lack of control and disordering. In conclusion, the story of the ‘salmon multiple’ of aquaculture is a story of fluidity and multiplicity where the ‘what is a salmon’ is performed through overlapping practices from the moment of fertilization to its final trip to the slaughterhouse. A practice approach operates a shift from what a thing is (and why) to how a thing is done within situated sociomaterial practices. Generally, describing a practice as ‘situated’ means considering the organization of the activities as emerging in situ from the dynamics of knowledgeable practicing. With reference to Suchman’s (2007) distinction between plans (ex-ante rationality) and situated action (contingency), we can say that a practice emerges (in time and space) as the effect of situated practicing. I illustrated it with the story of ‘what a salmon is’, and I wish to add and stress that also the researchers’ epistemic practices contribute to the empirical ontology of the multiple salmon and considering the researchers inside the practice they study means that the researchers make the salmon while the salmon makes the researchers. Not only can the salmon be described as a fluid entity that shifts its shape as it moves between practices (Laet & Mol, 2000), other well-known examples of empirical ontologies may be found in Mol’s (2002) work in relation to the multiple body in medicine with lower limb atherosclerosis, or Alzheimer’s disease (Moser, 2008), or anaemia (Mol & Law, 1994). For example, Annemarie Mol (2002) describes ethnographically a patient’s body and its disease moving from one hospital ward to another to see how they become different objects. 45 Teoria e Prática em Administração, volume 8, número 2 (special issue), ano 2018 Practices and Knowledges Gherardi p. 33-59 DOI: http://dx.doi.org/10.21714/2238-104X2018v8i2S-38857 Submission: Mar/05/18 –Second version: Mai/13/18 –Acceptance: Mai/15/18 She argues, in material-semiotic mode, that each practice generates its own material reality and not that there are different perspectives on a single disease. Teoria e Prática em Administração, volume 8, número 2 (special issue), ano 2018 Practices and Knowledges Gherardi p. A story about practices, a texture of practices, and their agencement For an empirical study of a practice and its connection with other interdependent practices within a texture, the definition of practice as an agencement has proved simple and useful (Gherardi, 2016). Agencement is a word which has the idea of agency in its root, and is currently used in French, as a synonym for ‘arrangement’, ‘fitting’ or ‘fixing’. It has been used as a philosophical term by Deleuze and Guattari (1987) with the sense of ‘in connection with’ and has been recently re-introduced into the social science vocabulary by Callon and colleagues (2013) to talk of economic performativity. It expresses clearly the word-play of the term agencement since it is ‘an actor’ in the sense of a sociotechnical assemblage and at the same time it has agence, agency. Similarly, when we look at a practice, we can see how the sociomaterial relations that tie bodies, artifacts, discourses, technologies, and rules together are performed within it and with other practices, and how agency is its effect. Within a practice, in its unfolding, neither humans, nor nonhumans, nor discourses have priority. If we describe the process of agencement as a process of heterogeneous engineering, we can say that all the resources necessary for practicing are the stuff of what is connected. It is difficult to enumerate the ingredients of a practice, since a resource for action becomes a resource only within an assemblage of relationships. In the language of actor-network theory, we should say that elements are performed in, by and through the relations in which they are located, and if the relations do not hold fast by themselves, they need to be performed. 46 Teoria e Prática em Administração, volume 8, número 2 (special issue), ano 2018 Practices and Knowledges Gherardi p. 33-59 DOI: http://dx.doi.org/10.21714/2238-104X2018v8i2S-38857 Submission: Mar/05/18 –Second version: Mai/13/18 –Acceptance: Mai/15/18 The concept of agencement can prove useful for a practice-based study, since in studying a practice the researcher may empirically follow and describe the process whereby humans, artifacts, rules, technologies, sensible knowledge, legitimacy and any other practice resource become connected thanks to a collective knowledgeable doing. It is not a final product (as the English translation in ‘assemblage’ suggests), but it calls for a process approach looking for temporality and becoming, agencement calls for ‘agencing’, as Cochoy (2014) prefers to name it. A story about practices, a texture of practices, and their agencement 33-59 DOI: http://dx.doi.org/10.21714/2238-104X2018v8i2S-38857 Submission: Mar/05/18 –Second version: Mai/13/18 –Acceptance: Mai/15/18 Teoria e Prática em Administração, volume 8, número 2 (special issue), ano 2018 Practices and Knowledges Gherardi p. 33-59 DOI: http://dx.doi.org/10.21714/2238-104X2018v8i2S-38857 Submission: Mar/05/18 –Second version: Mai/13/18 –Acceptance: Mai/15/18 more or less safely within the agencement that keeps a practice together. Learning safety means knowing how to behave as a competent member in a culture of safety practices. It means that within a practice learning is not distinct from knowing in practice. Working practices are specific to different occupations and professions, that are interdependent within the single construction site and that enter into conflicts and negotiations over the meaning and the multiple enactments of how to accomplish safe working and organizing practices. Moving along the connections among the working practices of one community and another interdepent one, we can explore how the culture of certain occupational practices are enacted when different communities of practice explain why accidents happen (Gherardi, Nicolini & Odella, 1998b). Similarly, when a firm recovers after a major accident (Gherardi, 2004), we can track how (and if) previous practices are challenged, changed, or reinforced, and who and what enter into a new agencement. Moving along the rhizomatic lines of connection within the texture of safety practices we can inquire on how an institutional field deals with safety regulations (Gherardi & Nicolini, 2002), and how the regulative practices (at national and international level) go back to the single construction site and to the individual novice learning the use of a risky tool. The end of this story about safety practices is that what counts as ‘safety’ within an historical context is the provisional and contested enactment of a texture of practices that acquires agency in their being connected and disconnected. In other words, when the researchers aim to inquire on the actual processes of organizing, they may trace how a flow of situated activities are connected into streams of action and the researchers may move along the threads of a texture of practices, from activities within a practice to practices connected to other practices. In fact, practices have no boundaries except those that the heuristic operation of a researcher establishes. Practice does not ‘exist’ in nature, researchers do not ‘find’ it, rather practice is always conceptually constructed. A story about practices, a texture of practices, and their agencement What we call ‘practice’ is a heuristic move that de-territorializes and re-territorializes the unfolding of a flow of practicing. When we put boundaries around ‘a’ practice, trying to see when (and where) it begins and where (when) it ends, we are doing a heuristic operation (an agential cut in Barad’s terms), since it is within practicing that connections are established and dissolved without a pre-defined order; and it is the process of agencement (of connecting with) that creates it. These connections are those of the rhizome, which has no beginning or end but is always in between, in motion. Therefore, the passage from the noun ‘practice’ to the verb ‘practicing’ implies not only a move towards a process view, but especially a passage to temporality and to the situated activity of agencement as the activity of establishing connections. But what is connected within a practice and how are practices connected? A story about the empirical study of safety in construction industry may illustrate how ‘safety’ is the sociomaterial object emergent from the agencement of a texture of practices (Gherardi & Nicolini, 2000; Gherardi, 2006). Like the salmon in the previous story about multiplicity, also safety knowledge and organizational safety learning are enacted in situated practices across a multiplicity of sites, and what we value as ‘safety’ within a society may be conceived as a collective competence developed alongside emergent practices within and across the boundaries of one organization, one industry, one organizational and institutional field. For describing empirically the agency that connects all the practices of ‘doing safety’ we can trace the sociomaterial enactments of knowing and learning at various point in time. For example, we can track a novice who enters a community of practices (Gherardi, Nicolini & Odella, 1998a) and how s/he learns what is safe working and organizing and what is risky. At the same time, we trace how the community teaches through words, discourses, and silences, and we follow how this knowledge is embodied, embedded in sociomaterial relations and is contingent and provisional, so that a practice is always practiced for another first time (Garfinkel, 1967). In a construction site, a specific working activity is performed 47 Teoria e Prática em Administração, volume 8, número 2 (special issue), ano 2018 Practices and Knowledges Gherardi p. A story about practices, a texture of practices, and their agencement Another story about agencement may be told taking the opportunity of a huge debate that the rumor about the introduction of a wireless bracelet (a newly-announced practice) at Amazon in Italy, where two new centers have been opened, for a total of 1,600 jobs. The bracelet, just patented in the US, has been designed to speed up the search for products stored in warehouses by employees, monitoring where they put their hands, vibrating to guide them in the right direction and actually controlling all their movements. These details are to be transmitted on the minicomputer to the employee's wrist to take the goods, put it in a box and switch to the next task. The news of the patent was taken from all newspapers, 48 and almost all the exponents of the government and the political parties reacted on social media, accusing the company of reducing their employees as new slaves of the capitalist system. The Speaker of the House Laura Boldrini, declared that "Working is not a crime," and called the proposal "degrading and offensive". Even the Prime Minister Paolo Gentiloni had a word in, saying that the challenge facing Italians was "quality jobs, not jobs with wristbands", and that Italy (differently for other countries) has labour laws that apply to every company. What did a wireless bracelet - a new tool that was not yet put into use - produce? It made evident the sociomaterial relations in a texture of practices, producing an agencement in which the reputation of a company (that turns employees, paid little, into human robots who work near real robots, carrying out repetitive packaging tasks as quickly as possible, with the goal of achieving the ambitious delivery targets set by Amazon) was materialized. Moreover, the wireless bracelet produced the visibility of retail services, that work with thin margins of profit, that minimize the cost of labor, that use work contracts that advise workers of their schedule time with little notice, that use algorithms to organize staffing according to the optimization of presence. We can see the bracelet’s performativity not only in its capacity to make visible the connections between working and organizing practices, but also in its capacity to bring to light a question of moral (and not only economic) value. The bracelet made audible/readable/tangible/knowable a societal issue: what is the value of work? A story about practices, a texture of practices, and their agencement What is the meaning of work in a life and in a society? The political and ethical materialization of a practice within a society (and differently from other societies) is made sayable. Within the study of work and organization one reason for a practice approach that leaves behind the assumption that actions spring from the intentionality and values of human beings, is that the focus on ‘a’ practice situated in any point within a texture of practices, enables the researchers to move along many lines of connections in any direction, following the connections in action. A texture of practices may be empirically explored and described either following the connections that from one practice move along radial lines, like in the web of a spider (as I prefer to say) or along the two moves of a zooming-in and zooming out that Nicolini (2010) suggests. Conclusion 49 49 Teoria e Prática em Administração, volume 8, número 2 (special issue), ano 2018 Practices and Knowledges Gherardi p. 33-59 DOI: http://dx.doi.org/10.21714/2238-104X2018v8i2S-38857 Submission: Mar/05/18 –Second version: Mai/13/18 –Acceptance: Mai/15/18 Teoria e Prática em Administração, volume 8, número 2 (special issue), ano 2018 Practices and Knowledges Gherardi p. 33-59 DOI: http://dx.doi.org/10.21714/2238-104X2018v8i2S-38857 Submission: Mar/05/18 –Second version: Mai/13/18 –Acceptance: Mai/15/18 The empirical study of practices may be approached from different angles and with different knowledge interests, and consequently practice as a knowledge object is multiple and fluid. Knowledge objects are characterized by their question-generating character, and they can never be fully attained since – as Knorr Cetina (2001: 190) writes - ‘epistemic objects are always in the process of being materially defined, they continually acquire new properties and change the ones they have’. In this essay, I have assumed the relation between practice and knowledge as my compass for arguing that knowing is a generative social practice. Within the literature on organizational learning and knowing, the knowledge object ‘practice’ has been modelled following the desire to avoid the cognitive formulation of knowledge as residing in people heads and, at the same time, to avoid the image of a commodified knowledge valued in economic terms. Therefore, practice can be considered as a figure of the discourse on knowing, learning and organizing, where learning is understood as competent sociomaterial participation in situated practices, knowing is embodied and entangled with doing, and practice takes the form of a mode of ordering heterogeneous materials, that achieve agency through their performative connections. Therefore, in saying that practices are situated modes of ordering and ‘agencing’ it is said that they are always emerging from practicing and, in their recursiveness, they become stabilized, institutionalized, and become objects of attachments. The question-generating power of formulating practice as the locus of learning and knowing is related to the inquiring into how knowing-in-practice is accomplished and how the heterogeneous elements are stitched together. One impetus for looking into the practice realm comes from conceiving practice as the container of elusive knowledges, embodied ways of knowing, pre-verbal and pre-individual forces that operate beyond the speaking subject and its presumed centrality. These kinds of questions are grounded in an Actor-network sensibility that harbors an onto-epistemology informed by relationality and performativity. Conclusion Thus, the object of knowledge ‘practice’ is displaced from a humanistic sociology in which actors are the main source of action (in the view of ‘actors and their practices’) to a post-humanistic formulation of practice theory as sociomateriality in which humans, materials, more-than-humans, discourses and knowledges are entangled within a practice, and practices are woven in a texture of practices. What keeps a practice or a texture of practices (temporally) connected 50 Teoria e Prática em Administração, volume 8, número 2 (special issue), ano 2018 Practices and Knowledges Gherardi p. 33-59 DOI: http://dx.doi.org/10.21714/2238-104X2018v8i2S-38857 Submission: Mar/05/18 –Second version: Mai/13/18 –Acceptance: Mai/15/18 or disconnected within an agencement? The glue may come from the power of association, communication and affect, when matter matters (in Barad’s words). Teoria e Prática em Administração, volume 8, número 2 (special issue), ano 2018 Practices and Knowledges Gherardi p. 33-59 DOI: http://dx.doi.org/10.21714/2238-104X2018v8i2S-38857 Submission: Mar/05/18 –Second version: Mai/13/18 –Acceptance: Mai/15/18 Teoria e Prática em Administração, volume 8, número 2 (special issue), ano 2018 Practices and Knowledges Gherardi p. 33-59 DOI: http://dx.doi.org/10.21714/2238-104X2018v8i2S-38857 Submission: Mar/05/18 –Second version: Mai/13/18 –Acceptance: Mai/15/18 or disconnected within an agencement? The glue may come from the power of association, communication and affect, when matter matters (in Barad’s words). The knowledge object ‘practice’ is constructed differently within different disciplinary traditions and different knowledge interests and there is no point in engaging in a war of epistemologies when we can learn to switch lenses and appreciate the dynamics of difference and differing. Practice is multifaceted: a theoretical starting point and an empirical focus for organizational inquiry. Practice as epistemology contributes to the empirical study of how we come to know what we know and how in knowing the object is always indeterminate and changing. Acknowledgements: I am entirely responsible for this reflection on the place of knowledge in practice theory. However, it would not have been possible without the kind invitation of Marcelo Bispo to reflect on my own contribution to practice-based studies and to disentangle what he calls my ‘hybrid’ approach. We discussed it at length, in Trento when he was visiting RUCOLA, in several international conferences when we met, by mail and by skype conversations, and still I do not see it. Nevertheless, I like the idea of hybridizing and creating new hybrid thoughts for the future. I thank Marcelo for his generosity as a scholar and a friend. 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L., Islam, G., & Mangia, G. (2016). Modes of knowing: Video research and the problem of elusive knowledges. Organizational Research Methods, 1094428116657394. Turner, B., (2009). The New Blackwell Companion to Social Theory, (pp. 451-474). Oxford: Blackwell Publishing. Toraldo, M. L., Islam, G., & Mangia, G. Teoria e Prática em Administração, volume 8, número 2 (special issue), ano 2018 Practices and Knowledges Gherardi p. 33-59 DOI: http://dx.doi.org/10.21714/2238-104X2018v8i2S-38857 Submission: Mar/05/18 –Second version: Mai/13/18 –Acceptance: Mai/15/18 Rorty, R. (1980). Philosophy and the mirror of nature. Oxford: Blackwell. (2016). Modes of knowing: Video research and the problem of elusive knowledges. Organizational Research Methods, 1094428116657394. Turner, B., (2009). The New Blackwell Companion to Social Theory, (pp. 451-474). Oxford: Blackwell Publishing. Weinberg, D. (2008). The Philosophical Foundations of Constructionist Research. In J. K. Holstein & J. Gubrium (Eds.), The handbook of constructionist research. New York: Guilford Press. Wittgenstein, L. (1968). Philosophical investigations. Oxford: Blackwell. Wolfe, C. (2010). What is posthumanism? Minneapolis: University of Minnesota Press. Wolfe, C. (2010). What is posthumanism? Minneapolis: University of Minnesota Press. Yakhlef, A. (2010). The corporeality of practice-based learning. Organization Studies, 31(4), 409-430. Yakhlef, A. (2010). The corporeality of practice-based learning. Organization Studies, 31(4), 409-430. 58 58 Teoria e Prática em Administração, volume 8, número 2 (special issue), ano 2018 Practices and Knowledges Gh di e Prática em Administração, volume 8, número 2 (special issue), ano 2018 Practices and Knowledges p. 33 59 DOI: http://dx.doi.org/10.21714/2238-104X2018v8i2S-38857 Submission: Mar/05/18 –Second version: Mai/13/18 –Acceptance: Mai/15/18 i In this section I refer the reader mainly to Turner (2009), The New Blackwell Companion to Social Theory, since the debate on the changed nature of knowledge in contemporary philosophies is quite extensive and cannot be presented extensively here. 59 59
https://openalex.org/W2904179168	http://periodicos.uem.br/ojs/index.php/Dialogos/article/download/40236/pdf	Portuguese	null	O contar sobre a cidade: a biografia e as memórias que distinguem o lugar	Diálogos	2,018	cc-by	9,904	O contar sobre a cidade: a biografia e as memórias que distinguem o lugar http://dx.doi.org/10.4025.dialogos.v22i2.40236 Marília Garcia Boldorini Roberta Barros Meira Universidade da Região de Joinville, UNIVILLE, Brasil. E-mail: rbmeira@gmail.com Resumo: Reflete-se sobre o papel das biografias na escrita das memórias da cidade com um paralelo entre Primavera em pleno outono: a jovem Olívia faz 80 anos!, de Wilson Gelbcke (2004), e Eu, Wittich Freitag, de Raquel S. Thiago (2000), para averiguar como memória, narrativas e paisagem cultural de Joinville aparecem nos discursos. Questiona- se a construção do contar biográfico, ressaltando as interferências dessas narrativas nas discussões de memória e patrimônio cultural, reforçando/negando os discursos oficiais da cidade. Como discurso oficial, tomamos como narrativa História de Joinville: crônicas da Colônia Dona Francisca, de Carlos Ficker (1965), importante influenciador do discurso que envolve Joinville. Resumo: Reflete-se sobre o papel das biografias na escrita das memórias da cidade com um paralelo entre Primavera em pleno outono: a jovem Olívia faz 80 anos!, de Wilson Gelbcke (2004), e Eu, Wittich Freitag, de Raquel S. Thiago (2000), para averiguar como memória, narrativas e paisagem cultural de Joinville aparecem nos discursos. Questiona- se a construção do contar biográfico, ressaltando as interferências dessas narrativas nas discussões de memória e patrimônio cultural, reforçando/negando os discursos oficiais da cidade. Como discurso oficial, tomamos como narrativa História de Joinville: crônicas da Colônia Dona Francisca, de Carlos Ficker (1965), importante influenciador do discurso que envolve Joinville. Telling about the city: biography and the memories that distinguish the place Abstract: We reflect about the role of biographies in the writing of city memories through a parallel between Primavera em pleno outono: a jovem Olívia faz 80 anos!, by Wilson Gelbcke (2004), and Eu, Wittich Freitag, by Raquel S. Thiago (2000), to examine how Joinville’s memory, narratives and cultural landscape appear in the speeches. We interrogate the construction of the biographic telling, emphasizing the interferences of these narratives in the discussions about memory and cultural heritage, reinforcing/denying the city official speeches. As official speech, we selected the narrative História de Joinville: crônicas da Colônia Dona Francisca, by Carlos Ficker (1965), important influencer of the speech that involves Joinville. El contar sobre la ciudad: biografía y las memorias que distinguen el lugar Resumem: Reflexionamos sobre el papel de las biografías en la escrita de las memorias de la ciudad con un paralelo entre Primavera em pleno outono: a jovem Olívia faz 80 anos!, de Wilson Gelbcke (2004), y Eu, Wittich Freitag, de Raquel S. Marília Garcia Boldorini Marília Garcia Boldorini Universidade da Região de Joinville, UNIVILLE, Brasil. E-mail: mariliaboldorini@gmail.com (Online) ISSN 1415-9945 (Impresso) (Online) ISSN 1415-9945 (Impresso) Introdução o local dessa circulação entre homem e mundo, dessa mistura” (BESSE, 2013, p. 34). Por conseguinte, a paisagem faz parte do nosso estar no mundo e consiste num elemento fundador das nossas identidades pessoais e coletivas. O homem, como o ser social que é, não vive sozinho. Sua vida desenrola-se considerando as relações que trava com aqueles que estão a seu redor. Essas relações, entretanto, não se restringem à interação homem-homem; elas acontecem no que se refere a tudo, seja material, seja imaterial, já que o homem também faz parte da natureza. Portanto, ele molda o espaço que habita, o qual, por sua vez, também interfere em sua constituição. Ambos recebem e sofrem influência igualmente de um e de outro, passando por transformações para a melhor adaptação mútua. Todavia, é importante ressaltar que essa paisagem que o homem molda e por ela é moldado é compartilhada em diferentes níveis. Esse compartilhamento ocorre na maioria das vezes sem problemas quando nos referimos a residências, por exemplo, mas a situação muda de figura quando esse espaço ultrapassa os muros e portões de casas e prédios e alcança espaços maiores, como a cidade e o país. As complicações acontecem nesses casos em razão da heterogeneidade dos grupos sociais que residem nesses espaços. Todos querem que os locais sejam configurados à sua maneira. Logo, esses lugares tornam-se um campo de tensões e embates com o propósito de privilegiar um único ponto de vista, que resulta na formatação almejada. Por essa característica, o ser humano acaba por criar vínculos com o território em que reside, com a intenção de instituir um lugar para pertencer e que lhe pertença. Nas palavras de Besse (2013, p. 38), “habitar é, por um lado, marcar (e organizar) um espaço e, por outro lado, ser marcado por ele. O lugar marca-nos e nós marcamos o lugar”. Sendo assim, a diversidade está presente nos variados regimes de espacialidade, e a paisagem também é atravessada por ela: “Cada pessoa, de acordo com a sua trajetória, consciência e experiência, vê as paisagens de forma diferente e única, sendo que nela se insere de determinada forma. Cada um constrói seus conceitos que vão refletir em suas ações e seus olhares” (VERDRUM; VIEIRA; PIMENTEL, 2016, p. Roberta Barros Meira Thiago (2000), para averiguar cómo memoria, narrativas e paisaje cultural de Joinville aparecen en los discursos. Cuestionase la construcción del contar biográfico, resaltando las interferencias de esas narrativas en las discusiones de memoria y patrimonio cultural, reforzando/negando los discursos oficiales de la ciudad. Como discurso oficial, tomamos como narrativa História de Joinville: crônicas da Colônia Dona Francisca, de Carlos Ficker (1965), importante influencia del discurso que envuelve Joinville 141 MG Boldorini; RB Meira. Diálogos, v.22, n.2, (2018) 140-159 Introdução A esse conjunto de relações existenciais mantidas pelos seres humanos com o mundo que os rodeia, relações essas experimentadas de diferentes modos, tanto em função da matriz cultural individual e coletiva quanto das temporalidades que exercem influência nos homens, se dá o nome de paisagem: “O homem está no mundo e o mundo está no homem: a paisagem é o nome e 142 MG Boldorini; RB Meira. Diálogos, v.22, n.2, (2018) 140-159 memória e identidade. Ademais, seus textos são dependentes de variáveis de tempo e espaço que interferem diretamente na vida exposta em suas páginas. Essa peculiaridade faz com que funcionem como uma base com características maleáveis para delineamentos do grupo, dos lugares, dos costumes, dos hábitos, entre outros, além de ser uma narrativa que se reconstrói pelos séculos, adaptando-se às visões demandadas pelas narrativas patrimoniais. 1 O autor do material é membro da Academia Joinvilense de Letras, nasceu em São Paulo (SP) em 1933 e mora em Joinville desde 1947. Após a aposentadoria, dedica-se exclusivamente à escrita, desde 1997, e é autor de obras juvenis, romances, poemas e biografias, além de também fazer as ilustrações dos próprios livros. p g ç p p 2 A autora da obra, assim como de outros livros, artigos científicos e matérias de jornal, é natural de Joinville e mestre em História pela Universidade Federal de Santa Catarina (UFSC). Suas pesquisas envolvem história, história de Santa Catarina e história regional, sobretudo nos seguintes temas: história, identidade, memória, colonização e imigração. É membro da Academia Joinvilense de Letras. 1 O autor do material é membro da Academia Joinvilense de Letras, nasceu em São Paulo (SP) em 1933 e mora em Joinville desde 1947. Após a aposentadoria, dedica-se exclusivamente à escrita, desde 1997, e é autor de obras juvenis, romances, poemas e biografias, além de também fazer as ilustrações dos próprios livros. 2 A autora da obra, assim como de outros livros, artigos científicos e matérias de jornal, é natural de Joinville e mestre em História pela Universidade Federal de Santa Catarina (UFSC). Suas pesquisas envolvem história, história de Santa Catarina e história regional, sobretudo nos seguintes temas: história, identidade, memória, colonização e imigração. É membro da Academia Joinvilense de Letras. 133). Entende-se então que a leitura da paisagem é uma construção contínua social e, ao mesmo tempo, particular. Nela se sobrepõem identidades, conhecimentos, memórias e sentimentos individuais, associados aos processos culturais e à carga simbólica que exercem interferência nos organismos. “As ações de perceber e representar a paisagem passam por valores estéticos, plásticos e emocionais em relação ao meio. E interpretar essas imagens e representações pressupõe a compreensão de determinada matriz cultural” (VERDRUM; VIEIRA; PIMENTEL, 2016, p. 133). Posto isso, a ideia deste artigo é traçar um paralelo comparativo entre textos biográficos com o intuito de averiguar em ambos os discursos principalmente a questão da paisagem e como esta aparece nas narrativas. Para tanto, selecionaram-se duas obras que bem representam discussões que envolvem memória, identidade e protagonismo versus coadjuvação. São elas: Primavera em pleno outono: a jovem Olívia faz 80 anos!, de Wilson Gelbcke1 (2004), e Eu, Wittich Freitag, de Raquel S. Thiago2 (2000). Nesse sentido, tem-se como pressuposto no presente estudo perceber de que maneira a memória, as narrativas e a paisagem cultural da cidade de Joinville (SC) são trazidas nos discursos literários de seus diferentes atores. Os discursos em foco nesse caso são textos biográficos, haja vista a biografia trabalhar basicamente com fontes documentais, bibliográficas, iconográficas e mnemônicas, servindo por isso como objeto de estudo tanto da literatura quanto da História. Pensou-se na investigação de biografias por elas serem uma fonte de pesquisa que engloba A primeira publicação mencionada, Primavera em pleno outono, conta a história de vida de Olívia Maia Mazzolli, professora e ex- funcionária da Receita Federal. Olívia é natural de Joinville, nasceu na década de 1920 e, 143 MG Boldorini; RB Meira. Diálogos, v.22, n.2, (2018) 140-159 lançamento, em 1965, funcionando como suporte para as narrativas que rondam a cidade. juntamente com o seu marido, atuou como voluntária em trabalhos sociais no auxílio de famílias em necessidade, por meio do Centro de Estudos e Orientação da Família (Cenef). 3 É válido ressaltar que o livro é o vencedor de um concurso promovido pela Fundição Tupy S.A. sobre a melhor história da sua comunidade, ou seja, a comunidade joinvilense. Ficker fez pesquisas tanto no Brasil quanto na Europa, considerando que Joinville enquanto colônia era comandada por uma empresa cuja sede ficava em terras europeias, e seu livro, de acordo com a introdução nele presente, constitui a história definitiva de Joinville. , ç p , 4 Atualmente a cidade é a maior de Santa Catarina, ficando à frente até mesmo da capital catarinense, Florianópolis, e se destaca no cenário nacional por conta de seu caráter industrial. Biografias e o discurso oficial corrente: memórias em disputa Já a segunda biografia analisada é Eu, Wittich Freitag, que narra episódios da vida do empresário e político Wittich Freitag. Nascido em Blumenau (SC), Wittich foi um importante empreendedor para Joinville, pois na cidade construiu e consolidou a primeira fábrica de refrigeradores da Região Sul brasileira, a Consul, marco para a industrialização joinvilense, e criou posteriormente a Empresa Brasileira de Compressores S.A. (Embraco). Atuou como vereador, deputado estadual e por duas vezes foi prefeito, exercendo seu trabalho sempre de Joinville. Guedes (2007) esclarece que a história de Joinville se iniciou ainda em 1846. As terras onde hoje está a cidade foram concedidas ao príncipe francês François Ferdinand Philipe como dote por ocasião de seu casamento com a princesa Francisca Carolina, irmã de D. Pedro II. Resultado de um empreendimento comandado pelo príncipe e executado pela Companhia Colonizadora de Hamburgo, chamada de Colônia Dona Francisca, em 9 de março de 1851 aportaram nas proximidades onde atualmente é o Mercado Público Municipal levas de um grupo de imigrantes alemães, suíços e noruegueses. Esse feito ficou conhecido como o marco inicial da fundação de Joinville enquanto município4. No intuito de averiguar se as biografias reforçam o discurso oficial corrente joinvilense, ou se de alguma maneira se posicionam de maneira contrária, basear-nos- emos no livro de Carlos Ficker História de Joinville: subsídios para a crônica da Colônia Dona Francisca3 (1965), um grande influenciador do discurso midiático que envolve Joinville, bem como suas políticas públicas. Sua escolha como discurso oficial deu-se porque o material é amplamente utilizado por pesquisadores locais desde seu No entanto, conforme as pesquisas de Ficker (1965), a data estabelecida como a oficial para a fundação de Joinville é apenas simbólica: “A Colônia Dona Francisca, já assim denominada pelos seus idealizadores em Hamburgo, teve o marco inicial de sua vida, a 22 de maio de 1850, quando desembarcados os 144 MG Boldorini; RB Meira. Diálogos, v.22, n.2, (2018) 140-159 quando exata, legítima, pura, baseada cientificamente em documentos devidamente autenticados” (FICKER, 1965, p. 14). seus primeiros moradores, às margens do Rio Cachoeira” (FICKER, 1965, p. 57-58). Segundo ele, uma sucessão de erros em documentos acabou tornando-se uma verdade: Tal postura colocaria de lado as mudanças que ocorreram no campo da história sobretudo após a circulação das ideias defendidas por movimentos como a chamada Escola dos Annales. Cardoso e Brignoli (1979, p. A empreitada, assim, não era fácil, se se quisesse escrever uma história real, exata, precisa, minuciosa, da ex-Colônia Dona Francisca. [...] Não é obra de sociólogo. É trabalho de cronista, de historiográfico, que relata os fatos, tão minuciosamente quanto possível e os situa no tempo e no espaço (CABRAL, 1965, p. 10-11). Biografias e o discurso oficial corrente: memórias em disputa 25) chamam a atenção para o fato, aclarando que não se tratava mais de construir a história “saltando de fato singular a fato singular”. Os objetos de análise deixaram de ser personagem de uma história marcadamente política ou militar que se centrava nas ações dos heróis ou dos gênios. A reconstituição da vida cotidiana e de uma história que não se apoiava só em fontes oficiais ensejaria os futuros novos estudos. Ou como bem diz Febvre (1989, p. 24): Sendo a obra [Colonização do Estado de Santa Catharina, de Jacinto de Matos] citada por muitos historiadores, o erro (e não somente este!) repete-se em todas as divulgações sobre a história de Joinville, com uma persistência incrível, tornando-se, finalmente, a mais pura verdade histórica (FICKER, 1965, p. 89). Sendo a obra [Colonização do Estado de Santa Catharina, de Jacinto de Matos] citada por muitos historiadores, o erro (e não somente este!) repete-se em todas as divulgações sobre a história de Joinville, com uma persistência incrível, tornando-se, finalmente, a mais pura verdade histórica (FICKER, 1965, p. 89). Por esse trecho, é possível perceber a força e o poder que as informações têm quando registradas, principalmente, em documentos oficiais e livros que se autointitulam de verdade histórica. Como o maior grupo de imigrantes aportou em Joinville em 1851, o grupo anterior acabou esquecido pela historiografia. Todavia, Ficker também recebe grande parcela do poder de contar a história correta de Joinville quando Oswaldo Cabral, do Instituto Histórico Brasileiro, passa na introdução da obra a ideia de que o historiador tem a função de escrever a historiografia oficial, pois só ele trabalha com os fatos tal e qual aconteceram: 24): Os textos, sem dúvida: mas todos os textos. E não só os documentos de arquivos em cujo favor se cria um privilégio [...]. Mas, também um poema, um quadro, um drama: documentos para nós, testemunha de uma história viva e humana, saturada de pensamentos e de ação em potência. Desde o prefácio, Ficker perpetua o conceito da colonização como sinônimo ao progresso: “Há um século, Joinville nascia como colônia de imigrantes europeus em terras de Santa Catarina” (FICKER, 1965, p. 13), correlacionando aos imigrantes adjetivos como “força de vontade”, “luta contínua” e “empenho” (FICKER, 1965, p. 13). A fala é Ficker compartilha dessa perspectiva, ao ressaltar: “Não aceitamos história, a não ser 145 MG Boldorini; RB Meira. corroborada por Cabral (1965, p. 11): O Autor, sem diminuir o valor do imigrante, que tem a coragem de enfrentar o desconhecido para instalar uma nova existência, não subestima o extraordinário concurso dos que o ajudaram na epopeia, já governo, já empresários e muito também o dos elementos nacionais envolventes, que abriram os braços para acolher os imigrantes, compreendendo- lhes as dificuldades com simpatia, reconhecendo-lhes a fibra lutadora e tudo fazendo para facilitar esta desejada aculturação de que é Joinville um dos mais estupendos e edificantes exemplos de que nos podemos orgulhar. O Autor, sem diminuir o valor do imigrante, que tem a coragem de enfrentar o desconhecido para instalar uma nova existência, não subestima o extraordinário concurso dos que o ajudaram na epopeia, já governo, já empresários e muito também o dos elementos nacionais envolventes, que abriram os braços para acolher os imigrantes, compreendendo- lhes as dificuldades com simpatia, reconhecendo-lhes a fibra lutadora e tudo fazendo para facilitar esta desejada aculturação de que é Joinville um dos mais estupendos e edificantes exemplos de que nos podemos orgulhar. Foi baseada nessa imigração que se estruturou a maior parte da historiografia local. Dessa forma, as origens de Joinville foram afirmadas sobre o imigrante europeu, procurando-se minimizar a participação daqueles que os antecederam, fossem eles indígenas, brasileiros ou negros (GUEDES, 2007), embora existam registros de sua presença no local antes mesmo da chegada dos novos colonizadores: “Não é exato, pois, afirmar-se que em 1851 as grandes zonas destinadas à colonização europeia, seriam ínvio e desconhecido sertão. Eram, ao contrário, bastante habitadas as cercanias” (FICKER, 1965, p. 32). Ficker (1965, p. 32) cita como moradores à época senhores de fazenda e escravos, além dos índios, aqui já associando os últimos à barbárie e à selvageria. A imagem que os colonizadores tinham dos grupos indígenas era a de que eles, por não serem civilizados conforme os moldes europeus habituais, não teriam cultura e, por isso, atrapalhariam o progresso que a colonização traria à região: “A reabertura do picadão e a sua reconstrução [da estrada que ligaria a colônia à Curitiba] deve-se ao principal fato de defesa contra os selvagens” (FICKER, 1965, p. 139). Sendo a história joinvilense contada pelo olhar da colonização principalmente germânica, não é estranho o fato de se terem escolhido como protagonistas de biografias que se passam na cidade duas personagens com tal característica. Biografias e o discurso oficial corrente: memórias em disputa Diálogos, v.22, n.2, (2018) 140-159 instalado naquele espaço, os índios, considerados bárbaros e não civilizados (RONCAGLIO, 2009), não houve sentimento de pertencimento ao território nem identificação com os habitantes nativos por todo o Brasil, e em Joinville aparentemente a situação não foi diferente. Conta Ficker (1965, p. 283), por exemplo: “O aborígene foi sempre o terror dos colonos. [...] Em 1836, uma família inteira foi aniquilada pelos bugres, no local onde, em 1852, o norueguês Peter Lyng instalou a Olaria, hoje esquina Rua do Príncipe e Rua São Pedro”. corroborada por Cabral (1965, p. 11): Tanto Olívia Maia Mazzolli quanto Wittich Freitag compartilhavam a descendência alemã, viveram em Joinville a maior parte de suas vidas – Freitag não era natural de Joinville, mas chegou à cidade por Por causa das relações predatórias da elite portuguesa no tocante à natureza e aos recursos naturais e da rejeição por parte dos europeus sofrida pelo grupo social que estava 146 MG Boldorini; RB Meira. Diálogos, v.22, n.2, (2018) 140-159 volta dos 20 anos e nunca mais a deixou – e na cidade constituíram carreira e família, obtendo sucesso em todos os aspectos de sua trajetória, ao menos de acordo com as suas biografias. Vê-se nas duas obras a questão do imigrante que deu certo, quase que uma panfletagem do discurso que ronda a imagem de Joinville aos olhos dos outros. colonos não apresentavam as características necessárias para desbravar nem para abrir clarões na mata virgem, derrubando árvores e preparando o chão para as primeiras plantações, ou seja, homens fortes e trabalhadores rurais experimentados, como era o caso dos brasileiros. Na obra o brasileiro é descrito como aquele trabalhador forte, experiente na agricultura e necessário para o desbravamento da mata virgem, ligando-o ao trabalho braçal puro e simples: “Os trabalhadores brasileiros, que prestaram serviços insuperáveis no desmatamento das florestas, roçando e queimando os terrenos dos colonos inexperientes” (FICKER, 1965, p. 98). Esse discurso da mão de obra primária prestada pelo brasileiro não difere do discurso que ronda a educação nacional, vide as mais recentes modificações sofridas pelo setor. Enquanto isso, sobre os colonos, Ficker associa-os a todo o momento ao progresso da colônia: Essa imagem é reforçada por Ficker (1965) em sua obra. Embora o escritor admita que havia habitantes em Joinville antes do projeto de colonização da cidade, mencionando sobretudo os escravos, a sua obra encerra-se com os seguintes dizeres: “Termina, com a exposição das razões mais evidentes do êxito da colonização e industrialização, a história de Joinville e a crônica da Colônia Dona Francisca” (FICKER, 1965, p. 439). Observa-se, portanto, a supervalorização do imigrante germânico, em detrimento das demais populações que compunham a região à época e que também colaboraram na criação e no desenvolvimento de Joinville, ou seja, uma campanha de colonização vitoriosa, apesar de Cabral salientar na introdução da obra a “absoluta isenção” (CABRAL, 1965, p. 11) com que Ficker se refere ao programa de imigração. Os 61 noruegueses [...] representaram [...] fator importante no desenvolvimento da colônia, por serem principalmente operários e artífices como carpinteiros, pedreiros e ferreiros. [...] Destacava-se a residência de nove noruegueses, que construíram a sua casa assobradada, com um acabamento profissional e em regime de coletividade, dividindo despesas e lucros em partes iguais (FICKER, 1965, p. 80). corroborada por Cabral (1965, p. 11): Os 61 noruegueses [...] representaram [...] fator importante no desenvolvimento da colônia, por serem principalmente operários e artífices como carpinteiros, pedreiros e ferreiros. [...] Destacava-se a residência de nove noruegueses, que construíram a sua casa assobradada, com um acabamento profissional e em regime de coletividade, dividindo despesas e lucros em partes iguais (FICKER, 1965, p. 80). Os 61 noruegueses [...] representaram [...] fator importante no desenvolvimento da colônia, por serem principalmente operários e artífices como carpinteiros, pedreiros e ferreiros. [...] Destacava-se a residência de nove noruegueses, que construíram a sua casa assobradada, com um acabamento profissional e em regime de coletividade, dividindo despesas e lucros em partes iguais (FICKER, 1965, p. 80). Em um ponto de sua narrativa, porém, Ficker (1965, p. 61) afirma que para a construção da colônia “empreitaram-se brasileiros, moradores da redondeza, que ofereceram seus serviços”, pois as famílias de A ideia, explícita nas três narrativas em foco neste artigo, de valorizar o imigrante olvidando-se dos indivíduos que na região já 147 MG Boldorini; RB Meira. Diálogos, v.22, n.2, (2018) 140-159 viviam antes da colonização, ou diminuindo- os, demonstra a disputa pela ocupação do território. Ao sobrepor uma representação a outras, verificamos que os interesses de certos grupos acabam prevalecendo, fazendo com que o mundo social seja construído de maneira parcial, e essa construção social perpassa também pela paisagem. Afinal de contas, a paisagem afirma o papel central das experiências sensoriais na fabricação de identidades. que neutralize os conflitos e mascare as contradições. Isso se alcança, então, por meio de representações que determinados grupos sociais têm do que acreditam simbolizar o todo. Entre esse todo, molda-se uma paisagem cultural que busca se harmonizar nas áreas centrais da cidade com uma história oficial formulada no passado e que procura dar as cartas no presente. Uma paisagem com sentimentos que é fruto de um contato real vindo de uma cidade que se pensa como uma ilha e não como um arquipélago. Afinal, como bem formula Ginzburg (2004, p. 113), “nenhum homem é uma ilha, nenhuma ilha é uma ilha”. Hoje em dia já se sabe que as identidades, independentemente de quais sejam, são fenômenos sociais, dinâmicos e dialéticos, são múltiplas e flexíveis no tempo e no espaço, estabelecidas em semelhanças e diferenças, mantidas e formadas não só por elementos sociais, coletivos e psíquicos, mas também por elementos simbólicos e materiais (CASTRO, 2008). corroborada por Cabral (1965, p. 11): Para Meneses (1984), toda identidade, pessoal ou coletiva, é sempre socialmente atribuída, socialmente mantida e só capaz de ser transformada também socialmente. Todos os valores, significações e papéis atribuídos necessitam de legitimidade social. Portanto, por ser resultado da construção de uma imagem forjada e supostamente instituída, é propícia a manipulações. Um mito bastante presente na obra de Ficker (1965) no que concerne à paisagem é a questão da natureza intocada. O autor abre sua obra com a imagem de que Joinville, antes da colonização, era uma floresta virgem e intacta, cujo propósito era servir de terreno para a construção de uma cidade que se edificaria por conta do progresso trazido pelos imigrantes que ali chegariam e a colonizariam. A página de abertura de seu livro, por exemplo, traz a figura de uma mata densa e a legenda: “Começou em 1843 a história da colonização desta área, vasta e fértil, coberta de florestas virgens...” (FICKER, 1965). Assim, na busca por uma identidade coletiva, para ser definidos sentimentos de unidade, continuidade e coerência, investe-se numa integração supostamente harmoniosa, Arruda (2006) defende a ideia de que a natureza, um dos componentes da paisagem cultural, consiste no pilar de sustentação da identidade nacional. Assim, trata-se de um 148 MG Boldorini; RB Meira. Diálogos, v.22, n.2, (2018) 140-159 suporte da identidade do espaço da nação. A natureza tem sido usada para a construção de singularidades e identidades regionais ou nacionais em larga escala e, desde a chegada dos europeus às Américas, ela é um dos principais temas para a produção de representações, discursos, símbolos e imagens sobre o país (ARRUDA, 2006; 2009). evidentes. Os dois tipos são testemunhas de fases de uma indissociável relação da história humana (DELPHIM, 2009). No caso específico de Joinville, a cidade iniciou-se como uma colônia de caráter basicamente agrícola: “[Em 1855] A indústria é representada por duas fábricas de cigarros, uma olaria, uma fábrica de louças de barro, 2 engenhos de arroz, um engenho de mandioca, duas moendas de milho e dois engenhos de açúcar” (FICKER, 1965, p. 149). 5 Considerada o primeiro documento escrito da história do Brasil e também o primeiro da literatura nacional, compondo a escola literária denominada de quinhentista, a Carta de Pero Vaz de Caminha consiste no registro das impressões do fidalgo português Caminha sobre a terra que mais tarde viria a ser chamada de Brasil. O texto pode ser visto integralmente na página virtual disponível em: Eleutério sempre foi um lutador. Depois de casar, deixou de ser caixeiro-viajante para trabalhar como gerente de indústria ervateira, liderada em Joinville por Abdon Batista e Procópio Gomes de Oliveira. Gerenciou o engenho durante muitos anos, enquanto a erva- mate tinha bom mercado (GELBCKE, 2004, p. 20). p g p <http://www.biblio.com.br/defaultz.asp?link=http://www.biblio.com.br/conteudo/perovazcaminha/carta.htm>. Acesso em: 15 out. 2017. Eleutério sempre foi um lutador. Depois de casar, deixou de ser caixeiro-viajante para trabalhar como gerente de indústria ervateira, liderada em Joinville por Abdon Batista e Procópio Gomes de Oliveira. Gerenciou o engenho durante muitos anos, enquanto a erva- mate tinha bom mercado (GELBCKE, 2004, p. 20). (FICKER, 1965, p. 231). iniciativa partiu de Antônio Sinke [...], para montar usina própria na Rua do Príncipe, esquina da Rua Cachoeira (hoje Princesa Isabel) [...]. Foi a erva-mate, inegavelmente, um fator econômico preponderante no desenvolvimento de Joinville. Da exportação passou-se para a construção de engenhos e, com a industrialização da erva-mate, tornou-se Joinville centro industrial e comercial, e mais importante praça do produto (FICKER, 1965, p. 310-311). A industrialização da cidade é motivo de orgulho para Ficker, assim como o é para Wittich Freitag, talvez por este ter sido responsável pela fundação de duas grandes empresas no município. Freitag conta que nos fins de semana em que a sua filha que morava em Curitiba (PR) vinha a Joinville, a família costumava ir jantar na Churrascaria Familiar e depois: “Na saída, lá pelas dez horas da noite, antes de voltarmos para casa, eu acabava levando todo mundo para a fábrica [referindo- se ao seu próprio empreendimento]. Geralmente era hora da saída dos operários. Eu me orgulhava daquela cena” (S. THIAGO, 2000, p. 65). No entanto, verifica-se ao longo do livro de Ficker (1965) que esse caráter essencialmente agrícola foi transformando-se ao longo do tempo, lenta e progressivamente, com intervalos prolongados, até a decadência da imagem agrícola para a cidade assumir a figura de um parque industrial. Conforme o autor, os trabalhos de implantação do povoado nascente resultaram na cidade, nas indústrias e na sua riqueza consequente: “O centro da Colônia Dona Francisca desenvolveu-se rapidamente com a construção de novas casas e a instalação de pequenas indústrias e ofícios diversos, por enquanto só para consumo interno [ainda em 1852]” (FICKER, 1965, p. 131-132). Logo, a paisagem pode ser empregada enquanto categoria de análise do espaço, de maneira a entender a relação que trava com os sujeitos que fazem parte dela, num processo de percepção no qual há a intersecção entre a esfera física, concreta e visual de um território e as memórias e os referenciais culturais individuais e coletivos (VERDRUM; VIEIRA; PIMENTEL, 2016). corroborada por Cabral (1965, p. 11): No caso brasileiro, por exemplo, Roncaglio (2009) relata que o país – com grande exuberância e abundância da natureza e riquezas naturais infinitas, como Pero Vaz de Caminha conta em sua famosa carta do descobrimento5, de 1500 (RONCAGLIO, 2009) – foi colonizado por povos de visão antropocêntrica e de concepção criacionista judaico-cristã, o que fez com que a natureza “se submetesse pacificamente” aos caprichos e desejos dos conquistadores e fossem implantados aqui o extrativismo predatório e a monocultura, os dois levados à última consequência. O mesmo caso ocorreu em Joinville e é descrito na obra de Ficker (1965). A caraterística agrícola da cidade também é vista na biografia de Olívia, que conta que por muitos anos um engenho de erva-mate, do qual seu pai era gerente, serviu de sustento à família: A respeito da indústria ervateira, na qual o pai de Olívia prestou seus serviços, consegue-se ver a ascensão ao longo do tempo: Herança do sistema colonial, tal exploração interferiu diretamente e interfere até hoje na paisagem cultural do território brasileiro, sobretudo pelo fato de que os limites entre paisagem natural e a paisagem resultante da ação humana se tornam cada vez mais Viera a Estrada Dona Francisca influir decisivamente na criação da indústria ervateira em Santa Catarina, instalando-se em 1877 três engenhos de erva-mate em Joinville. A 149 MG Boldorini; RB Meira. Diálogos, v.22, n.2, (2018) 140-159 (FICKER, 1965, p. 231). (FICKER, 1965, p. 231). Vê-se também que Ficker associa as três imagens básicas de seu livro – o imigrante, o progresso e a indústria –; cada elemento contribuindo para o desenvolvimento do outro: “Muitos imigrantes, chegados de zonas europeias já industrialmente desenvolvidas, trouxeram para cá as aptidões e a indispensável iniciativa de transformar, passo a passo, um território de mata virgem em uma zona das mais industrializadas do sul do Brasil” Igualmente, Santos (2004) afiança que o espaço é o resultado de uma práxis coletiva que reproduz as relações sociais, haja vista suas características e seu funcionamento, também pelo que oferece a alguns e recusa a outros e ainda pela seleção de localização feita entre as atividades e os homens. Assim, deve ser entendido como uma testemunha da 150 MG Boldorini; RB Meira. Diálogos, v.22, n.2, (2018) 140-159 história escrita por processos do passado e do presente e como um conjunto de formas representativas de relações sociais de outrora e dos dias atuais. são mencionados fatos que indicavam o progresso joinvilense em razão do seu povo trabalhador e honesto, herança talvez de seus colonizadores. Entre as várias passagens da obra de Freitag ressaltando a boa índole característica do grupo social alemão, já no prefácio se encontra que possivelmente esse foi o motivo de ele ter obtido sucesso em vida: “As suas origens ancestrais e herança cultural refletiram-se inconscientemente no modo de ser, nas atitudes e nos propósitos de vida” (BUSCHLE, 2000, p. 9). Como um dos registros dessas memórias e referenciais culturais, a literatura é uma das formas de representação tanto dessa identidade forjada quanto da paisagem cultural que deve sustentar essa paisagem. Por ser reflexo da sociedade, a paisagem cultural na literatura consiste numa representação em conformidade com a perspectiva do autor que a escreve, como afirma Schama (1996). Compartilham dessa ideia Verdrum, Vieira e Pimentel (2016, p. 138): De acordo com Santos (2004), como qualquer outra estrutura social, o espaço acaba por manter a estrutura que a sociedade em que nele está inserida reflete, seu dinamismo sendo consequência da cisão da sociedade global e igualmente sua distribuição sobre o território. Hoje, diante dos fatos e da história, compreendo muito bem que a nossa sociedade local, à época, compreendia apenas duas classes: a da elite, rica, dos coronéis, dos homens públicos e políticos, dos empresários... e a classe pobre, dos empregados e funcionários. Nossa família pertencia a esta, a classe pobre (MAIA in GELBCKE, 2004, p. 20). (FICKER, 1965, p. 231). Olívia consegue perceber esse fato na cidade em que reside: A narrativa é um sistema aberto à memória coletiva que se materializa na paisagem através do tempo, toda vez que um grupo determinado inscreve cotidianamente suas trajetórias sobre um suporte físico e material, deixando suas marcas e contribuindo para a manutenção das relações identitárias com o lugar. Hoje, diante dos fatos e da história, compreendo muito bem que a nossa sociedade local, à época, compreendia apenas duas classes: a da elite, rica, dos coronéis, dos homens públicos e políticos, dos empresários... e a classe pobre, dos empregados e funcionários. Nossa família pertencia a esta, a classe pobre (MAIA in GELBCKE, 2004, p. 20). Hoje, diante dos fatos e da história, compreendo muito bem que a nossa sociedade local, à época, compreendia apenas duas classes: a da elite, rica, dos coronéis, dos homens públicos e políticos, dos empresários... e a classe pobre, dos empregados e funcionários. Nossa família pertencia a esta, a classe pobre (MAIA in GELBCKE, 2004, p. 20). Constatam-se, dessa forma, nas duas biografias analisadas, as relações que ambas as personagens travam com o espaço em que estão inseridas, num misto de convivência e de reforço de dada soberania. São citados nos textos somente pontos centrais da cidade de Joinville, por onde circulavam os biografados: “Nos quatro anos de namoro, nos encontrávamos aos sábados, às três da tarde. Eu ia pela Rua Doutor João Colin e ela [a então futura esposa, Lilli] vinha da sua casa, na Rua Timbó” (THIAGO, 2000, p. 45-46). Também A biografada faz uma leitura particular de Joinville, entendendo o espaço como um campo de forças desiguais cujo domínio advém do poder econômico. Ela vê sua cidade diferentemente de Wittich Freitag, talvez por vir de uma classe social distinta da dele, além 151 MG Boldorini; RB Meira. Diálogos, v.22, n.2, (2018) 140-159 colonizadores, ter dado certo: “Uma sociedade que vai evoluindo numa absoluta integridade de justiça e moral, é que se fica sabendo do quanto estes costumes trazidos pela colonização alemã, têm feito em benefício das futuras épocas brasileiras” (FICKER, 1965, p. 367-368). da questão de gênero. Olívia define o espaço mais pelas diversas possibilidades econômicas concretas do que por outro motivo, mas não se abstém do fato de que os comportamentos pessoais contribuem para modelar o espaço. Essa seria a razão para a evolução espacial não ocorrer da mesma forma em todos os lugares. Dali partiu o sopro vivificador que, não obstante os contratempos e dificuldades encontradas, ou talvez exatamente porque encontrou esses obstáculos, fecundou os planos da Sociedade Colonizadora, de fundar uma cidade chamada “Joinville”, para garantir ao empreendimento colonial o mercado e o consumidor (FICKER, 1965, p. 103). Terminado o mandato e depois de tantos anos comandando os destinos da maior cidade de Santa Catarina, não digo que me sinto realizado, posto que ainda são muitas as carências que afligem nossa comunidade, mas posso dizer que voltei ao aconchego da minha família com a satisfação de haver contribuído para reduzir, nos limites das minhas possibilidades, as injustiças e distâncias sociais (S. THIAGO, 2000, p. 244). Terminado o mandato e depois de tantos anos comandando os destinos da maior cidade de Santa Catarina, não digo que me sinto realizado, posto que ainda são muitas as carências que afligem nossa comunidade, mas posso dizer que voltei ao aconchego da minha família com a satisfação de haver contribuído para reduzir, nos limites das minhas possibilidades, as injustiças e distâncias sociais (S. THIAGO, 2000, p. 244). linhas de força” (SANTOS, 2004, p. 165). tendo em vista que ele serve como um reforço das estruturas e relações sociais já existentes. Não se sabe o motivo que levou os alemães a terem tamanho destaque em relação à construção da cidade de Joinville, entretanto esse ponto é sentido por todos os lados. Ficker (1965), por exemplo, traz em sua obra um artigo do Jornal do Commercio de 1852, escrito pelo coronel Antônio João Vieira, em que mais uma vez esse pensamento é sobressalente: Quando o espaço é submetido a tal discussão, chega-se à conclusão de que a estrutura espacial é dependente direta da economia que a permeia, no entanto os fatores que compõem a sua organização não se limitam a isso. A questão política também possui papel essencial na discussão da organização espacial. Além da região do Mercado Público, outra área muito bem valorizada em Joinville fica num ponto específico da zona norte da cidade, próxima ao centro, onde se concentram as residências dos prefeitos que o município já teve, como é o caso de Freitag. Pode-se esperar com bons fundamentos que a Colônia Dona Francisca vingará e prosperará com a perseverança dos alemães, que nesta e n’outras províncias têm dado sobejas provas com a sua constância no trabalho, com o seu denodo, e com honestidade do seu proceder, do quanto valem e de quanto são apreciáveis para a colonização do nosso país (apud FICKER, 1965, p. 118-119). Sobre isso, Arantes (1994, p. 191) afirma que os habitantes da cidade se situam em determinados espaços urbanos. Nos espaços em comum no dia a dia são construídas as fronteiras e bordas simbólicas, as quais podem separar, aproximar, nivelar, hierarquizar os grupos sociais e suas mútuas relações, ordenando as categorias. Muito provavelmente essa seja a razão de as zonas de mais desenvolvimento e, por consequência, que recebem mais investimentos, serem aquelas voltadas à região do Mercado Público Municipal, considerado o berço de Joinville, onde se instalaram os primeiros imigrantes da cidade, que chegaram por aquela via. Sendo os imigrantes europeus de mais valor do que os habitantes de Joinville daquela época, a tendência é que a gestão da cidade se volte mais ao espaço que era ocupado por eles. (FICKER, 1965, p. 231). Diálogos, v.22, n.2, (2018) 140-159 linhas de força” (SANTOS, 2004, p. 165). (FICKER, 1965, p. 231). Conta ele que a introdução de “inteligência e capital” (FICKER, 1965, p. 103) mediante a segunda leva de imigrantes, formou o núcleo colonial Schroedersort, com a instalação de vendas, empórios, lojas e o estabelecimento de ofícios como seleiros, padeiros, ferreiros e tantos outros. Em razão disso, Schroedersort tornou-se o centro cultural, industrial e comercial da colônia, recebendo o nome de Joinville: Ao contrário de Olívia, Ficker (1965, p. 387) posiciona-se diferentemente quanto ao assunto, ao se referir aos pontos de lazer da cidade: “Em Joinville goza-se quanto possível e sem distinção de classes nem idades”. Wittich Freitag, conforme sua biografia, compartilha a opinião do escritor, ao falar sobre o fim do seu segundo mandato frente à prefeitura: Terminado o mandato e depois de tantos anos comandando os destinos da maior cidade de Santa Catarina, não digo que me sinto realizado, posto que ainda são muitas as carências que afligem nossa comunidade, mas posso dizer que voltei ao aconchego da minha família com a satisfação de haver contribuído para reduzir, nos limites das minhas possibilidades, as injustiças e distâncias sociais (S. THIAGO, 2000, p. 244). Terminado o mandato e depois de tantos anos comandando os destinos da maior cidade de Santa Catarina, não digo que me sinto realizado, posto que ainda são muitas as carências que afligem nossa comunidade, mas posso dizer que voltei ao aconchego da minha família com a satisfação de haver contribuído para reduzir, nos limites das minhas possibilidades, as injustiças e distâncias sociais (S. THIAGO, 2000, p. 244). Por essa perspectiva, diz Santos (2004) que o espaço não significa a mesma coisa para todos. Sendo assim, tratá-lo como se ele fosse dotado de uma representação única e comum seria uma espécie de violência contra o indivíduo que pertence a esse espaço, bem como as soluções todas fundamentadas nessa tese não fariam sentido: “Não se pode negar a tendência que tem a organização do espaço de fazer com que se reproduzam suas principais Não nos esqueçamos, contudo, da posição social que Freitag ocupava na cidade. Ficker (1965), igualmente, assume a postura de Freitag. Enfatiza, por toda a extensão da sua obra, o progresso da então Colônia Dona Francisca, num misto de orgulho por pertencer àquelas terras e de satisfação pelo projeto da Sociedade Colonizadora de Hamburgo, cujo propósito era erigir uma cidade por meio de 152 MG Boldorini; RB Meira. linhas de força” (SANTOS, 2004, p. 165). Essas fronteiras simbólicas podem ser reconhecidas quando se veem aflorar sociedades recreativas e culturais, por exemplo, ocorrências bastante comuns na historiografia de Joinville: Tal valorização pode ser mais bem compreendida quando se leva em conta a visão de Foucault (1994), o qual afirma que o espaço é fundamental em qualquer exercício de poder, Outro assunto que merece observação especial nas histórias das colônias, principalmente alemãs, será a influência que tiveram, na formação moral e cultural dos colonos, as sociedades culturais, recreativas e beneficentes 153 MG Boldorini; RB Meira. Diálogos, v.22, n.2, (2018) 140-159 que, desde a formação do primeiro povoado, se fundaram onde quer que grupos de colonos se estabelecessem (FICKER, 1965, p. 196). constituir um grupo autossuficiente e excluir todas as outras desse círculo de relações. Tal postura auxiliou para reforçar a solidariedade, o sentimento de pertença e de identidade, assim como de superioridade sobre todos os de fora do grupo. Essa ideia fundamenta a hipótese de os alemães serem tão bem valorizados na construção de Joinville em comparação aos demais grupos. Ficker (1965) dá grande destaque principalmente à Sociedade de Cultura, fundada na cidade em 1855 por um grupo de alemães e cuja intenção era “promover a prosperidade em todos os sentidos da agricultura e da indústria, ajudar os colonos recém-chegados” (FICKER, 1965, p. 166). Segundo o autor, era “realmente a primeira formação cultural de um organismo social em transformação, devido ao afastamento da terra natal e sob pressão de novas influências” (FICKER, 1965, p. 166). Embora existam lugares específicos para os grupos sociais, há também lugares que se superpõem, se entrecruzam, tornando-se um espaço comum e compartilhado por todos, independentemente da temporalidade em que ele está inscrito. É o caso das ruas centrais da cidade e das praças, lugares em que todas as variantes do município se encontram. Observa- se ainda que os pontos de encontro dificilmente mudam com o passar dos anos e em geral mantêm a mesma configuração. Na obra de Ficker, tem-se: Observa-se aqui que as sociedades culturais têm bastante importância para os germânicos, como bem explica Elias (1997). 8 Atualmente é conhecida por Rua das Palmeiras. p 7 Hoje sede do Museu Nacional de Imigração e Colonização. 8 6 Nobre francês, foi marido de Princesa Isabel, filha do imperador do Brasil D. Pedro II. Grandes festejos foram programados para a visita de Sua Alteza Real o Conde d’Eu6, a 12, 13 e 14 de dezembro de 1884 [...]. Formou-se um préstito de mais de 20 carruagens e carros de colonos, todos enfeitados e ornados com folhas de palmeiras e flores. Às seis horas da tarde, o cortejo chegou ao Palácio do Príncipe7. Na Alameda das Palmeiras8, que já nessa época apresentava um aspecto impressionante, os carros passaram entre alas do povo, colegiais e crianças festivamente trajadas (FICKER, 1965, p. 322). 9 Vindo da Alemanha, chegou a Joinville em 1854, de onde nunca mais saiu. Exerceu cargos públicos na cidade e fundou o jornal, todo escrito em alemão, Kolonie-Zeitung (Jornal da Colônia), cuja circulação durou quase 80 anos (FICKER, 1965). linhas de força” (SANTOS, 2004, p. 165). O autor, que estudou a fundo padrões do comportamento alemão dos séculos XIX e XX, afirma que, por causa da incerteza de status, das transformações das relações de poder e do desequilíbrio de poder entre grupos estabelecidos e grupos marginais, sobretudo no século XIX, houve a necessidade de se buscar, por parte dos alemães, uma identidade social. Grandes festejos foram programados para a visita de Sua Alteza Real o Conde d’Eu6, a 12, 13 e 14 de dezembro de 1884 [...]. Formou-se um préstito de mais de 20 carruagens e carros de colonos, todos enfeitados e ornados com folhas de palmeiras e flores. Às seis horas da tarde, o cortejo chegou ao Palácio do Príncipe7. Na Alameda das Palmeiras8, que já nessa época apresentava um aspecto impressionante, os carros passaram entre alas do povo, colegiais e crianças festivamente trajadas (FICKER, 1965, p. 322). Pensando nisso, conforme Elias (1997), os membros dos grupos similares passaram a formar uma rede de pessoas que sentiam que pertenciam ao mesmo círculo e que juntas exerciam suficiente poder para estar aptas a Wittich Freitag também faz Wittich Freitag também faz faz 154 MG Boldorini; RB Meira. Diálogos, v.22, n.2, (2018) 140-159 de um imóvel localizado à Rua Borba Gato, o primeiro manicômio da cidade: “Levaram-me para o prédio onde havia funcionado o Hospício Oscar Schneider, atrás do Cemitério Municipal. Por causa do grande número de prisões de pessoas naquele período [em decorrência da Campanha de Nacionalização], o local passou a servir de presídio” (S.THIAGO, 2000, p. 37). ponderações sobre o local, porém um pouco mais adiante da intitulada por Ficker como Alameda das Palmeiras: Ainda lembrando a juventude, recordo a Praça Nereu Ramos, na Rua do Príncipe, onde havia o footing, uma espécie de passeata das moças. Elas andavam para lá e para cá e nós, marmanjos, ficávamos parados, só na paquera! Em Joinville a maioria das moças era bastante tímida. Durante a passeata elas nos olhavam de um jeito muito gracioso, mas tímido. Era ali que praticamente iniciavam-se a maioria dos namoros (S. THIAGO, 2000, p. 44). Ficker (1965, p. Hoje em dia, o ribeirão Matias, devido ao desmatamento sistemático das florestas, nada tem de caudaloso, atravessando todo o centro da cidade, ocultando-se sob canalização de ferro e cimento, encabulado com todos os detritos que ajuda em sua escura e submersa caminhada, para desaguar, sempre modesto, no Rio Cachoeira. linhas de força” (SANTOS, 2004, p. 165). 64) também faz algumas análises sobre as modificações da paisagem: Para Olívia, a lembrança que tem da mesma rua envolve as comemorações do fim da Segunda Guerra Mundial, ponto de encontro de todos que eram contrários ao combate: Hoje em dia, o ribeirão Matias, devido ao desmatamento sistemático das florestas, nada tem de caudaloso, atravessando todo o centro da cidade, ocultando-se sob canalização de ferro e cimento, encabulado com todos os detritos que ajuda em sua escura e submersa caminhada, para desaguar, sempre modesto, no Rio Cachoeira. Então chegou oito de maio de 1945... Dia da Vitória! Alegria nas ruas, a guerra finalmente acabara. Espontaneamente, todas as samaritanas [da Cruz Vermelha] foram para a rua do Príncipe, onde havia um coreto, e lá cantaram hinos e fizeram discursos cívicos sobre os feitos dos pracinhas (MAIA in GELBCKE, 2004, p. 41). Então chegou oito de maio de 1945... Dia da Vitória! Alegria nas ruas, a guerra finalmente acabara. Espontaneamente, todas as samaritanas [da Cruz Vermelha] foram para a rua do Príncipe, onde havia um coreto, e lá cantaram hinos e fizeram discursos cívicos sobre os feitos dos pracinhas (MAIA in GELBCKE, 2004, p. 41). Outro assunto que exerce bastante influência na formatação das cidades e dos grupos sociais que nela habitam é a religiosidade. Sendo a religião uma das mais importantes marcas de ordenamento dos grupos sociais – no lançamento da pedra fundamental da igreja protestante Ottokar Doerffel9 diz que “o problema mais cruciante da Colônia é a falta de assistência religiosa” (FICKER, 1965, p. 186), fala na qual se observa o poder do discurso religioso em relação aos grupos sociais –, viu-se a Ademais, é possível ver por meio das transformações na paisagem diferentes usos para o mesmo espaço, conforme a temporalidade em que se inscreve, cada tempo correspondendo a uma específica prática para melhor uso do espaço de acordo com a necessidade da época. O narrador da biografia de Wittich Freitag, por exemplo, fala a respeito 155 MG Boldorini; RB Meira. Diálogos, v.22, n.2, (2018) 140-159 relevância de serem implantadas na colônia crenças religiosas. tradicional festa da cobertura de seu prédio. Defronte ao templo, houve as cerimônias, e deu-se um jantar no Salão Ravache, de acordo com as velhas tradições da Europa. Constata- se assim mais uma vez a disputa por territórios e fronteiras, mesmo que por vias simbólicas. linhas de força” (SANTOS, 2004, p. 165). Para resolver a questão, houve na Colônia Dona Francisca ao mesmo tempo a construção de templos tanto para os evangélicos protestantes como para os católicos. Todavia, provavelmente por contar com grande parcela da população – maioria na colônia em 1857: 1.484 evangélicos para 213 católicos (FICKER, 1965) –, ou por ser a religião original do grupo alemão, deu-se muito mais ênfase à religião protestante, com direito a festividades a fim de celebrar o lançamento da pedra fundamental da edificação, enquanto não havia nem sido enviado ainda à colônia um vigário católico: “Trabalhava-se simultaneamente nos templos das duas confissões, achando-se a casa de oração protestante já com a pedra fundamental assentada em comovente solenidade em julho do mesmo ano [1857]” (FICKER, 1965, p. 189). Outra disputa comum quando se fala de territorialidades ocorre em função do uso da língua, entendida como um dos pilares de um grupo social e base para a percepção de mundo dos indivíduos. Sendo o primeiro elemento de identificação de um grupo social e determinante da composição da identidade cultural, a língua teve bastante influência na Colônia Dona Francisca. Em seu início, em 1851, aportou na cidade a primeira grande leva de imigrantes, na qual havia três grupos distintos: suíço, alemão e norueguês. Por conta das diferenças linguísticas, cada grupo acabou desbravando uma região da colônia (FICKER, 1965). A língua igualmente esteve presente nas questões educacionais da colônia. Conta Ficker (1965, p. 178): Ficker (1965) conta que sempre se deu mais destaque ao protestantismo na colônia, enquanto referenciais católicos ficaram em segundo plano. Esse protagonismo é sentido até mesmo nos documentos oficiais da colônia: “Infelizmente não existem os mesmos documentos com relatos minuciosos dos festejos, como os encontrados nos fundamentados da igreja protestante” (FICKER, 1965, p. 189). Festejos relativos ao catolicismo só foram ocorrer em 1863, com a Uma das mais constantes preocupações do Diretor [Léonce] Aubé, foi a do ensino primário. Pela resolução do Governo Imperial, as aulas nas Colônias de principal população alemã, deveriam ser dadas nos dois idiomas [português e alemão]. Acontece que o professor de primeiras letras, enviado pela Presidência da Província, Sr. Carlos O. Schlappal, somente lecionava em língua portuguesa alegando que os seus diminutos vencimentos não lhe permitiam lecionar em duas línguas. O resultado não foi de surpreender: aulas do professor J. H. Auler, subvencionadas pela Sociedade Colonizadora (com 25$000 por 156 MG Boldorini; RB Meira. linhas de força” (SANTOS, 2004, p. 165). Diálogos, v.22, n.2, (2018) 140-159 apagada. Num trecho trazido de Crispim Mira, prosador, orador e jornalista, sobre Joinville de 1905, assim é dito: “Joinville, que trinta anos atrás era uma colônia exclusivamente alemã, tem agora vinte mil habitantes entre alemães, luso e teuto-brasileiros” (apud FICKER, 1965, p. 364), num nítido indicativo de que somente a parcela alemã da população angariou os méritos do desenvolvimento da cidade, além de que os negros e os chamados brasileiros, por exemplo, não são sequer mencionados. mês), tiveram uma frequência cada vez maior, enquanto que a escola do professor Schlappal acusava reduzido número de alunos. O reconhecimento da importância da língua pela gestão governamental também está presente nas biografias analisadas neste artigo. Wittich Freitag, por exemplo, conta que foi preso em Joinville em decorrência da língua alemã, que falava com seus companheiros de pensão, em um jogo de baralho, além de a língua ter sido proibida, assim como o italiano, por Getulio Vargas, na Campanha de Nacionalização. Já Olívia relata que, à época da campanha, estudava no Colégio Bom Jesus, escola tipicamente alemã de Joinville, em 1935, enquanto o Brasil “lutava para se libertar de ideias integralistas” (GELBCKE, 2004, p. 28). A biografada rapidamente esclarece como foi tal época no Colégio Bom Jesus. Anna Maria Harger, então diretora da escola, conforme ela, “foi submetida a fortes pressões” (MAIA in GELBCKE, 2004, p. 29) por causa da nacionalização do ensino e proibição do uso do idioma estrangeiro, em razão da Segunda Guerra Mundial. Também é interessante perceber o tom bucólico que é dado a Joinville por parte de Ficker (1965), que ressalta em inúmeras passagens como a cidade é pacata, ordeira e tranquila: “Nova e singelamente pura, Joinville é sem lhe dizer favores, cidadezinha de verdadeiro encanto” (FICKER, 1965, p. 365), e que seu habitante goza de uma “vida tranquila e pacífica” (FICKER, 1965, p. 270). Salienta-se, com isso, o progresso da cidade, direcionando-o à ordem e à modernização, discurso que aparece fortemente na mídia que circula pela região, embora Joinville seja atualmente o maior município de Santa Catarina e, por isso, tenha adquirido algumas características que envolvem sobretudo a violência, problemas típicos de uma cidade grande. Nos capítulos finais de seu livro, Ficker (1965) afirma: “Termina aqui [em 1901] a história colonial de Joinville e da Colônia Dona Francisca”, na clara intenção de demonstrar que ambas as histórias, da colônia e da cidade, se confundem. linhas de força” (SANTOS, 2004, p. 165). Não é mais possível saber o que é colônia e o que é cidade, muito embora boa parcela dessas histórias seja Considerações finais Propôs-se aqui uma nova maneira de Propôs-se aqui uma nova maneira de 157 MG Boldorini; RB Meira. Diálogos, v.22, n.2, (2018) 140-159 pensar a paisagem urbana, “como um todo coerente solidamente ajustado, que enquadra drasticamente as nossas atividades” (CARDOSO, 2013, p. 9). Tal qual uma narrativa que conta uma vida lógica, cronológica e coerente, contrariando a própria vida real, assim também acontece em relação às imagens paisagísticas que vemos ao longo dos textos. Elas aparecem como suporte, cenário para a história que se almeja contar. Por isso, devem estar em consonância com os demais elementos que compõem a narrativa, ajudando a manter, ao menos aparentemente, o sentido e a linearidade do texto biográfico. representação. construção de identidades CASTRO, Viviane Maria Cavalcanti de. O uso do conceito de identidade na arqueologia. 2008. Disponível em: <https://www.ufpe.br/clioarq/images/docume ntos/V23N1-2008/2008v1n23a9.pdf>. Acesso em: 9 set. 2016. representação. Percebeu-se, então, que o livro de Ficker (1965) ajudou a moldar o imaginário dos moradores de Joinville, assim como o da gestão municipal e o discurso midiático, forjando uma identidade local única e singular: a do colonizador alemão, aquele que trouxe a Joinville o progresso e o desenvolvimento industrial. Não obstante o apagamento de alguns grupos, sobretudo os chamados grupos minoritários, para a manipulação dessa identidade, a imagem de Joinville como uma cidade alemã é reforçada pelas biografias que se analisaram aqui, num claro indicativo que são essas as memórias, as do grupo mais potente, que devem ser perpetuadas e espalhadas. Viu-se com as análises aqui presentes que o homem molda o espaço e por ele é moldado, numa interferência recíproca. Todavia, o espaço é heterogêneo e deve comportar toda a diversidade nele presente, e essa convivência com o diferente na maioria das vezes não ocorre de maneira tranquila, o que acaba privilegiando alguns em detrimento de outros. Esse privilégio é em grande parte obtido pela posição econômica e política e acaba supervalorizando os chamados pertencentes à elite, ou seja, aqueles que detêm o poder simbólico. Ao mencionar o espaço, as narrativas promovem grande esforço de constituição da delimitação e individualização das culturas e do território quando em confronto com outros territórios, com vistas a sua homogeneização. Conforme as palavras de Sevcenko (1985), a literatura “aparece como um ângulo estratégico notável, para a avaliação das forças e dos níveis de tensão existentes”. Logo, deve-se sempre, ao tratar de obras literárias, considerar o meio que ela relata, pois a literatura é um dos meios de divulgação das representações do real à vista de experiências imaginárias acerca do mundo exterior. Todo material literário expressa contextos espaçotemporais e, como se viu, é um poderoso suporte para a O destaque dado a um grupo social em relação ao outro impacta também na configuração da identidade coletiva. À procura de uma identidade para o grupo única, homogênea e singular, a paisagem urbana tende a ser um dos suportes dessa 158 MG Boldorini; RB Meira. Diálogos, v.22, n.2, (2018) 140-159 Referências ARANTES, Antonio A. A guerra dos lugares: sobre fronteiras simbólicas e liminaridades no espaço urbano. Revista do Patrimônio Histórico Artístico Nacional, n. 23, p. 191-203, 1994. DELPHIM, Carlos Fernando de Moura. O patrimônio natural no Brasil. In: FUNARI, Pedro Paulo A.; PELEGRINI, Sandra C. A.; RAMBELLI, Gilson (Orgs.). Patrimônio cultural ambiental. São Paulo: Annablume, 2009. ARRUDA, Gilmar. “Minha terra tem palmeiras”: paisagem, patrimônio e identidade nacional. In: FUNARI, Pedro Paulo A.; PELEGRINI, Sandra C. A.; RAMBELLI, Gilson (Orgs.). Patrimônio cultural ambiental. São Paulo: Annablume, 2009. ELIAS, Norbert. Os alemães: a luta pelo poder e a evolução do habitus nos séculos XIX e XX. Tradução de Álvaro Cabral. Rio de Janeiro: Jorge Zahar, 1997. FEBVRE, Lucien. Combates pela história. Lisboa: Presença, 1989. ______. O chão de nossa história: natureza, patrimônio ambiental e identidade. Patrimônio e Memória, São Paulo, v. 2, n. 2, p. 110-125, 2006. FICKER, Carlos. História de Joinville: subsídios para a crônica da Colônia Dona Francisca. Joinville: Impressora Ipiranga, 1965. BESSE, Jean-Marc. Estar na paisagem, habitar, caminhar. In: CARDOSO, Isabel Lopes; TAVARES, André (Eds.). Paisagem património. Porto: Dafne/Chaia, 2013. p. 33- 53. FOUCAULT, Michel. Espaço e poder. Revista do Patrimônio Histórico e Artístico Nacional, n. 23, p. 138-145, 1994. BUSCHLE, Baltasar. Prefácio. 1999. In: S. THIAGO, Raquel. Eu, Wittich Freitag. Joinville: Movimento e Arte, 2000. 268 p. GINZBURG, Carlo. Nenhuma ilha é uma ilha: quatro visões da literatura inglesa. São Paulo: Companhia das Letras, 2004. CABRAL, Oswaldo. Introdução. In: FICKER, Carlos. História de Joinville: subsídios para a crônica da Colônia Dona Francisca. Joinville: Impressora Ipiranga, 1965. p. 9-14. GUEDES, Sandra Paschoal Leite de Camargo. A escravidão em uma colônia de “alemães”. In: SIMPÓSIO NACIONAL DE HISTÓRIA DA ASSOCIAÇÃO NACIONAL DE HISTÓRIA, 24., 2007, São Leopoldo. Anais... São Leopoldo, 2007. Disponível em: <http://anais.anpuh.org/wp- content/uploads/mp/pdf/ANPUH.S24.0464.pd f>. Acesso em: 23 set. 2017. CARDOSO, Ciro Flamarion; BRIGNOLI, Héctor Pérez. Os métodos da história. Rio de Janeiro: Graal, 1979. CARDOSO, Isabel Lopes. Paisagem e património: aproximações pluridisciplinares. In: ______; TAVARES, André (Eds.). Paisagem património. Porto: Dafne/Chaia, 2013. p. 7-15. MENESES, Ulpiano Bezerra de. Identidade cultural e arqueologia. Revista do Patrimônio Histórico e Artístico Nacional, n. 20, p. 33-36, 1984. 159 MG Boldorini; RB Meira. Diálogos, v.22, n.2, (2018) 140-159 SEVCENKO, Nicolau. Literatura como missão: tensões sociais e criação cultural na Primeira República. São Paulo: Brasiliense, 1985. RONCAGLIO, Cynthia. A ideia da natureza como patrimônio: um percurso histórico. Desenvolvimento e Meio Ambiente, Curitiba, n. 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https://openalex.org/W3130733579	https://oceanrep.geomar.de/id/eprint/52426/2/cp-17-397-2021-supplement.pdf	English	null	Last glacial inception trajectories for the Northern Hemisphere from coupled ice and climate modelling	Climate of the past	2,021	cc-by	806	Supplement of Clim. Past, 17, 397–418, 2021 https://doi.org/10.5194/cp-17-397-2021-supplement © Author(s) 2021. This work is distributed under the Creative Commons Attribution 4.0 License. Supplement of Last glacial inception trajectories for the Northern Hemisphere from coupled ice and climate modelling Taimaz Bahadory et al. Correspondence to: Lev Tarasov (lev@mun.ca) The copyright of individual parts of the supplement might differ from the CC BY 4.0 License. Supplement of Clim. Past, 17, 397–418, 2021 https://doi.org/10.5194/cp-17-397-2021-supplement © Author(s) 2021. This work is distributed under the Creative Commons Attribution 4.0 License. Supplement of Taimaz Bahadory et al. Correspondence to: Lev Tarasov (lev@mun.ca) Correspondence to: Lev Tarasov (lev@mun.ca) The copyright of individual parts of the supplement might differ from the CC BY 4.0 License. igure 1. Ensemble distribution of the NH seasonal maximum sea ice area during the inception period. b. Timing of the late-winter sea ic rea maximum against timing of the NA (black) and EA (red) ice sheet maximum volumes. Figure 1. Ensemble distribution of the NH seasonal maximum sea ice area during the inception period. b. Timing of the late-winter sea ice area maximum against timing of the NA (black) and EA (red) ice sheet maximum volumes. 1 Sea ice The Atlantic sea ice area variation pattern also follows the total NH sea ice area. While the maximum sea ice extent is less variable through time, the minimum sea ice extent reaches its largest extent around 114 ka, and its smallest extent between 107 and 105 ka. Regardless of the the glacial stage I are in (growth or shrink phase), the southward extent of the sea ice in the North Atlantic can never move below the 44°N (the same latitude as the jet-stream over the North Atlantic), which is reached shortly after entering the glacial period. The minimum extent, on the other hand, is well north of the 44°N, and therefore can freely oscillate with the cooling and warming of the stadial and interstadial phases (9th month in ﬁgure 2). The Atlantic sea ice area variation pattern also follows the total NH sea ice area. While the maximum sea ice extent is less variable through time, the minimum sea ice extent reaches its largest extent around 114 ka, and its smallest extent between 107 and 105 ka. Regardless of the the glacial stage I are in (growth or shrink phase), the southward extent of the sea ice in the North Atlantic can never move below the 44°N (the same latitude as the jet-stream over the North Atlantic), which is reached North Atlantic can never move below the 44°N (the same latitude as the jet-stream over the North Atlantic), which is reached 5 shortly after entering the glacial period. The minimum extent, on the other hand, is well north of the 44°N, and therefore can freely oscillate with the cooling and warming of the stadial and interstadial phases (9th month in ﬁgure 2). North Atlantic can never move below the 44°N (the same latitude as the jet-stream over the North Atlantic), which is reached 5 shortly after entering the glacial period. The minimum extent, on the other hand, is well north of the 44°N, and therefore can freely oscillate with the cooling and warming of the stadial and interstadial phases (9th month in ﬁgure 2). 2 Atlantic meridional heat transport 2 Atlantic meridional heat transport 3 Cross-continental correlations 4 Model bias for present-day precipitation 10 2 2 Figure 2. Monthly lowest latitude of the sea ice in the North Atlantic from 119 ka to 105 ka. Vertical axis shows each month, and the horizontal axis is the simulation year. Colors represent the lowest latitude. top ensemble mean, and bottom ensemble standard deviation. Figure 2. Monthly lowest latitude of the sea ice in the North Atlantic from 119 ka to 105 ka. Vertical axis shows each month, and the horizontal axis is the simulation year. Colors represent the lowest latitude. top ensemble mean, and bottom ensemble standard deviation. 3 3 Figure 3. Atlantic meridional northward heat transport top. ensemble mean and, bottom. standard deviation through the LGI. Figure 3. Atlantic meridional northward heat transport top. ensemble mean and, bottom. standard deviation through the LGI. 4 4 Figure 4. Correlation plots of maximum ice volume between NA and EA diagnostic sectors. Figure 4. Correlation plots of maximum ice volume between NA and EA diagnostic sectors. Figure 4. Correlation plots of maximum ice volume between NA and EA diagnostic sectors. Figure 4. Correlation plots of maximum ice volume between NA and EA diagnostic sectors. 5 Figure 5. Mean present-day annual precipitation bias of the reduced 55 member sub-ensemble relative to the NCEP reanalysis climatology. Figure 5. Mean present-day annual precipitation bias of the reduced 55 member sub-ensemble relative to the NCEP reanalysis climatology. Figure 5. Mean present-day annual precipitation bias of the reduced 55 member sub-ensemble relative to the NCEP reanalysis climatology. 6 6
https://openalex.org/W1489105307	https://europepmc.org/articles/pmc5711079?pdf=render	English	null	Evaluation of a novel secondary check tool for intensity‐modulated radiotherapy treatment planning	Journal of applied clinical medical physics	2,014	cc-by	5,414	JOURNAL OF APPLIED CLINICAL MEDICAL PHYSICS, VOLUME 15, NUMBER 5, 2014 JOURNAL OF APPLIED CLINICAL MEDICAL PHYSICS, VOLUME 15, NUMBER 5, 2014 Evaluation of a novel secondary check tool for intensity- modulated radiotherapy treatment planning Jonas D. Fontenota Department of Physics, Mary Bird Perkins Cancer Center, B jfontenot@marybird.com Received 21 March, 2014; accepted 5 May, 2014 Jonas D. Fontenota Department of Physics, Mary Bird Perkins Cancer Center, Baton Rouge, LA, USA jfontenot@marybird.com Received 21 March, 2014; accepted 5 May, 2014 The purpose of this study was to assess the accuracy and efficacy of an automated treatment plan verification, or “secondary check”, tool (Mobius3D), which uses a reference dataset to perform an independent three-dimensional dose verification of the treatment planning system (TPS) dose calculation and assesses plan quality by comparing dose-volume histograms to reference benchmarks. The accuracy of the Mobius3D (M3D) system was evaluated by comparing dose calculations from IMRT and VMAT plans with measurements in phantom geometries and with TPS calculated dose distributions in prostate, lung, and head and neck patients (ten each). For the patient cases, instances of DVH limits exceeding reference values were also recorded. M3D showed agreement with measured point and planar doses that was comparable to the TPS in phantom geometries. No statistically significant differences (p < 0.05) were noted. M3D dose distributions from VMAT plans in patient cases were in good agreement with the TPS, with an average of 99.5% of dose points showing γ5%,3mm < 1. The M3D system also identified several plans that had exceeded dose-volume limits specified by RTOG protocols for those sites. The M3D system showed dosimetric accuracy comparable with the TPS, and identified several plans that exceeded dosimetric benchmarks. The M3D system possesses the potential to enhance the current treatment plan verification paradigm and improve safety in the clinical treatment planning and review process. PACS number: 87.55.D-, 87.55.km, 87.55.Qr, PACS number: 87.55.D-, 87.55.km, 87.55.Qr, PACS number: 87.55.D-, 87.55.km, 87.55.Qr, Key words: intensity-modulated radiation therapy, volumetric-modulated arc therapy, treatment planning systems, quality assurance a Corresponding author: Jonas D. Fontenot, Mary Bird Perkins Cancer Center, 4950 Essen Lane, Baton Rouge, LA 70809, USA; phone: (225) 215 1337; fax: (225) 215 1376; email: jfontenot@marybird.com I. Introduction With conventional (e.g., unmodulated) treatment fields, the quality of each treatment planning system (TPS) calculation was verified through the use of independent treatment plan verifica tion (also referred to as “secondary check”) systems that typically apply simplistic scatter and pathlength corrections to calculations of dose to a single point for the purpose of independently verifying TPS monitor units.(1) However, the complexity of dose distributions has increased substantially since the initial adoption of such methods. Inverse planning and intensity modu lation of treatment fields have enabled the clinician to create highly conformal and irregular dose distributions that improve normal tissue sparing.(2,3) Subsequent developments in treat ment planning and delivery that have improved efficiency, such as volumetric-modulated arc therapy,(4) have further reduced the technical obstacles of utilization and resulted in marked proliferation of these technologies over the last 10 to 15 years.(5,6) In most clinics in the United States, the current approach to dosimetric verification of such plans is the combination of a “secondary check” calculation to independently evaluate the accuracy of the TPS dose cal culation algorithm, and a patient-specific quality assurance (QA) measurement in a dedicated 207 207 208 Fontenot et al.: Radiotherapy treatment plan verification 208 phantom to ensure data transfer fidelity and deliverability of the treatment plan. As the present work applies only to “secondary check” calculations, patient-specific QA measurements and approaches will not be discussed further. phantom to ensure data transfer fidelity and deliverability of the treatment plan. As the present work applies only to “secondary check” calculations, patient-specific QA measurements and approaches will not be discussed further. Despite increased complexity of treatment planning dose distributions, the methods and algo rithms employed by “secondary check” systems have remained largely unchanged: dose calcula tions to a single point using simple heterogeneity corrections. Such limitations inevitably lower the ability of those systems to detect clinically meaningful errors in the TPS calculation throughout the high-dose volume. An additional limitation of the current “secondary check” paradigm of calculating dose to a single point, aside from precluding a full three-dimensional verification of the TPS accuracy, also precludes an independent assessment of the quality of the treatment plans with respect to dosimetric benchmarks. As a result, there is a potential role for more sophisticated treatment planning dose verification tools to enhance current clinical practice. I. Introduction p gi p Recently, a new commercial secondary check tool has been developed (Mobius3D; Mobius Medical Systems, LP, Houston, TX). The system is intended to enhance the current secondary check paradigm by 1) performing an independent 3D calculation of the treatment plan within the patient CT geometry that allows for more comprehensive evaluation of TPS accuracy and its impact on the planning goals, and 2) evaluating of the quality of each treatment plan with respect to established dosimetric benchmarks. The potential benefit of such a system is to improve the value of secondary check calculations in validating TPS accuracy. Despite the potential of the system, clinical validation of the system was not heretofore reported. The purpose of this work was to evaluate the dosimetric accuracy and efficacy of the system for clinical use. The accuracy of the Mobius3D (M3D) system was evaluated by com paring its dose calculations of IMRT and/or VMAT plans with 1) measured doses in phantom geometries, and 2) dose calculations from a commercial TPS in actual patient data. Efficacy was evaluated by assessing the ability of the system to automatically review treatment plan quality and identify instances of dosimetric deviations beyond configured tolerances. Journal of Applied Clinical Medical Physics, Vol. 15, No. 5, 2014 A. Overview of Mobius3D M3D uses a collapsed cone convolution/superposition algorithm developed by the manufacturer to calculate dose in the patient or phantom geometry. Similar to convolution/superposition algorithms used by other commercial treatment planning systems, the M3D algorithm models the essential elements of the linear accelerator treatment head (e.g., MLC, jaws, flattening filter) and calculates the dose at each point within the patient by convolving the energy fluence with a dose deposition kernel. Because the algorithm is implemented on a graphical processing unit (GPU), calculations are purported to require less time compared to the existing TPSs that utilize a similar dose calculation algorithm.(7) M3D operates on DICOM objects (CT images, RT Dose, RT Structure, and RT Plan) exported from the TPS following the completion of a patient treatment plan. Upon receiving the neces sary files, the system associates the various objects under a patient-specific entry and extracts the necessary treatment field information from the RT plan file. The treatment field information is then passed to the dose calculation algorithm, which uses this information to calculate the three-dimensional dose distribution within the CT dataset associated with the plan. The dose calculation algorithm arrives precommissioned with a standard reference dataset specific to each linear accelerator manufacturer and model. The user has the ability to customize the model using a subset of site-specific depth-dose values and off-axis ratios, though the stock reference model was utilized for this work. Following dose calculation, the dose distribution calculated by M3D is compared with the TPS dose extracted from the RT Dose file using dose-volume histograms (DVH) of the structures associated with the CT dataset, isodose overlays on the CT dataset, and a 3D comparison of the dose matrices using the gamma metric.(8) Both calculated 209 209 Fontenot et al.: Radiotherapy treatment plan verification sets of DVH profiles are automatically checked against reference DVH limits by using regular expressions of regions-of-interest (ROI) names to identify relevant structures within the plan and looking up available RTOG protocol dose limits for those structures. The user may also edit, remove, or add additional DVH limits. sets of DVH profiles are automatically checked against reference DVH limits by using regular expressions of regions-of-interest (ROI) names to identify relevant structures within the plan and looking up available RTOG protocol dose limits for those structures. The user may also edit, remove, or add additional DVH limits. B. Phantom plans The accuracy of the M3D system was evaluated in a phantom by comparing its dose calcula tions for IMRT and VMAT plans with measurements previously reported by our group.(9) The four structure sets provided by Task Group 119 of the American Association of Physicists in Medicine(10) (prostate, C-shape, multitarget, and head and neck) were copied to a cylindrical solid water phantom, and IMRT and VMAT plans were constructed to meet the dosimetric goals specified in TG-119 using a commercial treatment planning system (Pinnacle3, Philips Medical Systems, Fitchburg, WI). Additional details regarding the treatment planning param eters and results are described elsewhere.(9) Point-dose measurements were performed in high- and low-dose regions using an A1SL cylindrical type ionization chamber. Planar dose measurements were performed in the sagittal and coronal planes of the phantom using radio chromic film. Additional details of the experimental geometry and dosimetry techniques are also described elsewhere.(9) i The treatment planning data (CT images, RT Dose, RT Structure, and RT Plan files) from the TG-119 plans was exported to the M3D server (running version 1.2.1 of the software), which then performed its own calculation of the three-dimensional dose distribution within the phantom resulting from the planned treatment fields. The sagittal and coronal planar doses corresponding to the film plane of each plan were extracted from the M3D and TPS dose dis tributions and registered to the planar dose measured with film using in-house code (MATLAB, MathWorks, Natick, MA). The gamma metric(8) was used to quantify the agreement between the calculated (M3D and TPS) and measured planar doses using criteria of 3% dose difference and 3 mm distance to agreement. M3D and TPS point doses were compared with measured values by taking the mean dose of a region of interest (ROI) encompassing the volume of the chamber at the location of the measurement. Percentage differences between point doses were computed using the formula recommended in AAPM Task Group 119. Differences in agreement between the M3D and TPS calculations with measured planar and point doses was assessed for significance (p < 0.05) using the Wilcoxon signed-rank test. Journal of Applied Clinical Medical Physics, Vol. 15, No. 5, 2014 C. Patient plans p The accuracy of the M3D system was also evaluated by comparing its dose calculations from VMAT plans in actual patient data with that calculated by the TPS. The study utilized clinical VMAT treatment planning data for ten patients with prostate cancer, ten patients with lung cancer, and ten patients with head and neck cancer, which included simultaneous irradiation of regional lymphatics. All VMAT plans consisted of one (prostate patients) or two arcs (all others), utilized an energy of 6 MV, a collimator angle of 45°, a leaf motion constraint of 2 mm (lung patients) or 4 mm (all others) per degree of gantry rotation, and were constructed for delivery on an Elekta linear accelerator (Infinity; Elekta AB, Stockholm, Sweden). The plans were exported to the M3D server, which performed its own calculation of the three-dimensional dose distribution within the patient CT data resulting from the planned treatment fields. As one of its evaluation metrics, M3D calculates the percentage of points showing gamma values less than one using default criteria of 5% dose difference and 3 mm distance to agreement. The 5% dose threshold follows the recommendation of AAPM Task Group 40(11) for the agreement between primary and verification calculations when using sophisticated algorithms, substantial field blocking, or heterogeneity corrections. This choice is further supported by the recent AAPM Task group 114 report,(1) which recommended a 3% tolerance between similar algorithms used to calculate dose in the patient geometry for non-IMRT fields. The average percentage of points showing Journal of Applied Clinical Medical Physics, Vol. 15, No. 5, 2014 210 Fontenot et al.: Radiotherapy treatment plan verification 210 gamma less than one was computed for each site and over all sites. Instances of DVH values exceeding reference values were also recorded. gamma less than one was computed for each site and over all sites. Instances of DVH values exceeding reference values were also recorded. Journal of Applied Clinical Medical Physics, Vol. 15, No. 5, 2014 A. Phantom plans Percentage differences between the calculated (M3D and TPS) and measured point doses for the TG-119 structure sets are shown in Table 1. In general, the TPS and M3D showed similar agreement with point doses measured with an ionization chamber. For IMRT point dose compari sons, Mobius3D showed slightly better agreement compared with the measurement (-0.6% vs. -0.8%, p = 1); however, taking the average agreement irrespective of the sign of the differences showed slightly better agreement with measurement in favor of the TPS (1.5% vs. 2.2%, p = 0.13). However, neither of these differences was found to be statistically significant. Despite similar means, Mobius3D showed a larger standard error in point-dose differences; all TPS doses were within 3% of the measured dose, whereas M3D point doses showed differences of between 3% and 5% in four cases. For VMAT point-dose comparisons, Mobius3D again showed slightly better average agreement with measurement (-1.6 vs. -1.9, p = 0.71), with slightly better agreement in favor of the TPS when neglecting the sign difference (2.0% vs. 2.3%, p = 0.28). Again, neither of these differences was found to be statistically significant. For VMAT plans, the TPS and Mobius3D showed similar standard errors in point-dose differences.i Results of the comparison between the planar dose calculations and the film measurements are shown in Table 2. In general, the TPS and M3D showed similar agreement with planar doses measured with radiochromic film. For IMRT plans, the TPS showed slightly better aver age agreement with measurement (96.9% vs. 96.2%). Conversely, M3D showed slightly better average agreement with measurement (97.5% vs. 97.0%) for VMAT plans. However, neither of these differences was found to be statistically significant (p = 0.20 and 0.06 for IMRT and VMAT, respectively). The standard error of the agreement with film measurements was similar Table 1. Measured point doses and percent differences between doses measured and calculated by the treatme planning system (TPS) and Mobius3D (M3D) for IMRT and VMAT treatment plans of the AAPM Task Group 1 structure sets. Percent differences are displayed as the mean ± standard error (N = 5). Table 1. Measured point doses and percent differences between doses measured and calculated by the treatment planning system (TPS) and Mobius3D (M3D) for IMRT and VMAT treatment plans of the AAPM Task Group 119 structure sets. Percent differences are displayed as the mean ± standard error (N = 5). Table 1. A. Phantom plans Measured point doses and percent differences between doses measured and calculated by the treatment planning system (TPS) and Mobius3D (M3D) for IMRT and VMAT treatment plans of the AAPM Task Group 119 structure sets. Percent differences are displayed as the mean ± standard error (N = 5). IMRT VMAT Measured % diff Measured % diff Location Dose (cGy) TPS M3D Dose (cGy) TPS M3D Multitarget Central target 213.9±0.3 -0.4±0.1 -0.6±0.1 219±0.2 0.3±0.1 1.5±0.1 Superior target 118.5±0.8 -0.6±0.4 -3.3±0.4 108±0.2 -0.1±0.1 -0.5±0.1 Inferior target 59.8±0.5 -2.8±0.2 -0.1±0.2 53.7±0.2 -1.2±0.1 -1.2±0.1 Prostate PTV 182.6±0.2 -0.8±0.1 -0.2±0.1 184.3±0.3 -0.6±0.2 0.2±0.2 Rectum 134.4±0.5 -1.6±0.3 2.5±0.3 144.0±0.3 -1.5±0.2 1.7±0.2 Bladder 138.8±0.8 1.3±0.4 -1.2±0.4 129.4±0.8 -2.8±0.4 -4.2±0.4 Head and neck PTV 207.1±0.1 -2.9±0.0 -3.5±0.0 198.0±0.3 -4.2±0.1 -3.5±0.1 Spinal cord 124.1±1.4 -1.4±0.7 -2.0±0.7 127.4±0.9 -4.0±0.4 -2.8±0.4 C-shape Central core 53.2±0.3 -0.9±0.2 -3.4±0.2 44.0±0.3 -2.0±0.1 -4.0±0.1 Outer target 212.0±0.3 1.8±0.1 5.5±0.1 202.0±0.7 -2.8±0.3 -3.5±0.3 Average -0.8±0.5 -0.6±0.9 -1.9±0.5 -1.6±0.7 \|Average\| 1.5±0.3 2.2±0.6 2.0±0.5 2.3±0.5 Fontenot et al.: Radiotherapy treatment plan verification 211 211 Table 2. The percentage of calculated treatment planning system (TPS) and Mobius3D (M3D) dose points with gamma (γ) values less than one when compared with dose points measured with radiochromic film for IMRT and VMAT plans of the AAPM Task Group 119 structure sets. Percentage of points are shown as the mean ± standard error (N = 3). for both the TPS and M3D for VMAT plans. For both dose calculations and delivery types, agreement with film was highest for the mock prostate plans (range: 99.7%–100%) and lowest for the mock head and neck plans (range: 90.9%–95.6%). Table 2. The percentage of calculated treatment planning system (TPS) and Mobius3D (M3D) dose points with gamma (γ) values less than one when compared with dose points measured with radiochromic film for IMRT and VMAT plans of the AAPM Task Group 119 structure sets. Percentage of points are shown as the mean ± standard error (N = 3). % points γ3%,3mm < 1 IMRT VMAT Film Plane TPS M3D TPS M3D Multitarget Coronal 98.6±0.3 96.0±0.6 97.4±0.2 99.0±0.2 Sagittal 98.6±0.6 96.4±0.7 98.1±0.3 98.5±0.3 Prostate Coronal 99.7±0.2 99.8±0.1 100±0.0 99.9±0.0 Sagittal 99.5±0.2 99.9±0.1 99.9±0.1 100±0.0 Head and neck Coronal 95.6±0.4 94.3±0.4 90.9±0.2 91.8±0.2 Sagittal 91.6±0.2 91.4±0.3 94.2±0.3 94.8±0.1 C-shape Coronal 95.5±0.8 96.7±0.8 98.7±0.1 98.4±0.3 Sagittal 96.3±0.8 94.8±0.6 96.8±0.1 97.4±0.6 Average 96.9±1.0 96.2±1.0 97.0±1.1 97.5±1.1 for both the TPS and M3D for VMAT plans. A. Phantom plans For both dose calculations and delivery types, agreement with film was highest for the mock prostate plans (range: 99.7%–100%) and lowest for the mock head and neck plans (range: 90.9%–95.6%). for both the TPS and M3D for VMAT plans. For both dose calculations and delivery types, agreement with film was highest for the mock prostate plans (range: 99.7%–100%) and lowest for the mock head and neck plans (range: 90.9%–95.6%). Journal of Applied Clinical Medical Physics, Vol. 15, No. 5, 2014 B. Patient plans The percentage of M3D-calculated dose points in patient cases showing a gamma values less than one (i.e., γ5%,3mm < 1) when compared with the TPS is shown in Table 3. A representative result is shown in Fig. 1 for a head and neck patient. On average, 100% (range: 99.9%–100%), 99.7% (range: 99.0%–100%), and 98.7% (range: 93.2%–99.9%) of M3D dose points showed gamma values less than one for prostate, lung, and head and neck plans, respectively. For the lung and head and neck cases, the anatomical region most typically associated with regions of gamma failure was the trachea and esophagus, each of which contained significant volumes of air (see Fig. 1). In these volumes, M3D calculated a slightly (approximately 5%–7%) lower in dose to air compared with surrounding tissue, as compared with the TPS. Theoretical consid erations and previous publications(12,13) suggest that absorbed dose should be lower in air than surrounding tissue; however, it is likely that the TPS interpolates the dose in this region due to the comparative lack of clinical relevance of the absorbed dose to air. Doses calculated by M3D in low-density lung tissue were found to be within tolerance criteria of the TPS calculation. M3D also reported several instances of TPS-calculated or M3D-calculated (or both) DVHs exceeding the default dosimetric benchmarks. For the prostate patients, dose volumes that had been potentially exceeded were identified for the femoral heads (limits specified in RTOG 0822), bladder (RTOG 0126), and penile bulb (RTOG 0126). For the lung cases, dose volumes that had been potentially exceeded were identified for the spinal cord (RTOG 0623) and its planning organ-at-risk volume (PRV) and the esophagus (RTOG 0920). Finally, for the head and neck patients, dose volumes that had been potentially exceeded were identified for the one or both parotid glands (RTOG 0912) and the mandible (RTOG 0225). In all cases, the final dose distributions and dose volumes had previously been thoroughly reviewed and approved as clinically acceptable by the radiation oncologist; nevertheless, these warnings provided a useful tool for ensuring that dose-volume limits had previously been reviewed and approved during the clinical treatment planning process. 212 Fontenot et al.: Radiotherapy treatment plan verification 212 Table 3. IV. DISCUSSION The findings of this work are noteworthy in the potential for improving safety and quality in radiation oncology treatment planning, a process that occurs every day in nearly every radiation oncology clinic in the world. In addition to automatically evaluating the dosimetric accuracy and quality of treatment plans, the use of a dose calculation model precommissioned with a refer ence, or stock, dataset also offers unique and significant advantages. To a first approximation, fundamental beam data characteristics (percentage depths doses and off-axis ratios) are similar for a given linear accelerator manufacturer and model. It is, therefore, reasonable to assume that a single commissioning dataset would be adequate for verification of all treatment plans from a given linear accelerator model (e.g., Varian iX, Elekta Synergy). In addition to being easier to adopt into clinical practice, a unified reference model also serves to independently verify the TPS beam model and the integrity of its commissioning data. The significance of the latter observation is potentially very high. A recent study by Nelms et al.(14) described eight cases where traditional patient-specific QA measurements had failed to detect a clinically meaning ful dosimetric error; of the eight cases, all resulted from errors in the TPS model, algorithm, or configuration, as opposed to data transfer or deliverability issues. Thus, there is evidence that enhanced tools for treatment plan verification could have a meaningful impact on patient safety in radiation oncology.i The purpose of treatment plan verification or “secondary checks” is to catch errors or mis takes in the treatment plan that could result in harm to the patient. Of potential concern is that the collapsed cone convolution/superposition (CCCS) dose algorithm used by M3D is similar to that used by other TPSs, leading to the hypothesis that such an approach would not be capable of detecting inherent flaws in the TPS algorithm. However, the dosimetric accuracy of convolution-based algorithms for treatment planning purposes is well documented by a large body of literature under a variety of conditions, compared with measurements and Monte Carlo simulations.(15-19) Hence, any errors in the dose calculation are likely to result from 1) the spe cific implementation of the algorithm within a particular system, the permutations of which are large, or 2) a software malfunction (i.e, a “bug”) resulting from an architectural deficiency under specific parameters. B. Patient plans The percentage of dose points calculated by the treatment planning system (TPS) with gamma (γ) values less than one when compared with those calculated by Mobius3D (M3D) for VMAT plans for cancers of the prostate, lung, and head and neck. % points γ5%,3mm < 1 Patient # Prostate Lung Head and Neck 1 100 99.9 99.4 2 99.9 98.8 99.3 3 100 99.8 99.3 4 100 99.5 99.9 5 100 100 93.2 6 100 100 99.2 7 100 99.8 99.1 8 100 100 99.1 9 100 100 99.1 10 100 99.0 99.0 Average 100±0.0 99.7±0.1 98.7±0.6 212 Fontenot et al.: Radiotherapy treatment plan verification 12 Fontenot et al.: Radiotherapy treatment plan verification 212 212 Table 3. The percentage of dose points calculated by the treatment planning system (TPS) with gamma (γ) values less than one when compared with those calculated by Mobius3D (M3D) for VMAT plans for cancers of the prostate, lung, and head and neck. % points γ5%,3mm < 1 Patient # Prostate Lung Head and Neck 1 100 99.9 99.4 2 99.9 98.8 99.3 3 100 99.8 99.3 4 100 99.5 99.9 5 100 100 93.2 6 100 100 99.2 7 100 99.8 99.1 8 100 100 99.1 9 100 100 99.1 10 100 99.0 99.0 Average 100±0.0 99.7±0.1 98.7±0.6 Journal of Applied Clinical Medical Physics, Vol. 15, No. 5, 2014 Fig. 1. Isodose distributions and dose profiles (a) from the treatment planning system (solid lines) and Mobius3D (dashed lines) for head and neck Case #6. Regions of blue and purple colorwash in the sinus and airway denote regions where M3D underpredicted the TPS dose by greater than the gamma criteria. Dose-volume histograms (b) for relevant regions of interest calculated by the TPS (solid) and M3D (dashed). (Adapted from the M3D user interface.) Fig. 1. Isodose distributions and dose profiles (a) from the treatment planning system (solid lines) and Mobius3D (dashed lines) for head and neck Case #6. Regions of blue and purple colorwash in the sinus and airway denote regions where M3D underpredicted the TPS dose by greater than the gamma criteria. Dose-volume histograms (b) for relevant regions of interest calculated by the TPS (solid) and M3D (dashed). (Adapted from the M3D user interface.) Journal of Applied Clinical Medical Physics, Vol. 15, No. 5, 2014 Journal of Applied Clinical Medical Physics, Vol. 15, No. 5, 2014 213 213 Fontenot et al.: Radiotherapy treatment plan verification Journal of Applied Clinical Medical Physics, Vol. 15, No. 5, 2014 IV. DISCUSSION As noted by AAPM Task Group 53,(20) a modern planning system may be the result of 30–50 person-years of work, consisting of 1 million lines of code or more. Even well-designed and implemented software systems will usually contain at least one software error in every 100–1000 lines of code,(21) some of which will produce significant errors under certain conditions. In such cases, the role of the secondary check is to identify circumstances where the TPS implementation has produced an errant result. This can be achieved either by using a different class of algorithm or by using a different implementation, with the probability of two independent implementations of an algorithm producing identical errors being exceedingly small. In this case, the CCCS algorithm within M3D was developed in-house and, therefore, uses different approaches to each step of the algorithm (e.g., beam model parameterization, fluence transport, ray tracing, TERMA calculation, output factor determination), meaning it is unlikely that a calculation error in a TPS using a CCCS algorithm would be replicated by M3D. This work also had several limitations. Both the TPS and M3D dose distributions were extracted from the M3D software. The TPS dose was separately verified to match that taken directly from the clinical server; however, M3D does not contain planar dose export tools that easily facilitate registration of a single dose plane with a measured film image. Thus, while the TPS and M3D dose matrices were always coregistered, aligning them with the film image required data manipulation with custom MATLAB (MathWorks) scripts. The point-dose agree ment was also found to be sensitive to the exact dimensions and placement of the ROI cor responding to the active chamber volume in the CT dataset, particularly for lower dose points located in high-dose gradient regions. However, as the TPS and M3D and dose distributions showed similar features, a slight change in film registration or ROI shape/location produced a change in agreement between each calculation and measurement that was similar in direction Fontenot et al.: Radiotherapy treatment plan verification 214 214 and magnitude. Thus, while agreement of each system with measurement could potentially be improved slightly, the difference in the agreement between the two dose distributions and the measurement would not be expected to change. Finally, it is important to note that only a single TPS and linear accelerator model was examined in this work. V. Conclusions The M3D system showed dosimetric accuracy comparable with the TPS and identified several plans that exceeded dosimetric benchmarks. The M3D system possesses the potential to enhance the current treatment plan verification paradigm and improve safety in the clinical treatment planning and review process. IV. DISCUSSION Future work should focus on validation of M3D for other clinical vendors. The ability of the system to detect known problems in treatment plans, such as those described in the work of Nelms et al.,(14) should also be assessed. Acknowledgments The author would like to thank Nathan Childress for technical insights regarding the function the Mobius3D treatment plan verification system. Portions of this study were supported by research grants from Mobius Medical Systems, LP, and Elekta, Ltd. Journal of Applied Clinical Medical Physics, Vol. 15, No. 5, 2014 References 1. Stern RL, Heaton R, Fraser MW, et al. Verification of monitor unit calculations for non-IMRT clinical radiotherapy: report of AAPM Task Group 114. Med Phys. 2011;38(1):504–30. p p y ( ) 2. Cozzi L, Fogliata A, Bolsi A, Nicolini G, Bernier J. Three-dimensional conformal vs. intensity-modulated radiotherapy in head-and-neck cancer patients: comparative analysis of dosimetric and technical parameters. 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Accuracy of two heterogeneity dose calcula tion algorithms for IMRT in treatment plans designed using an anthropomorphic thorax phantom. Med Phys. 2007;34(5):1850–57. ( ) 18. Davidson SE, Popple RA, Ibbott GS, Followill DS. Technical note: Heterogeneity dose calculation accuracy in IMRT: study of five commercial treatment planning systems using an anthropomorphic thorax phantom. Med Phys. 2008;35(12):5434–39. 19. Hasenbalg F, Neuenschwander H, Mini R, Born EJ. Collapsed cone convolution and analytical anisotropic algorithm dose calculations compared to VMC++ Monte Carlo simulations in clinical cases. Phys Med Biol. 2007;52(13):3679–91. 20. Fraass B, Doppke K, Hunt M, et al. American Association of Physicists in Medicine Radiation Therapy Committee Task Group 53: quality assurance for clinical radiotherapy treatment planning. Med Phys. 1998;25(10):1773–829. ( ) 21. Jacky J and White CP. Testing a 3-D radiation therapy planning program. Int J Radiat Oncol Biol Phys. 1990;18(1):253–61.
https://openalex.org/W2009776573	https://bmcbioinformatics.biomedcentral.com/counter/pdf/10.1186/1471-2105-11-S6-S3	English	null	Assessing constancy of substitution rates in viruses over evolutionary time	BMC bioinformatics	2,010	cc-by	5,150	Correspondence: u-melcher-4@alumni.uchicago.edu Department of Biochemistry & Molecular Biology, Oklahoma State University, Stillwater OK 74078, USA PROCEEDINGS Open Access © 2010 Melcher; 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. Ulrich Melcher From Seventh Annual MCBIOS Conference. Bioinformatics: Systems, Biology, Informatics and Computation Jonesboro, AR, USA. 19-20 February 2010 Abstract Background: Phylogenetic analyses reveal probable patterns of divergence of present day organisms from common ancestors. The points of divergence of lineages can be dated if a corresponding historical or fossil record exists. For many species, in particular viruses, such records are rare. Recently, Bayesian phylogenetic analysis using sequences from closely related organisms isolated at different times have been used to calibrate divergences. Phylogenetic analyses depend on the assumption that the average substitution rates that can be calculated from the data apply throughout the course of evolution. Results: The present study tests this crucial assumption by charting the kinds of substitutions observed between pairs of sequences with different levels of total substitutions. Datasets of aligned sequences, both viral and non- viral, were assembled. For each pair of sequences in an aligned set, the distribution of nucleotide interchanges and the total number of changes were calculated. Data were binned according to total numbers of changes and plotted. The accumulation of the six possible interchange types in retroelements as a function of distance followed closely the expected hyperbolic relationship. For other datasets, however, significant deviations from this relationship were noted. A rapid initial accumulation of transition interchanges was frequent among the datasets and anomalous changes occurred at specific divergence levels. Conclusions: The accumulation profiles suggested that substantial changes in frequencies of types of substitutions occur over the course of evolution and that such changes should be considered in evaluating and dating viral phylogenies. Melcher BMC Bioinformatics 2010, 11(Suppl 6):S3 http://www.biomedcentral.com/1471-2105/11/S6/S3 Melcher BMC Bioinformatics 2010, 11(Suppl 6):S3 http://www.biomedcentral.com/1471-2105/11/S6/S3 Analysis Each of the six datasets was analyzed for the numbers of each type of the six interchanges for one strand for each pair of sequences in the dataset and the total numbers of positions evaluated for each pair was recorded. Posi- tions where either member of the pair lacked a residue were ignored. For datasets with open reading frames on one strand only, that strand was used for analysis. When ORFs were on both strands, as in TYLCV, the genomic strand was used. Analysis was accomplished by a short program written in Future Basic (Staz Software, Bay St. Louis, Missouri; source code available as addi- tional file 3). The program tabulated the numbers of each type of interchange in each pair of sequences and the total number of differences in each pair. For this analysis three viral datasets and three non-viral datasets were chosen. Available complete genome sequences of virus species in the Tobamovirus genus (family Virgaviridae) served as one dataset. These viruses have single strands of positive sense RNA as their encap- sidated genomes. Wheat streak mosaic virus is a single viral species with multiple sequence representatives of moderately wide diversity. Its genome consists also of a single strand of positive sense RNA. To contrast with these viruses, the study included isolates of Tomato yel- low leaf curl virus of which there are several closely related species distinguished by geographic location of original isolation. The genomes of these viruses are sin- gle-stranded circular DNA molecules that have open reading frames on both their genomic and their anti- genomic strands. A series of eucaryotic retroelements were chosen as coding regions with relaxed evolutionary constraints. Coding regions for each of the two subunits of ribulose-bisphosphate carboxylase were included since that encoding RbcL is plastid localized and that for RbcS is encoded in the nuclear genome. Plastid and nucleus are expected to have different mutational profiles. Results of the pairwise comparison were imported into Microsoft Excel. The total number of evaluated inter- changes, normalized by the number of positions compared were converted to t-distances using the Jukes- Cantor correction for multiple substitutions [18]. For each of the six interchange types, values were converted to numbers of each type per knt compared. The results were sorted according to t-distances. The numbers of interchanges were then binned according to t-distance to give between 30 and 60 well populated bins. Background hosts, implying codivergence of virus with host. This implication is consistent with conclusions from analysis of viral hallmark genes that viruses existed at the time that cellular life evolved [8]. The two views appear to conflict [2,9]. Substitution frequencies required for the codivergence hypothesis need to be about 104 fold less than those observed in many viral species [2,7]. A recent review [10] suggests, by way of reconciliation of the two views, that different evolutionary mechanisms are at work in viral speciation than in the evolution of viral strains of individual species. Phylogenetic analyses reveal probable patterns of diver- gence of present day organisms from common ances- tors. The points of divergence of lineages can be dated if a corresponding historical or fossil record exists. For many species, in particular viruses, such records are rare. Substitution frequencies calculated from analysis of strains of single viral species suggest that many viruses are evolving so rapidly that any traces of distant evolu- tionary events should be obscured [1,2]. On the other hand, evidence has been presented [3-7] that phyloge- netic trees of larger taxa often mirror those of their Evolutionary theory has recognized that not all resi- dues in a nucleotide or amino acid sequence are subject to the same evolutionary constraints [11]. It also has allowed for the possibility that some kinds of substitu- tions occur more frequently than others [12] and that Melcher BMC Bioinformatics 2010, 11(Suppl 6):S3 http://www.biomedcentral.com/1471-2105/11/S6/S3 Page 2 of 6 selected from the EST database, isolates of Wheat streak mosaic virus and the related Oat necrotic mottle virus (WSMV), members of the Tobamovirus genus and iso- lates of Tomato yellow leaf curl virus (TYLCV). Lists of the accession numbers used are presented in additional file 1. Sequences were aligned manually using Se-Al [17] with reference to the amino acid sequences encoded. In the case of tobamoviruses, previously constructed align- ments [5] were used as guide. Alignments are available as fasta format in additional file 2. selected from the EST database, isolates of Wheat streak mosaic virus and the related Oat necrotic mottle virus (WSMV), members of the Tobamovirus genus and iso- lates of Tomato yellow leaf curl virus (TYLCV). Lists of the accession numbers used are presented in additional file 1. Sequences were aligned manually using Se-Al [17] with reference to the amino acid sequences encoded. Background In the case of tobamoviruses, previously constructed align- ments [5] were used as guide. Alignments are available as fasta format in additional file 2. overall substitution rates may speed up or slow down along selected lineages [13]. Phylogenetic applications [14,15] allow incorporation of these possibilities for var- iation into the models to be tested. One application, DAMBE, allows one to examine the relative rates of accumulation of transitions and transversions [16], but not to distinguish between transitions or among trans- versions. However, no phylogenetic model-testing appli- cations allow the individual substitution frequencies to vary relative to one another over the course of evolu- tion. Constancy is important particularly when phyloge- netic trees are constructed and dated using only recent dates as references, such as in BEAST [14]. The present study was undertaken to answer questions about varia- tion of the relative proportions of the different substitu- tion types over evolutionary time. Do interchanges noted between recently diverged sequence pairs have distributions of interchange types similar to those of dis- tantly diverged pairs? If they are not the same, do the relative rates of types of interchange change gradually or abruptly over divergence time? Does coding ability of a strand influence relative rates of types of interchanges? Are other factors involved? Analysis Under- populated bins were removed from consideration. For each interchange type the mean numbers of inter- changes per knt in each bin were plotted as a function of t-distance. For each each t-distance bin, the standard deviations were also calculated for each interchange type and plotted as half error bars. Microsoft Excel, through its “solver” tool, was used also to estimate the goodness of fit of observed values to a hyperbolic equa- tion [19] Methods Figs. 1, 2, 3, 4, 5, 6, show the accumulation of nucleo- tide interchanges between pairs of sequences as a func- tion of the divergence t-distance between the sequences. Absent other considerations, the relationships should be hyperbolic with an initial nearly linear phase followed by a leveling-off as previously interchanged sites undergo additional substitutions. Datasets Six datasets were assembled from sequences available in GenBank/EMBL/DDBJ for coding regions for the large subunit of ribulose bisphosphate carboxylase (rbcL) from red algae, the small subunit of the same enzyme (rbcS) from cereals, reverse transcriptase (RT) coding sequences from retroelements of a diversity of sources Melcher BMC Bioinformatics 2010, 11(Suppl 6):S3 http://www.biomedcentral.com/1471-2105/11/S6/S3 Page 3 of 6 0 20 40 60 80 100 120 0 0.2 0.4 0.6 0.8 1 1.2 1.4 t-Distance GA TA CG TG CA TC Figure 1 Nucleotide substitution profile of tobamoviral genomes. Levels of types of nucleotide interchanges between pairs of tobamoviral genome sequences at varying levels of divergence (t-distance). Half error bars are standard deviations 0 20 40 60 80 100 120 0 0.2 0.4 0.6 0.8 1 1.2 1.4 t-Distance GA TA CG TG CA TC 0 10 20 30 40 50 60 70 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 t-Distance TC GA TA CG TG CA Figure 3 Nucleotide substitution profile of Tomato yellow leaf curl virus genomes See Figure 1 legend for details. Figure 1 Nucleotide substitution profile of tobamoviral genomes. Levels of types of nucleotide interchanges between pairs of tobamoviral genome sequences at varying levels of divergence (t-distance). Half error bars are standard deviations Figure 3 Nucleotide substitution profile of Tomato yellow leaf curl virus genomes See Figure 1 legend for details. The Tobamovirus data set was chosen to focus on speciation events. For comparison, multiple isolates of the same virus species were tested for two viruses (Figs. 2 and 3). Limitation of the dataset to members of the same species resulted in a narrowing of the range of t- distances plotted. For isolates of WSMV (Fig. 2), transi- tions accumulated eight times as rapidly as transversions in the initial period of divergence. Indeed, the transition accumulation curves appeared non-hyperbolic, being characterized by an apparent lag phase. C<>G inter- changes were rare during this period. On further accu- mulation of substitutions, the T<>C interchanges appeared to plateau after more limited divergence times than did G<>A interchanges. Between t-distances of 0.12 and 0.28, a significantly anomalous transient shift in interchange frequency occurred for C<>A interchanges. Viral interchange accumulation profiles For the dataset of species from the Tobamovirus genus (Fig. 1), transitions accumulated 3.5 times as rapidly as transversions in the initial period of divergence. On further accumulation of substitutions during divergence, the T<>C interchanges appeared to plateau at a lower level than G<>A interchanges. Transitions and transver- sions had similar rates of accumulation during this per- iod of divergence. At the largest divergence distances, the number of T<>A transversion interchanges could not be distinguished from the number of T<>C transi- tion interchanges. Not surprisingly, the complementary T<>G and C<>A interchanges had practically indistin- guishable accumulation profiles, while C<>G inter- changes were least frequent. Between t-distances of 0.45 and 0.50, significant anomalous shifts in interchange fre- quency occurred for T<>A, G<>A and T<>C interchanges. WSMV is a virus with a single-stranded positive sense RNA genome. To contrast with WSMV, TYLCV, a virus with a circular ambisense DNA molecule as genome, 0 20 40 60 80 100 120 140 0 0.2 0.4 0.6 0.8 1 1.2 t-Distance GA TA CG TG CA TC Figure 2 Nucleotide substitution profile of Wheat streak mosaic virus genomes See Figure 1 legend for details. 0 25 50 75 100 125 150 175 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 t-distance TC GA TA CG TG CA Figure 4 Nucleotide substitution profile of retroelements of diverse eucaryotes See Figure 1 legend for details. 0 20 40 60 80 100 120 140 0 0.2 0.4 0.6 0.8 1 1.2 t-Distance GA TA CG TG CA TC Figure 2 Nucleotide substitution profile of Wheat streak mosaic virus genomes See Figure 1 legend for details. 0 20 40 60 80 100 120 140 0 0.2 0.4 0.6 0.8 1 1.2 t-Distance GA TA CG TG CA TC 0 25 50 75 100 125 150 175 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 t-distance TC GA TA CG TG CA Figure 4 Nucleotide substitution profile of retroelements of diverse eucaryotes See Figure 1 legend for details. 0 25 50 75 100 125 150 175 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 t-distance TC GA TA CG TG CA Figure 4 Nucleotide substitution profile of retroelements of diverse eucaryotes See Figure 1 legend for details. Figure 2 Nucleotide substitution profile of Wheat streak mosaic virus genomes See Figure 1 legend for details. Viral interchange accumulation profiles Figure 4 Nucleotide substitution profile of retroelements of diverse eucaryotes See Figure 1 legend for details. Melcher BMC Bioinformatics 2010, 11(Suppl 6):S3 http://www.biomedcentral.com/1471-2105/11/S6/S3 Page 4 of 6 pressure (Fig. 4). With the exception of non-significant fluctuations at high divergence levels, the accumulation profiles of the different types of interchanges conformed to the expected hyperbolic pattern. Correlation coeffi- cients ranged from 0.988 for T<>C to 0.997 for C<>G interchanges. T<>A transversion interchanges joined the two transitions (T<>C and G<>A) as the most frequent type of substitution in this dataset. As with the viral profiles, C<>G interchanges accumulated to the smallest extent. 0 5 10 15 20 25 30 35 40 45 50 0.00 0.05 0.10 0.15 0.20 0.25 0.30 t-Distance GA TA CG TG CA TC Figure 5 Nucleotide substitution profile of RbcS coding regions from cereals See Figure 1 legend for details. As additional controls, datasets of coding regions for plastid and nuclear subunits of ribulose-bisphosphate carboxylase (RbcL and RbcS, respectively) were also examined. As for viral sequences, initial interchanges between recently diverged pairs of RbcS from cereals (Fig. 5) were 4.7 times as likely to be transitions than to be transversions and G<>A transitions began to plateau at a lower level of divergence distance than did T<>C transitions. T<>G interchanges were the least frequent at all levels of divergence and significantly less frequent than the complementary C<>A interchange, except at the very shortest divergence distances. Figure 5 Nucleotide substitution profile of RbcS coding regions from cereals See Figure 1 legend for details. was chosen (Fig. 3). Despite the virus’ DNA genome, mutation rates, both inferred [20] and experimentally estimated [21], have been reported to be of the order of those for RNA viruses. Lag phases were apparent for several inerchange types. T<>C interchange accumula- tion was more prominent than others, including G<>A interchanges, in the initial period of divergence. Indeed, accumulation of G<>A transition interchanges resembled closely that of T<>A transversions. C<>G and C<>A transversion interchanges accumulated much less rapidly than transversions involving T. Although C<>G interchanges appeared to have plateaued between 0.19 and 0.21 t-distance units, additional such inter- changes were noted in more diverged pairs. For RbcL coding regions from algae (Fig. 6), T<>C transition and T<>A transversion interchanges were the most prominent throughout the period of divergence, with T<>C exceeding T<>A except in the most diverged pairs. Viral interchange accumulation profiles At the highest divergence levels these two accounted for four-fold as many interchanges as for the other four combined. As with the viral profiles, C<>G interchanges accumulated to the smallest extent. An apparent anomalous change occurred between 0.11 and 0.12 t-distance units where C<>A transversion inter- changes were more prominent among distant pairs than with less distant pairs. This transition appeared to have been compensated by fewer T<>C interchanges at greater distances. Non-viral interchange accumulation profiles The reverse transcriptase coding regions of nuclear ret- roelements from a variety of eukaryotes were assembled into a dataset to provide a highly diverged protein-cod- ing gene set that may be under reduced selective Discussion The interchanges summarized in this work are repre- sented as X<>Y to make clear that no attempt has been made to determine the direction of substitutions, whether X changed to Y or Y changed to X. Such deter- mination requires confident knowledge of ancestral sequences. This knowledge was lacking in most instances. Trial runs using a consensus sequence as common ancestor resulted in separation of the six curves into twelve curves. Often the reciprocal exchange values were widely different. Investigation into mechan- isms responsible for anomalies and differences will need to analyze the full set of 12 types. However, such separation was not needed to observe the existence of anomalies. Of greatest relevance to the issue of dating viral phy- logenetic trees is the nature of the curves representing the accumulation of substitutions over evolutionary time. Fig. 4 shows that, for a dataset where selective pressures are likely to have been slight during evolu- tion, the curves are very consistent with the expected hyperbolic relationships. The other datasets generated graphs with irregular appearances, including lag phases and abrupt changes in slope. It is intriguing that these slope changes appear to occur at specific divergence levels. That they may represent substitutions associated with speciation events merits investigation. They are likely associated with a shift in base composition. The apparent lag phases are likely the result of a non-linear relationship between t-distance and time. Between recently diverged pairs some types of interchanges (usually transitions) happen frequently at a limited number of sites. These interchanges occur over a small time span thus artificially expanding the x-axis at early “times” of divergence and resulting in apparent lag phases. This interpretation is consistent with the view that substitution frequencies are much higher for intrastrain divergence than for interspecies divergence. Thus, it may not be valid to apply substitution fre- quencies calculated from recently diverged pairs to evolution of species. If substitutions were to occur with equal frequency on coding and non-coding strands, one would expect that, because of complementarity, T<>A and C<>G inter- changes would be detected equally frequently as would C<>A and T<>G interchanges. T<>A exchanges were observed to be prominent in the tobamovirus, retroele- ment and TYLCV datasets, while C<>G interchanges were minor in all but the RbcS dataset. On the other hand, C<>A and T<>G interchange levels had practically indistinguishable accumulation profiles in the tobamo- virus dataset. Interchange comparisons Table 1 compares the percentages of the total inter- changes represented by each of the six types of inter- change, obtained for the data sets analyzed in this study at a t-distance of 0.10, with two classic sets of data, one on globin genes [22] and one on pseudogenes [23]. A t- distance of 0.1 was chosen for the comparison to avoid influence of highly frequent mutations (t-distance <0.5) and the influence of substitution saturation seen at higher t-distances. The classic percentages had been summed over all distances, but nonetheless provide a useful comparison. For the pseudogenes, presumably subject to relaxed evolutionary constraints on substitu- tions, only G<>A transition interchanges were notice- ably higher than other types. G<>A interchanges were not the predominant type for five of the six datasets analyzed in the current study. The exception was the 0 10 20 30 40 50 60 0 0.03 0.06 0.09 0.12 0.15 0.18 0.21 t-Distance TC GA TA CA CG TG Figure 6 Nucleotide substitution profile of RbcL coding regions from red algae See Figure 1 legend for details. 0 10 20 30 40 50 60 0 0.03 0.06 0.09 0.12 0.15 0.18 0.21 t-Distance TC GA TA CA CG TG Figure 6 Nucleotide substitution profile of RbcL coding regions from red algae See Figure 1 legend for details. Melcher BMC Bioinformatics 2010, 11(Suppl 6):S3 http://www.biomedcentral.com/1471-2105/11/S6/S3 Page 5 of 6 Table 1 Substitution Profiles of Selected Viral and Non-viral Genes Substitution Globina Pseudogenesb rbcLc Tobamov.c rbcSc Retroel.c TYLCVc WSMVc G <> A 32.4 30.1 10.7 20.0 25.0 18.6 21.7 38.0 T <> C 13.1 9.2 42.9 18.2 31.8 19.7 26.4 40.4 A <> T 9.2 9.1 36.2 18.3 9.2 22.8 19.8 7.6 A <> C 14.5 11.5 2.0 15.1 12.8 14.4 9.6 5.3 G <> C 24.5 9.5 2.1 12.2 16.2 8.1 7.2 1.4 G <> T 6.5 10.5 6.1 16.2 5.0 16.4 15.2 7.4 a [1] b [2] c At t-distance = 0.10; see Figs. 1, 2, 3, 4, 5, 6. Table 1 Substitution Profiles of Selected Viral and Non-viral Genes had this feature while for the TYLCV only T<>C inter- changes showed this property. The same transitions played a much diminished role in pairs that diverged from one another over a longer period of time. Interchange comparisons Similar early spurts of T<>C interchanges occurred also in evo- lution of both Rbc coding regions, with G<>A inter- changes also being prominent early in RbcS. The result suggests that there are subsets of sites that can readily tolerate transitions and that substitutions at these sites approach saturation after only short periods of evolu- tion. If the suggestion is correct, then such changes in substitution frequency need to be taken into account when calibrating phylogenetic trees to determine dates of divergence. Initial rates of divergence may be sub- stantially higher than those after saturation of these sites has been reached. tobamoviral sequences where the G<>A percentage was slightly higher than that for T<>C. G<>C interchanges were relatively rare in all datasets except for globin and the RbcS ones. In globin genes their percentage was sec- ond only to the G<>A transition interchange. The com- plementary substitution pair A<>C and G<>T had similar percentages in pseudogene, retroelement and Tobamovirus datasets, but were biased in the other sub- stitution classes. tobamoviral sequences where the G<>A percentage was slightly higher than that for T<>C. G<>C interchanges were relatively rare in all datasets except for globin and the RbcS ones. In globin genes their percentage was sec- ond only to the G<>A transition interchange. The com- plementary substitution pair A<>C and G<>T had similar percentages in pseudogene, retroelement and Tobamovirus datasets, but were biased in the other sub- stitution classes. Discussion This was also true for retroelements, RbcL and, at low to moderate divergence levels, for WSMV, but not for TYLCV or RbcS. In the viral datasets, among sequence pairs that had undergone only limited divergence, one or both transi- tion interchanges were by far the most predominant type. For tobamoviruses and WSMV both transitions Melcher BMC Bioinformatics 2010, 11(Suppl 6):S3 http://www.biomedcentral.com/1471-2105/11/S6/S3 Page 6 of 6 9. Ramsden C, Holmes EC, Charleston MA: Hantavirus evolution in relation to its rodent and insectivore hosts: no evidence for co-divergence. Mol Biol Evol 2008, 26:143-153. Conclusions Additional file 1: Accession numbers of sequences usedAccession and gi numbers of sequences used in the study 15. Pond SLK, Frost SDW, Muse SV: HyPhy: hypothesis testing using phylogenies. Bioinformatics 2005, 21:676-679. 16. Xia X, Xie Z: Tetrapod phylogeny and data exploration using DAMBE. The Phylogenetic Handbook Cambridge: Cambridge University PressSalemi M, Vandamme A-M 2003. Additional file 2: Multiple sequence alignmentsFile, when unzipped, consists of a folder with aligned fasta files for each of the six datasets employed in this study. 17. Se-Al. [http://tree.bio.ed.ac.uk/software/seal/]. Additional file 3: Source code for pairwise comparisonCode, when compiled in Future Basic, which was used to calculate numbers of interchanges of each type for pairs of aligned sequences. Output was input to Microsoft Excel for further processing. 18. Jukes TH, Cantor CR: Evolution of protein molecules. Mammalian Protein Metabolism New York: Academic PressMunro MN 1969, III. 18. Jukes TH, Cantor CR: Evolution of protein molecules. Mammalian Protein Metabolism New York: Academic PressMunro MN 1969, III. 19. John EG: Simplified curve fitting using spreadsheet add-ins*. Int J Engng Ed 1998, 14:375-380. 20. Duffy S, Holmes EC: Phylogenetic evidence for rapid rates of molecular evolution in the single-stranded DNA begomovirus tomato yellow leaf curl virus. J Virol 2008, 82:957-965. 21. Ge L, Zhang J, Zhou X, Li H: Genetic structure and population variability of tomato yellow leaf curl china virus. J Virol 2007, 81:5902-5907. Acknowledgements The author acknowledges National Science Foundation support of the Plant Virus Ecology Network (IOS-0639139), whose discussions contributed to the background for the work reported and support of the Oklahoma Agricultural Experiment Station whose Director has approved the manuscript for publication. y , 22. Gojobori T, Li WH, Graur D: Patterns of nucleotide substitution in pseudogenes and functional genes. J Mol Evol 1982, 18:360-369. 23. Li WH, Wu CI, Luo CC: Nonrandomness of point mutation as reflected in nucleotide substitutions in pseudogenes and its evolutionary implications. J Mol Evol 1984, 21:58-71. This article has been published as part of BMC Bioinformatics Volume 11 Supplement 6, 2010: Proceedings of the Seventh Annual MCBIOS This article has been published as part of BMC Bioinformatics Volume 11 Supplement 6, 2010: Proceedings of the Seventh Annual MCBIOS Conference. Bioinformatics: Systems, Biology, Informatics and Computation. The full contents of the supplement are available online at http://www.biomedcentral.com/1471-2105/11?issue=S6. doi:10.1186/1471-2105-11-S6-S3 Cite this article as: Melcher: Assessing constancy of substitution rates in viruses over evolutionary time.. BMC Bioinformatics 2010 11(Suppl 6):S3. doi:10.1186/1471-2105-11-S6-S3 Cite this article as: Melcher: Assessing constancy of substitution rates in viruses over evolutionary time.. BMC Bioinformatics 2010 11(Suppl 6):S3. Conference. Bioinformatics: Systems, Biology, Informatics and Computation. The full contents of the supplement are available online at http://www.biomedcentral.com/1471-2105/11?issue=S6. Competing interests The author declares that he has no competing interests. Conclusions These observations suggest strongly that the probabil- ities of particular substitutions are different at different stages of evolution. Thus, a single probability applied to an entire dataset, such as those analyzed for Figs. 1, 2, 3 and 5, 6 should produce unreliable results in phyloge- netic analysis. This study therefore supports the conclu- sion [10] that different evolutionary rules may apply to recent divergences than apply to divergences that give rise to speciation and similar distant past events. 10. Gibbs AJ, Fargette D, Garcia-Arenal F, Gibbs MJ: Time–the emerging dimension of plant virus studies. J Gen Virol 2010, 91:13-22. 11. Swofford DL, Olsen GJ, Waddell PJ, Hillis DM: Phylogenetic inference. Molecular Systematics Sunderland, Masssachusetts: Sinauer AssociatesHillis DM Moritz C, Mable BK , 2 1996, 407-514. 12. Gojobori T, Ishii K, Nei M: Estimation of average number of nucleotide substitutions when the rate of substitution varies with nucleotide. J Mol Evol 1982, 18:414-423. 12. Gojobori T, Ishii K, Nei M: Estimation of average number of nucleotide substitutions when the rate of substitution varies with nucleotide. J Mol Evol 1982, 18:414-423. 13. Thorne JL, Kishino H, Felsenstein J: An evolutionary model of maximum likelihood alignment of DNA sequences. J Mol Evol 1991, 33:114-124. g 14. Drummond AJ, Rambaut A: BEAST: Bayesian evolutionary analysis by sampling trees. BMC Evol Biol 2007, 7:214. 14. Drummond AJ, Rambaut A: BEAST: Bayesian evolutionary analysis by sampling trees. BMC Evol Biol 2007, 7:214. Additional file 1: Accession numbers of sequences usedAccession and gi numbers of sequences used in the study Additional file 2: Multiple sequence alignmentsFile, when unzipped, consists of a folder with aligned fasta files for each of the six datasets employed in this study. Additional file 3: Source code for pairwise comparisonCode, when compiled in Future Basic, which was used to calculate numbers of interchanges of each type for pairs of aligned sequences. Output was input to Microsoft Excel for further processing. Additional file 1: Accession numbers of sequences usedAccession and gi numbers of sequences used in the study Additional file 2: Multiple sequence alignmentsFile, when unzipped, consists of a folder with aligned fasta files for each of the six datasets employed in this study. Additional file 3: Source code for pairwise comparisonCode, when compiled in Future Basic, which was used to calculate numbers of interchanges of each type for pairs of aligned sequences. Output was input to Microsoft Excel for further processing. Additional file 1: Accession numbers of sequences usedAccession and gi numbers of sequences used in the study Additional file 2: Multiple sequence alignmentsFile, when unzipped, consists of a folder with aligned fasta files for each of the six datasets employed in this study. Additional file 3: Source code for pairwise comparisonCode, when compiled in Future Basic, which was used to calculate numbers of interchanges of each type for pairs of aligned sequences. Output was input to Microsoft Excel for further processing. References 1. Duffy S, Shackelton LA, Holmes EC: Rates of evolutionary change in viruses: patterns and determinants. Nat Rev Genet 2008, 9:267-276. 1. Duffy S, Shackelton LA, Holmes EC: Rates of evolutionary change in viruses: patterns and determinants. Nat Rev Genet 2008, 9:267-276. 1. Duffy S, Shackelton LA, Holmes EC: Rates of evolutionary change in viruses: patterns and determinants. Nat Rev Genet 2008, 9:267-276. 2. Harkins G, Delport W, Duffy S, Wood N, Monjane A, Owor B, Donaldson L, Saumtally S, Triton G, Briddon R, et al: Experimental evidence indicating that mastreviruses probably did not co-diverge with their hosts. Virol J 2009, 6:104. 3. Gibbs A: Evolution and origins of tobamoviruses. Philos Trans R Soc Lond B Biol Sci 1999, 354:593-602. 3. Gibbs A: Evolution and origins of tobamoviruses. Philos Trans R Soc Lond B Biol Sci 1999, 354:593-602. 4. Gibbs A, Po T, Liang-yi K, Ying-chun T, Randles J: Classification of several tobamoviruses isolated in China on the basis of the amino acid composition of their virion proteins. Intervirology 1982, 18:160-163. 4. Gibbs A, Po T, Liang-yi K, Ying-chun T, Randles J: Classification of several tobamoviruses isolated in China on the basis of the amino acid composition of their virion proteins. Intervirology 1982, 18:160-163. 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 y 5. Lartey RT, Voss TC, Melcher U: Tobamovirus evolution: gene overlaps, recombination, and taxonomic implications. Mol Biol Evol 1996, 13:1327-1338. Submit your next manuscript to BioMed Central and take full advantage of: 6. Kang HJ, Bennett SN, Sumibcay L, Arai S, Hope AG, Mocz G, Song JW, Cook JA, Yanagihara R: Evolutionary insights from a genetically divergent hantavirus harbored by the European common mole (Talpa europaea). PLoS One 2009, 4:e6149. 7. Wu B, Melcher U, Guo X, Wang X, Fan L, Zhou G: Assessment of codivergence of mastreviruses with their plant hosts. BMC Evol Biol 2008, 8:335. 8. Koonin EV, Wolf YI, Nagasaki K, Dolja VV: The complexity of the virus world. Nat Rev Microbiol 2009, 7:250. 8. Koonin EV, Wolf YI, Nagasaki K, Dolja VV: The complexity of the virus world. Nat Rev Microbiol 2009, 7:250.
https://openalex.org/W3046197737	https://www.beilstein-journals.org/bjoc/content/pdf/1860-5397-16-205.pdf	English	null	A new method for the synthesis of diamantane by hydroisomerization of binor-S on treatment with sulfuric acid	Beilstein journal of organic chemistry	2,020	cc-by	3,529	Abstract A new method was developed for the direct synthesis of the second representative of the homologous series of diamond- like hydrocarbons, diamantane, in 65% yield by hydroisomerization of the norbornadiene dimer, endo-endo- heptacyclo[8.4.0.02,12.03,8.04,6.05,9.011,13]tetradecane (binor-S) on treatment with concentrated sulfuric acid (98%). In the presence of H2SO4 of lower concentration (75–80%), the reaction stops after the hydrogenation step giving endo-endo- pentacyclo[7.3.1.12,5.18,10.03,7]tetradecane in 68% yield with excellent selectivity (100%). Address: Received: 25 July 2020 Accepted: 01 October 2020 Published: 12 October 2020 Associate Editor: H. Ritter © 2020 Aminov and Khusnutdinov; licensee Beilstein-Institut. License and terms: see end of document. Rishat I. Aminov* - rishaminov@gmail.com * Corresponding author A new method for the synthesis of diamantane by hydroisomerization of binor-S on treatment with sulfuric acid Rishat I. Aminov* and Ravil I. Khusnutdinov Open Access Beilstein J. Org. Chem. 2020, 16, 2534–2539. https://doi.org/10.3762/bjoc.16.205 Received: 25 July 2020 Accepted: 01 October 2020 Published: 12 October 2020 Associate Editor: H. Ritter © 2020 Aminov and Khusnutdinov; licensee Beilstein-Institut. License and terms: see end of document. Full Research Paper Full Research Paper Address: Institute of Petrochemistry and Catalysis, Russian Academy of Sciences, pr. Oktyabrya 141, Ufa, 450075, Russian Federation Email: Rishat I. Aminov* - rishaminov@gmail.com * Corresponding author Keywords: binor-S; diamantane; hydroisomerization; sulfuric acid; tetrahydrobinor-S Beilstein J. Org. Chem. 2020, 16, 2534–2539. https://doi.org/10.3762/bjoc.16.205 Beilstein J. Org. Chem. 2020, 16, 2534–2539. https://doi.org/10.3762/bjoc.16.205 Introduction duced on an industrial scale (prepared by AlBr3 or AlCl3-in- duced skeletal isomerization of a petrochemical monomer, hydrogenated dicyclopentadiene) [1], have been studied rather extensively, the chemical behavior of diamantane, the second member of the diamandoid homologous series, has been poorly studied. The main cause of this situation is the lack of facile methods for its synthesis. Among the highly diverse polycyclic and cage compounds, an important place is occupied by diamond-like compounds called diamondoids, whose lower representatives belong to the homol- ogous series C4n+6H4n+12. Owing to the rigid structure, diamon- doids typically have high thermal stability and high reactivity compared with aliphatic and alicyclic saturated hydrocarbons and show peculiar chemical behavior. In the literature, diamantane (1) is prepared by skeletal isomerization of strained С14Н20 polycyclic hydrocarbons [2-7]. In particular, the most suitable initial compounds for the preparation of diamantane are three isomeric polycyclic hydrocarbons C14H20 3а–с, which are obtained Crude oil is known to be the main natural source of diamon- doids. In the oil and gas field exploration, the presence of diamondoids is used to evaluate the field maturity. Whereas the synthesis and chemical reactivity of adamantane, the first member of the diamondoid homologous series, which is pro- 2534 2534 Beilstein J. Org. Chem. 2020, 16, 2534–2539. ([2]/[H2SO4] = 1:10–50) during 7–15 h affords a mixture of endo-endo-pentacyclo[7.3.1.12,5.18,10]tetradecane (tetrahy- drobinor-S, 3c) and diamantane (1) (Table 1). An increase in the sulfuric acid ratio to binor-S (2) ([2]/[H2SO4] = 1:20–50) and rising the temperature to 40 °С lead to decreased product yield due to resinification. When the H2SO4 ratio to binor-S (2) is 1:5, the conversion of compound 2 decreases to 10%. On the other hand, when the reactions are carried out in CS2 or with- out any solvent, the selectivity to diamantane (1) increases to 100%, with the maximum yield being 65% (Table 1, entry 12). A portion of binor-S (2) is converted to resinous products. When the reaction was ultrasonically assisted, the reaction time decreased to 2 h with the yield of diamantane (1) being retained (62%). by hydrogenation of the norbornadiene dimer, hepta- cyclo[8.4.0.02,12.03,8.04,6.05,9.011,13]tetradecane (binor-S, 2). Binor-S is hydrogenated in the presence of a platinum catalyst (Н2PtCl6, PtO2) in glacial acetic acid under high pressure conditions at 70 °С and 200 psi of H2 [8,9]. Introduction In the presence of superacid catalysts, such as B(OSO2CF3)3, CF3SO3H/SbF5 1:1, CF3SO3H/B(OSO2CF3)3 1:1 [10], NaBH4/CF3SO3H [11], or zeolite Y in the NaH form (NaY) [12], hydrocarbons 3a–c isomerize to diamantane in up to 99% yield (Scheme 1). As can be seen from Scheme 1, the synthesis of diamantane (1) from binor-S (2) is a two-step process, in which the hydrogena- tion performed in the first step is most complex and has always been an obstacle to the generation of large amounts of diaman- tane. In view of the foregoing, we set ourselves the task to develop a one-pot method for the synthesis of diamantane (1) from binor-S (2). In order to answer the question of what is the hydrogen source in the hydroisomerization of binor-S (С14H16, 2) containing 4 hydrogen atoms less than diamantane (С14H20, 1), we carried out a series of control experiments using deuterated sulfuric acid (98%) in cyclohexane (С6H12, experiment A), in deuter- ated cyclohexane (C6D12, experiment B), or in carbon disulfide (CS2, experiment C). Results and Discussion In this study, we developed a new method for the synthesis of pentacyclo[7.3.1.14,12.02,7.06,11]tetradecane (diamantane, 1) by skeletal hydroisomerization of endo-endo-hepta- cyclo[8.4.0.02,12.03,8.04,6.05,9.011,13]tetradecane (binor-S, 2) on treatment with sulfuric acid (Scheme 2). In experiment А, the major isomer 1-D2, which is formed upon hydroisomerization of binor-S (2), contains two deuterium atoms. Two more hydrogen atoms are probably provided by cyclohexane. Unexpectedly, the reaction also gave undeuter- ated diamantane (1), which may be due to deuterium exchange with hydrogen of cyclohexane under the action of D2SO4. The reaction selectivity and the yield of diamantane (1) considerably depend on the reaction conditions and the solvent nature. Indeed, at 20–40 °C, hydroizomerization of binor-S (2) in cyclohexane in the presence of 98% sulfuric acid Scheme 1: Isomerization of 3а–с to diamantane (1). Reaction conditions: (a) CoBr2·2PPh3–BF3·OEt2, 110 °C, 12 h; (b) Pt, H2 (200 psi), 70 °C, 3 h; (c) superacidic catalysts or NaY–NaH. Scheme 1: Isomerization of 3а–с to diamantane (1). Reaction conditions: (a) CoBr2·2PPh3–BF3·OEt2, 110 °C, 12 h; (b) Pt, H2 (200 psi), 70 °C, 3 h; (c) superacidic catalysts or NaY–NaH Scheme 1: Isomerization of 3а–с to diamantane (1). Reaction conditions: (a) CoBr2·2PPh3–BF3·OEt2, 110 °C, 12 h; (b) Pt, H2 (200 psi), 70 °C, 3 h; (c) superacidic catalysts or NaY–NaH. Scheme 2: Isomerization of binor-S (2) to diamantane (1). Scheme 2: Isomerization of binor-S (2) to diamantane (1). Scheme 2: Isomerization of binor-S (2) to diamantane (1). 2535 Beilstein J. Org. Chem. 2020, 16, 2534–2539. Table 1: Hydroisomerization of binor-S (2) in the presence of sulfuric acid. entry ratio solvent temp. Results and Discussion [°C] time [h] product ratio [%]a [2]/[H2SO4] 2 3 1 1 1:50 cyclohexane 40 7 2 10 23 2 1:50 cyclohexane 20 7 3 26 10 3 1:20 cyclohexane 40 7 3 52 28 4 1:20 cyclohexane 20 7 12 46 22 5 1:20 cyclohexane 20 15 – 55 31 6 1:10 cyclohexane 20 7 22 41 36 7 1:10 cyclohexane 20 15 16 47 34 8 1:5 cyclohexane 40 15 56 31 2 9 1:20 carbon disulfide 20 7 21 – 36 10 1:20 carbon disulfide 20 15 15 – 44 11 1:10 carbon disulfide 20 7 24 – 52 12 1:10 carbon disulfide 20 15 – – 65 13 1:5 carbon disulfide 40 7 78 – 10 14 1:5 carbon disulfide 20 7 90 – – 15 1:10 – 20 7 – – 8 16b 1:10 cyclohexane 20 2 9 64 26 17b 1:10 carbon disulfide 20 2 – 18 62 18b 1:10 – 20 2 – – 6 aDetermined by GC using C12H26 as the internal standard. bThe reaction was conducted under ultrasonic irradiation. Table 1: Hydroisomerization of binor-S (2) in the presence of sulfuric acid. ble 1: Hydroisomerization of binor-S (2) in the presence of sulfuric ac aDetermined by GC using C12H26 as the internal standard. bThe reaction was conducted under ultrasonic irradiation. Scheme 3: Selective synthesis of tetrahydrobinor-S (3c) from binor-S (2). The major product 1-D3, which is formed in experiment B with D2SO4 in C6D12 contains three deuterium atoms. The expected isomer with four deuterium atoms is formed in a minor amount. Evidently, binor-S (2) acts as the hydrogen source for the isomer C14H17D3, 1-D3. Our attempt to carry out the deutera- tion of diamantane (1) with D2SO4 in carbon disulfide for 7 h at 20 °C was unsuccessful. Evidently, the deuterium exchange, re- sulting in the formation of diamantanes 1-D7 and 1-D8 contain- ing 7 and 8 deuterium atoms, occurs at the hydroisomerization step (experiment C). Scheme 3: Selective synthesis of tetrahydrobinor-S (3c) from binor-S (2). were unsuccessful, with the starting binor-S (2) being recov- ered unchanged. The reaction of hydrocarbon 2 with hydro- chloric acid proceeds with the addition of HCl to the cyclo- propane ring and results in the formation of a mixture of mono- and dichloro derivatives, the synthesis of which has been re- ported [13,14]. Results and Discussion When sulfuric acid is replaced by an ionic liquid prepared from triethylamine and sulfuric acid [15], the reaction follows a different route: Starting binor-S (2) is converted to two isomeric hexacyclic hydrocarbons, hexa- cyclo[8.4.0.02,7.03,14.04,8.09,13]tetradec-5-ene (4а) and hexa- cyclo[6.6.0.0.2,6.05,14.07,12.09,13]tetradec-3-ene (4b), which are important precursors for the synthesis of triamantane [10,11,16- 24] (Scheme 4). As shown by further studies, when the sulfuric acid concentra- tion decreases to 75–80%, the reaction stops at the intermediate step giving endo-endo-pentacyclo[7.3.1.12,5.18,10.03,7]tetrade- cane (tetrahydrobinor-S, 3с; Scheme 3). It should be empha- sized that the reaction selectively gives only one of the possible isomers, hydrocarbon 3с, which is confirmed by 1H and 13C NMR spectral data. The 13C NMR spectrum of compound 3с shows five characteristic carbon signals at 33.44, 35.64, 37.84, 38.30, and 40.49 ppm, coinciding with the reported values [13]. Since 75–80% H2SO4 contains 20–25% water, the participation of water as a hydrogen source in the reaction cannot be ruled out either. Experimental General procedures and materials: 1H and 13С NMR spectra were measured on a Bruker Avance-III 400 Ascend instrument (400 MHz for 1Н and 100 MHz for 13С in CDCl3). Mass spec- tra were run on a Shimadzu GCMS-QP2010Plus mass spec- trometer (SPB-5 capillary column, 30 m × 0.25 mm, helium as the carrier gas, temperature programming from 40 to 300 °С at 8 °C/min, evaporation temperature of 280 °С, ion source tem- perature of 200 °С, and ionization energy of 70 eV). The elemental composition of the samples was determined on a Carlo Erba 1106 elemental analyzer. The course of the reaction and the purity of the products were monitored by gas liquid chromatography on a Shimadzu GC-9A, GC-2014 instrument [2 m × 3 mm column, SE-30 silicone (5%) on Chromaton N-AW-HMDS as the stationary phase, temperature program- ming from 50 to 270 °С at 8 °C/min, helium as the carrier gas (47 mL/min)]. The sonication was carried out with an ultrasound generator IL10–0.63 (INLAB LTD) for 180 min at a frequency of 22 kHz with a submerged 15 mm diameter titanium horn, with output power 150 W. The reactions were carried out in a 100 × 35 mm glass reactor equipped with a jacket to maintain the required temperature (20 °C). Preparation of hexacyclo[8.4.0.02,7.03,14.04,8.09,13]tetradec- 5-ene (4a) and hexacyclo[6.6.0.0.2,6.05,14.07,12.09,13]tetradec- 3-ene (4b): Heptacyclo[8.4.0.02,12.03,8.04,6.05,9. 011,13]tetrade- cane (2, 0.368 g, 2 mmol) was charged into a glass reactor (V = 100 mL) and dissolved in cyclohexane. Then, [Et3NH]+[HSO4]− (1.99 g, 10 mmol) was added and the reaction mixture was stirred at 40 °С for 8 h. Then the reactor was cooled to room temperature, the reaction mixture extracted with petroleum ether, and filtered through a silica gel layer (with petroleum ether as the eluent). Hexa- cyclo[8.4.0.02,7.03,14.04,8.09,13]tetradec-5-ene (4а) and hexa- cyclo[6.6.0.0.2,6.05,14.07,12.09,13]tetradec-3-ene (4b) (45:55). Colorless oil; 78% yield; 4a: 1H NMR (400 MHz, CDCl3) δ 1.04 (d, J = 7.2 Hz, 2H), 1.41 (d, J = 7.6 Hz, 2H), 1.95 (s, 2H), 2.09 (d, J = 7.2 Hz, 4H), 2.21 (d, J = 7.2 Hz, 2H) 2.56 (s, 2H), 5.87 (s, 2H); 13С NMR (100 MHz, CDCl3) δ 26.27 (C11, C12), P r e p a r a t i o n o f d i a m a n t a n e : H e p t a - cyclo[8.4.0.02,12.03,8.04,6.05,9. Conclusion Thus, we developed a new one-pot method for the synthesis of diamantane (1) by hydroisomerization of binor-S (2) on treat- ment with concentrated sulfuric acid (98%) in carbon disulfide Attempts to perform hydroisomerization of binor-S (2) to diamantane (1) on treatment with nitric or orthophosphoric acid 2536 Beilstein J. Org. Chem. 2020, 16, 2534–2539. Scheme 4: Isomerization of binor-S (2) to hydrocarbons 4а and b. Scheme 4: Isomerization of binor-S (2) to hydrocarbons 4а and b. 1:1 ethyl acetate/cyclohexane mixture. The characteristic data and graphical spectra of diamantane are almost identical with the literature data [25]. or cyclohexane. It was found that both, sulfuric acid and cyclo- hexane can serve as the main hydrogen sources. In the presence of H2SO4 with a lower concentration (75–80%), the reaction stops at the step of formation of endo-endo-penta- cyclo[7.3.1.12,5.18,10.03,7]tetradecane (3c) in 68% yield. P r e p a r a t i o n o f e n d o - e n d o - p e n t a - cyclo[7.3.1.12,5.18,10.03,7]tetradecane (tetrahydrobinor-S, 3c): Heptacyclo[8.4.0.02,12.03,8.04,6.05,9. 011,13]tetradecane (2, 0.368 g, 2 mmol) was charged into a glass reactor (V = 100 mL) and dissolved in cyclohexane (10 mL). Then, 75–80% sulfuric acid (1.96 g, 20 mmol) was added in portions with vigorous stirring. When the whole amount of H2SO4 has been added, the reaction mixture was stirred at 20 °С for 7 h. After completion of the reaction, 10% NaOH was added to the reaction mixture, the organic part was separated, and filtered through a silica gel layer (with petroleum ether as the eluent). The sol- vent was distilled off and the residue was recrystallized from a 1:1 ethyl acetate/cyclohexane mixture. Colorless crystals; 68% yield; mp 104–106 °C; 1H NMR (400 MHz, CDCl3) δ 0.95–0.98 (m, 4H), 1.38 (s, 8H), 1.66–1.71 (m, 4H), 1.99–2.01 (m, 2H), 2.12–2.16 (m, 2H); 13С NMR (100 MHz, CDCl3) δ 33.42 (С6, С9, C13, C14), 35.63 (С1, С2, C7, C8), 37.82 (С5, С10), 38.27(С3, С12), 40.47 (С4, С11); EIMS (70 eV, m/z): 188 [M]+ (100), 187 (35), 159 (24), 145 (23), 131 (38), 117 (25), 105 (39), 91(82), 79 (57), 67 (29), 41 (47) %; Anal. calcd for C14H20: С, 89.29; H, 10.71; found: С, 89.14; H, 10.86. 21.Kafka, Z.; Vodicka, L. 21.Kafka, Z.; Vodicka, L. Preprint 23.Kafka, Z.; Vodicka, L. Sb. Vys. Sk. Chem.-Technol. Praze, D: Technol. Paliv 1986, 54, 65–74. 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Experimental 011,13]tetradecane (2, 0.368 g, 2 mmol) and the solvent were charged into a glass reactor (V = 100 mL). Then, concentrated (98%) sulfuric acid (1.96 g, 20 mmol) was added in portions with vigorous stirring. When the whole amount of H2SO4 has been added, the reaction mix- ture was stirred at 20 °С for 15 h. After completion of the reaction, 10% NaOH was added to the reaction mixture, the organic phase was separated, and filtered through a silica gel layer (with petroleum ether as the eluent). The sol- vent was distilled off and the residue was recrystallized from a 2537 Beilstein J. Org. 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Funding The results were obtained with the financial support of the Russian Ministry of Education and Science (project no. 2019- 05-595-000-058) on unique equipment at the 'Agidel' Collec- tive Usage Center (Ufa Federal Research Center, Russian Academy of Sciences), by the Scholarship of the President of the Russian Federation to young scientists and postgraduates (SP-1601.2018.1) and carried out within the RF state assign- ment, reg. no. АААА-А19-119022290009-3. Sb. Vys. Sk. Chem.-Technol. Praze, D: Technol. Paliv 1985, 51, 247–255. Sb. Vys. Sk. Chem.-Technol. Praze, D: Technol. Paliv 1985, 51, 247–255. 20.Kafka, Z. Sb. Vys. Sk. Chem.-Technol. Praze, D: Technol. Paliv 1991, 59, 79–90. 20.Kafka, Z. Sb. Vys. Sk. Chem.-Technol. Praze, D: Technol. Paliv 1991, 59, 79–90. References 536–538. doi:10.1016/j.tetlet.2014.12.006 –538. doi:10.1016/j.tetlet.2014.12.006 1. Murray, R. K.; Morgan, T. K.; Babiak, K. A. J. Org. Chem. 1975, 40, 1079–1083. doi:10.1021/jo00896a019 5.Aminov, R. 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Please note that the reuse, redistribution and reproduction in particular requires that the authors and source are credited. The license is subject to the Beilstein Journal of Organic Chemistry terms and conditions: (https://www.beilstein-journals.org/bjoc) The definitive version of this article is the electronic one which can be found at: https://doi.org/10.3762/bjoc.16.205 License and Terms This is an Open Access article under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0). Please note that the reuse, redistribution and reproduction in particular requires that the authors and source are credited. The license is subject to the Beilstein Journal of Organic Chemistry terms and conditions: (https://www.beilstein-journals.org/bjoc) The definitive version of this article is the electronic one which can be found at: https://doi.org/10.3762/bjoc.16.205 License and Terms 2539
https://openalex.org/W4380287867	https://zenodo.org/records/8027188/files/BioValue_Transformation-Action-Workshop-I-Final-Report_compressed.pdf	English	null	Transformation Action Workshop I Milestone 2 - Transformation Dynamics	Zenodo (CERN European Organization for Nuclear Research)	2,023	cc-by	3,550	Transformation Action Workshop I Milestone 2 Transformation Dynamics ransformation Action Workshop I estone 2 ransformation Dynamics hnical erences Project Acronym BioValue Project Full Name Biodiversity value in Spatial Planning Leveraging Multi-Level and Transformative Change Project ID 101060790 Milestone ID M_02_4.2 (Version 1) Milestone Type TAW I Report Lead Partner IST Author(s) Margarida B. Monteiro (IST-ID) Karla E. Locher-Krause (UFZ) Contributor(s) Enzo Falco (UniTrento), Jenny Schmidt (CoKnow), Matteo Marchese (Comune di Trento), Sofia santos (Municipio de Mafra), Yuanzao Zhu (UFZ) Date 12 May 2023 s Project Acronym BioValue Project Full Name Biodiversity value in Spatial Planning Leveraging Multi-Level and Transformative Change Project ID 101060790 Milestone ID M_02_4.2 (Version 1) Milestone Type TAW I Report Lead Partner IST Author(s) Margarida B. Monteiro (IST-ID) Karla E. Locher-Krause (UFZ) Contributor(s) Enzo Falco (UniTrento), Jenny Schmidt (CoKnow), Matteo Marchese (Comune di Trento), Sofia santos (Municipio de Mafra), Yuanzao Zhu (UFZ) Date 12 May 2023 TAW I Objectives Specific objectives: ▪Identification of current activities, interventions and practices that impact biodiversity ▪Identification of current activities, interventions and practices that impact biodiversity ▪Outline considering subsystems and what needs to be phased in and out, and how this change can be supported ▪Identify the current state in transition process T d l b h X C i l l f li i ▪Outline considering subsystems and what needs to be phased in and out, and how this change can be supported ▪Identify the current state in transition process Timing & Location: Executive Summary This report delivers the results of the 1st Transformation Action Workshop (TAW) that had the objective of a first understanding and exploration of the arenas4transf processes of transition, and its dynamics, supported by BioValue. Despite international and European policies in place to halt biodiversity loss, the effect of multi-level, and multi-sector, direct and indirect drivers of change contribute to continuing negative trends. As biodiversity is impacted by many different sectors, the main challenge consists in balancing a wide range of interests and value systems across different political levels, negotiating different interests while ultimately seeking to improve, or at least maintain, biodiversity. The TAWs are a series of spaces of collective thinking to co- create action-oriented knowledge and transformative pathways throughout the arena4transf processes. Specific objectives of theTAWs are: The main goal of BioValue is to safeguard and enhance biodiversity through transformative change in spatial policymaking, planning practices and infrastructure development, upscaling opportunities for valuing biodiversity in support of EU strategic actions on biodiversity, in particular the EU Biodiversity Strategy 2030. To address this, BioValue adopts three complementary instrumental perspectives relevant to spatial planning processes: spatial planning and management instruments (SP&MI), environmental assessment instruments (EAI), and economic and financial instruments (E&FI). i. Support the structure of the transformation processes of the arenas4transf ii. Formulate needs and opportunities iii. Help co-creation and discussion among the arenas4transf iv. Facilitate knowledge brokerage between the arenas4transf v. Advance improvements for transformation of joint application of the three instrumental perspectives The instrumental perspectives will support the structuring of the research in three case studies (in Portugal, Italy, and Germany) to explore and experiment BioValue research frameworks with stakeholders in action. The case studies will work as arenas for transformation (arenas4transf), as ‘experimental’ areas of the capacity of the three instruments to create transformative change for biodiversity value enhancement. These cases represent distinct spatial planning systems and cultures, as well as for scale and biodiversity- related situations. Three more TAWs are expected to occur, in BioValue months 15 (in Italy), 23 (in Germany), and 30 (in Portugal). Three more TAWs are expected to occur, in BioValue months 15 (in Italy), 23 (in Germany), and 30 (in Portugal). The TAW I report constitutes BioValue Milestone 2 and it is a project public resource. Transformation Action Workshop I [understand and explore the arenas4transf transition processes and its dynamics supported by BioValue] T f ti A ti W g March 7th 2023 from 9:00 to 12:30 (CEST), Aalborg University, Copenhagen, Denmark Description: g March 7th 2023 from 9:00 to 12:30 (CEST), Aalborg University, Copenhagen, Denmark Description: This workshop takes the form of a discussion within each arena4transf context in order to map the dynamics of the local system aimed at change and worked as the first step of the arenas4transf to prioritise interventions addressing transformative change in spatial policymaking, planning practices and infrastructures development to upscale opportunities for valuing biodiversity. It also worked as a testing momentum for the workshop approach, and to capacitate the arenas4transf partners to learn about the workshop approach and be able to replicate it in their contexts. This workshop takes the form of a discussion within each arena4transf context in order to map the dynamics of the local system aimed at change and worked as the first step of the arenas4transf to prioritise interventions addressing transformative change in spatial policymaking, planning practices and infrastructures development to upscale opportunities for valuing biodiversity. It also worked as a testing momentum for the workshop approach, and to capacitate the arenas4transf partners to learn about the workshop approach and be able to replicate it in their contexts. MV MAFRA FERSINA RIVER Observe and facilitate mainstreaming of biodiversity in rewetting as a policy option for the Mecklenburg- Vorpommern (MV) under the Climate Act. Consider the multi-level aspects of planning while bringing together different actors from different sectors of society in the co-creation of the desirable future of the peatlands. Promote a planning system in Mafra that is focused on protecting and valuing biodiversity and natural values beyond current legislations/regulations, while recognizing the high touristic pressure. Consider natural heritage, ecological structure and green infrastructure in the next planning cycle. Promote a planning system that incorporates the principles of ecological transition into the Fersina River, while recognizing the diversity of territorial characteristics. Support the development of a coding system to include the protection of biodiversity into territorial development. Observe and facilitate mainstreaming of biodiversity in rewetting as a policy option for the Mecklenburg- Vorpommern (MV) under the Climate Act. Consider the multi-level aspects of planning while bringing together different actors from different sectors of society in the co-creation of the desirable future of the peatlands. MAFRA Promote a planning system in Mafra that is focused on protecting and valuing biodiversity and natural values beyond current legislations/regulations, while recognizing the high touristic pressure. g March 7th 2023 from 9:00 to 12:30 (CEST), Aalborg University, Copenhagen, Denmark Description: Consider natural heritage, ecological structure and green infrastructure in the next planning cycle. FERSINA RIVER Promote a planning system that incorporates the principles of ecological transition into the Fersina River, while recognizing the diversity of territorial characteristics. Support the development of a coding system to include the protection of biodiversity into territorial development. FERSINA RIVER TAW I Structure purpose: Use of the X-Curve1 as the support tool to map the dynamics of the system and to talk about change duration: 3h30min activities: y y g duration: 3h30min activities: ▪1: Identification of activities, interventions and practices that impact biodiversity (arena) ~2h ▪2: Reflection of the mapped dynamics (plenary) ~45min ▪3: How to move forward – replication of the workshop (arena) ~15min [1 credit & source of inspiration:X-Curve booklet available in https://transitionshub.clima kic.org/publications/x-curve-a-sensmaking-tool-to-foster-collective-narratives-on-system-change [1 credit & source of inspiration:X-Curve booklet available in https://transitionshub.climate- kic.org/publications/x-curve-a-sensmaking-tool-to-foster-collective-narratives-on-system-change/ ] [1 credit & source of inspiration:X-Curve booklet available in https://transitionshub.climate- kic.org/publications/x-curve-a-sensmaking-tool-to-foster-collective-narratives-on-system-change/ ] activity 1: identification of activities, interventions and practices that impact biodiversity Based on the arenas4transf expected outcome, reflect on the dynamics of the system that you recognize the existence or see in your arena – e.g., activities/practices, resources, actors. Make sure the focus is on observed activities/practices and not possible (future) interventions ▪What needs to be organized/changed/adapted/modified? ▪What needs to be built/developed? ▪What is the end goal? ▪What stands out?What does it mean? activity 2: reflection of the mapped dynamics Overall discussion considering the arenas4transf results: ▪What are common or similar elements? ▪What deserves more attention based on the arenas4transf expected outcome? activity 3: how to move forward ▪What deserves more attention based on the arenas4transf expected outcome? ▪How can we replicate this workshop in our contexts? activity 1: identification of activities, interventions and practices that impact biodiversity Based on the arenas4transf expected outcome, reflect on the dynamics of the system that you recognize the existence or see in your arena – e.g., activities/practices, resources, actors. Make sure the focus is on observed activities/practices and not possible (future) interventions ▪What needs to be organized/changed/adapted/modified? ▪What needs to be built/developed? ▪What is the end goal? ▪What stands out?What does it mean? activity 2: reflection of the mapped dynamics Overall discussion considering the arenas4transf results: ▪What are common or similar elements? ▪What deserves more attention based on the arenas4transf expected outcome? activity 3: how to move forward ▪What deserves more attention based on the arenas4transf expected outcome? ▪How can we replicate this workshop in our contexts? Based on the arenas4transf expected outcome, reflect on the dynamics of the system that you recognize the existence or see in your arena – e.g., activities/practices, resources, actors. Make sure the focus is on observed activities/practices and not possible (future) interventions ▪What needs to be organized/changed/adapted/modified? ▪What needs to be built/developed? ▪What is the end goal? ▪What stands out?What does it mean? activity 2: reflection of the mapped dynamics Overall discussion considering the arenas4transf results: ▪What are common or similar elements? ▪What deserves more attention based on the arenas4transf expected outcome? ▪What deserves more attention based on the arenas4transf expected outcome? ▪How can we replicate this workshop in our contexts? ▪How can we replicate this workshop in our contexts? TAW I Structure - workgroups - ▪ Arena MV representative I ▪ Arena MV representative II (online) ▪ WP1 representative ▪ WP3 representative (online) ▪ WP4 representative ▪ Arena Mafra representative I ▪ Arena Mafra representative II ▪ WP1 representative ▪ WP3 representative ▪ Arena Fersina River representative I ▪ Arena Fersina River representative I ▪ WP1 representative ▪ WP2 representative ▪ WP3 representative s - ▪ Arena MV representative I ▪ Arena MV representative II (online) ▪ WP1 representative ▪ WP3 representative (online) ▪ WP4 representative ▪ Arena Mafra representative I ▪ Arena Mafra representative II ▪ WP1 representative ▪ WP3 representative ▪ Arena Fersina River representative I ▪ Arena Fersina River representative II ▪ WP1 representative ▪ WP2 representative ▪ WP3 representative ▪ Arena MV representative I ▪ Arena MV representative II (online) ▪ WP1 representative ▪ WP3 representative (online) ▪ WP4 representative ▪ Arena MV represen ▪ Arena MV represen ▪ WP1 representative ▪ WP3 representative ▪ WP4 representative ▪ Arena MV representative I ▪ Arena MV representative II (online) ▪ WP1 representative ▪ WP3 representative (online) ▪ WP4 representative ▪ Arena Mafra representative I ▪ Arena Mafra representative II ▪ WP1 representative ▪ WP3 representative ▪ Arena Fersina River representative I ▪ Arena Fersina River representative II ▪ WP1 representative ▪ WP2 representative ▪ WP3 representative ▪ Arena Fersina River representative I ▪ Arena Fersina River representative I ▪ WP1 representative ▪ WP2 representative ▪ WP3 representative TAW I Results - MV - EXPECTED OUTCOME MAPPED DYNAMICS X-CURVE Observe and facilitate mainstreaming of biodiversity in rewetting as Mecklenburg-Vorpommern (MV) under the Climate Act. Consider th planning while bringing together different actors from different secto creation of the desirable future of the peatlands. Land ownership (private, state) Income for land to farmers by state Flubereining Use it to create areas for rewetting? Explore possible ways within the existing laws Awareness raising & communication (add biodiv. activity 2: reflection of the mapped dynamics To facilitate planning process) CO2 market and certificates Action plan nature based climate protection (4bilion, ANK) Market for tourism (e.g., bird watching, beavers) Market for paludiculture Co-benefits for biodiversity conservation Observe and facilitate mainstreaming of biodiversity in rewetting as a policy option for the Mecklenburg-Vorpommern (MV) under the Climate Act. Consider the multi-level aspects of planning while bringing together different actors from different sectors of society in the co- creation of the desirable future of the peatlands. EXPECTED OUTCOME MAPPED DYNAMICS X-CURVE Land ownership (private, state) Income for land to farmers by state Flubereining Use it to create areas for rewetting? Explore possible ways within the existing laws Awareness raising & communication (add biodiv. To facilitate planning process) CO2 market and certificates Action plan nature based climate protection (4bilion, ANK) Market for tourism (e.g., bird watching, beavers) Market for paludiculture Co-benefits for biodiversity conservation Land ownership (private, state) Income for land to farmers by state MAPPED DYNAMICS X-CURVE Flubereining Use it to create areas for rewetting? Awareness raising & communication (add biodiv. To facilitate planning process) CO2 market and certificates Explore possible ways within the existing laws Market for tourism (e.g., bird watching, beavers) Co-benefits for biodiversity conservation Market for paludiculture REVISED EXPECTED OUTCOME Promote a planning system in Mafra that is focused on protecting and valuing biodiversity and nature beyond current legislations/regulations of the municipal ecological structure, while recognizing the high touristic pressure. Consider natural capital in the green infrastructure in the next planning cycle – built green infrastructure to adapt to the high touristic pressure. R MAPPED DYNAMICS X-CURVE Heritage network Collaboration with other municipalities Channeling the existing projects within the municipality Lack of support/ guidelines for GI implementation Different understandings of legislation and if it is working well or not Coordination & collaboration issues (planning, heritage, environment) High transaction costs in communication; different guidelines & requests Different dynamics (spatial planning always behind schedule) Lack of support/ guidelines for GI implementation MAPPED DYNAMICS X-CURVE Coordination & collaboration issues (planning, heritage, environment) Different understandings of legislation and if it is working well or not Collaboration with other municipalities REVISED EXPECTED OUTCOME Promote a planning system that incorporates the principles of ecological transition in the Fersina River, while recognizing the diversity of spatial characteristics. activity 2: reflection of the mapped dynamics How much can the federal state influence CAP funding pro/con peat soil rewetting? Vision for the future: The Green Finger Plan (built green infrastructure to adapt to the high touristic pressure) – to be confirmed/discussed in Mafra workshop. Vision for the future: The Green Finger Plan (built green infrastructure to adapt to the high touristic pressure) – to be confirmed/discussed in Mafra workshop. Need for citizens (landowners) to recognize the full value (economic and environment of green infrastructure). Need for citizens (landowners) to recognize the full value (economic and envir Spatial planning dynamics cannot keep pace with changing situations. p p g y p p g g ▪ Definition of “quality of life” and recognition of the value of land should be consistent between public and private actors. ▪ Definition of “quality of life” and recognition of the value of land should be consistent between public and private actors. Fersina River: ▪ Sense of place/belonging: more spaces for deliberation to raise people's awareness connection of people with the river is restricted by current regulations. ▪ Sense of place/belonging: more spaces for deliberation to raise people's awareness need to be created/developed. Also, connection of people with the river is restricted by current regulations. p p y g ▪ There are a lot of tools not making real impact. Exist to exist, are not being used. e are a lot of tools not making real impact. Exist to exist, are not being used. ▪ Promote a less strict regulation (water banks accessibility). ▪ Need for more flexible legislation?To promote relations p.e. ▪ How to cope with the competition of functions in the area of the river? E.g., Establishment of strategies to deal with the high land use competition. g p ▪ Establish options to decrease urban inequality (bypass). g p ▪ Establish options to decrease urban inequality (bypass). ish options to decrease urban inequality (bypass) ▪ Analyze new developments (e.g., infrastructure, scientific tools) and their potentiality (new hospital Trento, touristic routes- University,TUT biodiversity values) ▪ Integrate different initiatives and projects: PUMS sustainable mobility, underground bypass), identity connection with the river. ▪ Integrate different initiatives and projects: PUMS sustainable mobility, underground railway project (recovery found bypass), identity connection with the river. A d i h l l i ( f ) g p j y, g y p j ( y bypass), identity connection with the river. activity 2: reflection of the mapped dynamics Support the development of a coding system that incorporates biodiversity protection into spatial development by focusing on developing an intervention project on the Fersina River as a pilot project that integrates the implementation of biodiversity protection policies MAPPED DYNAMICS X-CURVE Urban inequality – bypass Local strict regulation – water banks acessibility Local perception (car use) PUP-PRG Responsibility, disconnection, communication Overcome legal perception related to land value Land use competivity (high land cost) Underground railway project (recovery fund-bypass) TUT valuation of biodiversity values – scientific tools - details PUMS – sustainable mobility Città e Il Fiume Identity connection with river Touristic routes Hydroeletric production (stakeholder) New Hospital Trento (stakeholder) Inter- administrative communication & cohesion Urban inequality – bypass PUP-PRG Responsibility, disconnection, communication MAPPED DYNAMICS X-CURVE Inter- administrative communication & cohesion Overcome legal perception related to land value TUT valuation of biodiversity values – scientific tools - details Underground railway project (recovery fund-bypass) Hydroeletric production (stakeholder) PUMS – sustainable mobility Città e Il Fiume Identity connection with river New Hospital Trento (stakeholder) MV: MV: TAW I Results - Reflections & questions per arena4transf - MV: ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ Mafra: ▪ ▪ ▪ ▪ Fersina R ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ Find priorisation areas for rewetting where negative impact is low. p g g p Bringing together actors with different knowledge (e.g., moor center, farmers, …). Bridging sectoral thinking and planning. Bringing together actors with different knowledge (e.g., moor center, farmers, …). Bridging sectoral thinking and planning. Bringing together actors with different knowledge (e.g., moor center, farmers, …). Bridging sectoral thinking and planning Market for Schwarzerle (land wood from peat soils). rket for Schwarzerle (land wood from peat soils). p Explore potential avenues for paludiculture (within the Common Agricultural policy - CAP) Initiatives to maintain measures underAktionsprogramm Natürlicher Klimaschutz (ANK). Explore potential avenues for paludiculture (within the Common Agricultural policy - CAP) Initiatives to maintain measures underAktionsprogramm Natürlicher Klimaschutz (ANK). Farmers as key actors: finding co-benefits for agrifood system. Are there any actions that could/should be taken to enhance benefits of rewetting for biodive made in a ‘nature-positive’ way? s that could/should be taken to enhance benefits of rewetting for biodiversity? Can rewetting b itive’ way? How much can the federal state influence CAP funding pro/con peat soil rewetting? How much can the federal state influence CAP funding pro/con peat soil rewetting? activity 2: reflection of the mapped dynamics ▪ As next step: promote and raise awareness to change local perceptions (e g for car use) bypass), identity connection with the river. ▪ As next step: promote and raise awareness to change local perceptions (e.g., for car use). bypass), identity connection with the river. ▪ As next step: promote and raise awareness to change local perceptions (e.g., for car use). yp ), y As next step: promote and raise awareness to change local perceptions (e.g., for car use). activity 2: reflection of the mapped dynamics activity 3: how to move forward TAW I Results -Global - Ownership Integrated valuation (economic, cultural, etc.) Absence of economic value of green land Ownership Integrated valuation (economic, cultural, etc.) Absence of economic value of green land Ownership Integrated valuation (economic, cultural, etc.) Absence of economic value of green land Absence of economic value of green land Ownership Integrated valuation (economic, cultural, etc.) activity 2: reflection of the mapped dynamics activity 3: how to move forward TAW I Results -Global - There were six main aspects that were mentioned by the three arenas, and the main message can be expressed as: ▪ Perceptions: importance of understanding the different perceptions at stake, working towards change in promoting shared thinking about the importance of biodiversity for territorial development and spatial planning systems. ▪ Perceptions: importance of understanding the different perceptions at stake, working towards change in promoting shared thinking about the importance of biodiversity for territorial development and spatial planning systems. ▪ Regulations: current regulations are restrictive in nature and may not be expressing, in a positive way, different policy options for spatial transformation that cope with biodiversity and nature. There is the need to overcome current practices of ‘working in silos’ and to promote cross-sectoral approaches. ▪ Ownership: Is important to shift from thinking of biodiversity and natural capital as restrictive in a way for policymakers/landowners to take ownership of their territories and thus recognize the possible uses of valued biodiversity. ▪ Governance: governance systems need to promote relational approaches in order to promote cooperation and collaboration among different decision-making levels. ▪ Awareness: more needs to be done to raise awareness on biodiversity and nature, in order to, in a positive and informed way, consider/integrate biodiversity value in spatial planning policies and more local practices. ▪ Awareness: more needs to be done to raise awareness on biodiversity and nature, in order to, in a positive and informed way, consider/integrate biodiversity value in spatial planning policies and more local practices. ▪ Valuation: overall recognition that ‘green does not have an economic value’ and does not ‘represent development’, and the importance of overcoming current perceptions to recognize value in all of its dimensions (economic, social and natural).
https://openalex.org/W2407131011	https://icm-experimental.springeropen.com/counter/pdf/10.1186/2197-425X-3-S1-A909	English	null	INTENSIVE CARE ADMISSION IN PATIENTS WITH HELLP SYNDROME IN A TERTIARY REFERRAL HOSPITAL	Intensive care medicine experimental	2,015	cc-by	809	Introduction Every year, 500.000 mothers die from pregnancy-related complications, and 99% of these deaths occur in low- and middle-income countries [1]. These maternal deaths occur from complications associated with pre-eclampsia or eclampsia; hemolysis elevated liver enzymes, and low platelet (HELLP) syndrome, or other hypertensive disorder of pregnancy [2]. Intensive care admission in patients with hellp syndrome in a tertiary referral hospital From ESICM LIVES 2015 Berlin, Germany. 3-7 October 2015 (p <0.0001), and bilirubin (p = 0.040) and lower platelet count (p = 0.005). Conclusions The patients with HELLP syndrome should be treated in intensive care unit, especially after cesarean delivery. Disseminated intravascular coagulation is major risk factor affecting maternal outcome. References 1. World Health Organization: The World Health Report 2005: make every mother and child count. Geneva, World Health Organization; 2005. All 77 women with HELLP syndrome who sought care for delivery and postpartum assessment at the emergency department, and who were treated in our intensive care unit between January 2007 and July 2012 were identified, retrospectively. Findings were analyzed according to surviving and non-surviving patients. g p y mother and child count. Geneva, World Health Organization; 2005. 2. Duley L: The global impact of pre-eclampsia and eclampsia. Semin Perinatol 2009, 33:130-7. 2. Duley L: The global impact of pre-eclampsia and eclampsia. Semin Perinatol 2009, 33:130-7. doi:10.1186/2197-425X-3-S1-A909 Cite this article as: Gedik et al.: Intensive care admission in patients with hellp syndrome in a tertiary referral hospital. Intensive Care Medicine Experimental 2015 3(Suppl 1):A909. doi:10.1186/2197-425X-3-S1-A909 Cite this article as: Gedik et al.: Intensive care admission in patients with hellp syndrome in a tertiary referral hospital. Intensive Care Medicine Experimental 2015 3(Suppl 1):A909. esults Maternal mortality rate was 14% and perinatal death ccurred in 24 of 81 fetuses and newborn (30%). The most common cause of maternal complications was disse- minated intravascular coagulation in 22 patients (29%), cute renal failure in 19 patients (25%), and postpartum emorrhage in 16 patients (21%). Compared with surviv- ng women, non-surviving women had higher mean values or international normalized ratio (p < 0.0001), levels of erum aspartate aminotransferase (p < 0.0001), serum ala- ine aminotransferase (p < 0.0001), lactate dehydrogenase Submit your manuscript to a journal and beneﬁ t from: 7 Convenient online submission 7 Rigorous peer review 7 Immediate publication on acceptance 7 Open access: articles freely available online 7 High visibility within the ﬁ eld 7 Retaining the copyright to your article Submit your next manuscript at 7 springeropen.com askent University, Ankara, Turkey ll list of author information is available at the end of the article © 2015 Gedik et al.; 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. Gedik et al. Intensive Care Medicine Experimental 2015, 3(Suppl 1):A909 http://www.icm-experimental.com/content/3/S1/A909 Authors’ details 1B k U To identify risk factors affecting maternal outcome among women with Hemolysis, Elevated Liver Enzymes, and Low Platelets (HELLP) syndrome who required transfer for critical care. 1Baskent University, Ankara, Turkey. 2Inonu University, Malatya, Turkey. 3Ersin Arslan State Hospital, Gaziantep, Turkey. 4Malatya State Hospital, Malatya, Turkey. 1Baskent University, Ankara, Turkey Full list of author information is available at the end of the article Submit your manuscript to a journal and beneﬁ t from: 1Baskent University, Ankara, Turkey Full list of author information is available at the end of the article 1Baskent University, Ankara, Turkey Full list of author information is available at the end of the article © 2015 Gedik et al.; 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. © 2015 Gedik et al.; 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. Results Maternal mortality rate was 14% and perinatal death occurred in 24 of 81 fetuses and newborn (30%). The most common cause of maternal complications was disse- minated intravascular coagulation in 22 patients (29%), acute renal failure in 19 patients (25%), and postpartum hemorrhage in 16 patients (21%). Compared with surviv- ing women, non-surviving women had higher mean values for international normalized ratio (p < 0.0001), levels of serum aspartate aminotransferase (p < 0.0001), serum ala- nine aminotransferase (p < 0.0001), lactate dehydrogenase Submit your manuscript to a journal and beneﬁ t from: 7 Convenient online submission 7 Rigorous peer review 7 Immediate publication on acceptance 7 Open access: articles freely available online 7 High visibility within the ﬁ eld 7 Retaining the copyright to your article Submit your next manuscript at 7 springeropen.com Submit your manuscript to a journal and beneﬁ t from:
https://openalex.org/W4389246021	https://www.nature.com/articles/s41467-023-43600-9.pdf	English	null	Spatial transcriptomics deconvolution at single-cell resolution using Redeconve	Nature communications	2,023	cc-by	14,148	Spatial transcriptomics deconvolution at single-cell resolution using Redeconve Zixiang Zhou 1,2,3, Yunshan Zhong 1,3, Zemin Zhang 1,2 & Xianwen Ren 1 Received: 26 October 2022 Accepted: 14 November 2023 Check for updates Computational deconvolution with single-cell RNA sequencing data as refer- ence is pivotal to interpreting spatial transcriptomics data, but the current methods are limited to cell-type resolution. Here we present Redeconve, an algorithm to deconvolute spatial transcriptomics data at single-cell resolution, enabling interpretation of spatial transcriptomics data with thousands of nuanced cell states. We benchmark Redeconve with the state-of-the-art algo- rithms on diverse spatial transcriptomics platforms and datasets and demonstrate the superiority of Redeconve in terms of accuracy, resolution, robustness, and speed. Application to a human pancreatic cancer dataset reveals cancer-clone-speciﬁc T cell inﬁltration, and application to lymph node samples identiﬁes differential cytotoxic T cells between IgA+ and IgG+ spots, providing novel insights into tumor immunology and the regulatory mechanisms underlying antibody class switch. Spatial transcriptomics (ST) technologies provide new tools to identify the cellular organization and interactions of biological samples, which is pivotal to biomedical studies. Multiple ST technologies have been developed and applied to mouse and human brains, lymph node, heart, etc., providing novel insights into cellular communication net- works underlying different conditions. However, sequencing-based ST technologies, e.g., the 10x Genomics Visium platform and Slide-seq1, are essentially of a spot-by-gene matrix structure, needing additional data to provide the cellular information. While the commercial emer- gence of imaging-based ST technologies, e.g., seqFISH+2, MERFISH3, 10x Genomics Xenium4, and NanoString CosMx5, provides subcellular resolution, these technologies are limited by low gene throughput, with hundreds of customized genes detected, making their discovery potential unparallel to whole transcriptome-wide spatial technologies. Therefore, integrative analysis of whole transcriptome-wide ST data together with matched single-cell RNA sequencing (scRNA-seq) data is of high signiﬁcance for biological discoveries. deconvolution-based methods, e.g., CARD10, RCTD11, cell2location12, DestVI13, SpatialDWLS14, SPOTlight15, STRIDE16, CellDART17, Celloscope18, DSTG19, and Stereoscope20, which try to reconstruct the ST observations by modeling the experimental process as sampling from different combinations of single cells. Mapping-based methods are superior to the current deconvolution-based methods regarding their single-cell resolution as the resolution of current deconvolution methods is limited to tens of cell types. However, mapping-based methods may introduce artiﬁcial biases during the mapping process due to the absence of strong constraint on the reconstruction accuracy of the ST observations. Article https://doi.org/10.1038/s41467-023-43600-9 1Changping Laboratory, Yard 28, Science Park Road, Changping District, Beijing, China. 2Biomedical Pioneering Innovation Center (BIOPIC), Peking University, 100871 Beijing, China. 3These authors contributed equally: Zixiang Zhou, Yunshan Zhong. e-mail: renxwise@cpl.ac.cn Results d To evaluate the performance of Redeconve in estimating the absolute abundance of cells within ST spots, we applied Redeconve to three datasets: Mouse Brain, PDAC and Human Breast Cancer Xenium, in which the cell counts were obtained by nucleus counting based on image segmentation12,22,23. Without any priori information, the results of Redeconve showed high conformity with the “ground-truth” cell counts (Fig. 1e), similar to those methods with cell counts (or cell density) as priori knowledge e.g., cell2location and Tangram (Supple- mentary Fig. 5). We used Shannon entropy to estimate the potential number of different cell states within each spatial spot (see Methods for details about using perplexity as a metric). Redeconve revealed high spot heterogeneity by showing that some spots had complex cellular composition while others had a relatively simple one. In con- trast, the entropy of other methods is uniformly high, showing that each spot had been composed of almost all the cell types in reference, which is unrealistic (Supplementary Fig. 13). High accuracy, resolution, robustness, efﬁcacy, and scalability of Redeconve We applied Redeconve to multiple ST datasets from various platforms (10x Visium, Slide-seq v2, ST, etc.)and compared the performance with other methods. We ﬁrst compared the consistency of results among different methods at the cell-type resolution based on a human breast cancer dataset. The results suggested that deconvolution-based methods including Redeconve had higher consistency with each other than mapping-based methods (Fig. 1b), indicating the relative superiority and robustness of deconvolution-based methods. This observation is conﬁrmed on additional ST datasets (Supplementary Fig. 1). Different from previous deconvolution-based methods which only reported cell-type-level results, Redeconve can further dictate ﬁne-grained cell states at single-cell resolution (Fig. 1c and Supple- mentary Fig. 2). On a ST dataset from a human breast cancer sample, Redeconve resolved 249 different cell states from 9 major cell types (Fig. 1c). On a ST dataset from mouse cerebellum, Redeconve resolved 1000 different cell states from 14 major cell types (Fig. 1c). In contrast, the resolution of previous deconvolution methods is limited by the clustering results of sc/snRNA-seq data. Single-cell resolution is unique to Redeconve compared with previous deconvolution algorithms Then we examined whether the current deconvolution-based algo- rithms could be upgraded to single-cell resolution by switching the required cell types to thousands of single cells as Redeconve does. Among all the methods we evaluated, only cell2location and DestVI completed the task but took a rather long time compared with the cell- type inputs (Supplementary Fig. 14) while other algorithms reported errors. Although single-cell inputs improved the reconstruction accu- racy of cell2location on the ST data of a human lymph node sample based on the 10x Genomics Visium platform, cell2location did not reach improvement on the human pancreatic tumor and mouse brain datasets, and DestVI failed on all three evaluations (Supplement Fig. 15). In contrast, Redeconve outperformed cell2location and DestVI on almost all spots of the evaluated datasets (Fig. 2a). When switching the inputs from cell types to single cells, DestVI achieved well sparsity regarding the different cell states within each spot (measured by perplexity according to Shannon entropy), similar to the performance of Redeconve. But cell2location reported extremely high perplexity for most spots, indicating overpredicted presence of almost all cell types and thus high false positive rate (Fig. 2b). Therefore, changing inputs from cell types to single cells cannot upgrade the performance of current algorithms to levels parallel to that of Redeconve, and the superiority of Redeconve analysis is mainly derived from algorithmic innovation. Redeconve: a quadratic programming model for single-cell deconvolution of ST data Redeconve uses scRNA-seq or single-nucleus RNA-seq (snRNA-seq) as reference to estimate the abundance of different cell states in each spot of ST data (Fig.1a). Different from previous deconvolution methods, Redeconve does not need to group single cells into clusters and then do deconvolution. Instead, Redeconve treats each cell of the sc/snRNA-seq data as a speciﬁc cell state serving as reference to esti- mate the cellular composition of ST data. The direct usage of sc/ snRNA-seq data as reference is conceptually direct and computation- ally efﬁcient, with the potential to handle the heterogeneity of ST data. However, direct usage of sc/snRNA-seq data as reference will intro- duce a new challenge, i.e., collinearity. That is, multiple single cells have similar proﬁles of gene expression, prohibiting the accurate estimation of the abundance of individual cell states. We introduce a biologically reasonable heuristic by assuming that similar cells have similar abundance within ST spots, and thus mathematically introduce a regularization term in the deconvolution model based on non- negative least regression. Solving this regularized deconvolution model by quadratic programming will produce robust estimation of the cellular composition at single-cell resolution for each spot of ST data. Spatial transcriptomics deconvolution at single-cell resolution using Redeconve It is urgently needed to develop a deconvolution-based algorithm with single-cell resolution to fully release the biological information hidden in ST data. In this study, we develop an algorithm, named as Redeconve21, to estimate the cellular composition of ST spots. Different from previous deconvolution-based algorithms, Redeconve introduces a regularizing term to solve the collinearity problem of high-resolution deconvolu- tion, with the assumption that similar single cell states have similar abundance in ST spots. This algorithmic innovation not only improves the deconvolution resolution from tens of cell types to thousands of single cell states, but also greatly improve the reconstruction accuracy of ST data, enabling illustration of the nuanced biological mechanisms hidden in the ST data. Stringent comparison with the state-of-the-art Multiple effective and efﬁcient algorithms have been proposed for integrative analysis of whole-transcriptome ST and scRNA-seq data. The current algorithms can be categorized to two groups: (1) mapping- based methods, e.g., NovospaRc6, Tangram7, Celltrek8, and CytoSPACE9, which map single cells to the positions of ST data according to gene expression similarity or related measures; and (2) Nature Communications\| (2023) 14:7930 1 Article https://doi.org/10.1038/s41467-023-43600-9 algorithms including cell2location, CARD, DestVI, CellTrek, NovoS- paRc, and Tangram demonstrates the superiority of Redeconve in terms of reconstruction accuracy, cell abundance estimation per spot, sparseness of the reconstructed cellular composition, cell state reso- lution, and computational speed. Application to human pancreatic cancer data reveals novel insights into tumor-inﬁltrating CD8 + T cells, and application to human lymph node data reveals new clues for the regulatory factors of IgA+ and IgG+ B cells. example, only snRNA-seq data are accessible for brain samples), Redeconve still outperforms other methods (Fig. 1d). Pairwise com- parison between Redeconve and other methods further shows the superiority of Redeconve on almost all spots regarding the recon- struction accuracy (Supplementary Figs. 7–12). Because Redeconve conducts deconvolution analysis spot by spot, parallel computation is enabled and thus Redeconve demonstrates superior computation speed compared with current deconvolution algorithms (Fig. 1f and Supplementary Fig. 6). Nature Communications\| (2023) 14:7930 Evaluating the impact of cell-type resolution on deconvolution by simulation In addition to the robustness and resolution superiority, Redec- onve also improves the reconstruction accuracy of gene expression per spot, and the improvement is independent on similarity measures such as cosine similarity, Pearson’s correlation, and Root Mean Square Error (RMSE) between the true ST gene expression proﬁle and the reconstructed gene expression vector (Fig. 1d, and Supplementary Figs. 3–4). Redeconve also reached high accuracy of estimated cell abundance (based on a ground truth by nucleus counting, Fig. 1e and Supplementary Fig. 5), and superior computational speed (Fig. 1f and Supplementary Fig. 6). When suitable reference is provided, e.g., matched scRNA-seq data, Redeconve can reach >0.8 cosine accuracy for most ST spots (Fig. 1d). With no suitable reference available (for To evaluate how the cell-type resolution of reference data impacts the deconvolution analysis, we devised a series of simulation experiments to showcase the performance differences of Redeconve and the state- of-the-art algorithms. We constructed three pseudo-bulk RNA-seq datasets by averaging the gene expression data of individual cells based on scRNA-seq data from the PDAC24, human lymph node12,25 and human testis26 datasets separately (Fig. 2c and Methods). Then we applied Redeconve and cell2location, the only alternative method capable of this task. With direct comparison with ground-truth, the results indicate that Redeconve performs substantially better than cell2location, as evidenced by its signiﬁcantly higher accuracy Nature Communications\| (2023) 14:7930 2 Article https://doi.org/10.1038/s41467-023-43600-9 lementary Figs. 16–18). When examining the relationship en accuracy and number of clusters in single-cell reference, onve showed an increase in accuracy when the number of rs grows, while cell2location experienced a sharp drop (Fig. 2d). uggests that Redeconve is capable of handling large-scale scRNA- ta more effectively and can use ﬁner-grained clusters to increase acy instead of becoming confused. Furthermore, simulation experiments also corroborate the validity of using perplexity as a metric of sparsity (Supplementary Table 1 and Methods). Evaluating the estimating accuracy of cell-type proportion by 10x Genomics Xenium data as ground truth Single-cell ST platforms, such as MERFISH3, Xenium4 and CoxMx5, are commercially emerging as a powerful tool for the high-resolution experiments also corroborate the validity of using perplexity as a metric of sparsity (Supplementary Table 1 and Methods). (Supplementary Figs. 16–18). When examining the relationship between accuracy and number of clusters in single-cell reference, Redeconve showed an increase in accuracy when the number of clusters grows, while cell2location experienced a sharp drop (Fig. 2d). https://doi.org/10.1038/s41467-023-43600-9 Redeconve out- performed other algorithms on most cell types (13/20 in top one), especially for cancer, ductal, endocrine cells, and demonstrated comparable performance to the best performers on the remaining of cell types (20/20 in top three, Supplementary Fig. 23a, b). In addition, the performance of Redeconve, cell2location, and Tangram was robust to cell type abundance variations in scRNA-seq data, while the per- formances of DestVI, CARD, and NovoSpaRc were positively correlated with cell type abundances (p-value < 0.05) (Supplementary Fig. 23c). mapping of the precise location of single cells, but are limited by the number of genes proﬁled during experiments because customized probes speciﬁc to target genes need to be designed and synthesized before experiments. The high resolution of these platforms provides natural ground truth to evaluate the performance of Redeconve. Here, we used a human breast cancer Xenium dataset generated by 10x Genomics4 to evaluate the performance of Redeconve regarding reconstruction of ST spot expression proﬁles, cell type proportion predictions and abundance of individual cell states. This dataset encompasses not only Xenium data containing coordinates and expression proﬁles of segmented single cells, but also matched scRNA- seq (including 5’, 3’ and scFFPE-seq) and Visium data, enabling us to generate ground truths for Visium spots regarding cell abundances and cell type proportions (See Methods for details). 3906 Visium spots overlapped with the Xenium data were extracted for comparative analysis (Fig. 3a). Compared with the state-of-the-art algorithms including cell2location, DestVI, CARD, NovoSpaRc, CellTrek, and Tangram, Redeconve demonstrated superior cosine similarities between the predicted cell type proportions and the ground truths for most of the Visium spots (Fig. 3b). Specially, Redeconve exhibited superior performance on more than 60% and 70% of spots compared to alternative deconvolution-based or mapping-based methods, respectively (Supplementary Fig. 19). Redeconve, cell2location and Tangram demonstrated comparable performance in estimating the absolute cell abundance within Visium spots, as evidenced by high Pearson’s correlation with the ground-truth cell counts indicated by the overlapped cell counts according to the Xenium data, but the performance of Redeconve was more robust to the selection of scRNA- seq references (Fig. 3c and Supplementary Fig. 20). Similarly, the performance of Redeconve in reconstructing the expression proﬁles of different Visium spots was also more robust to the selection of different scRNA-seq references compared with the state-of-the-art algorithms (Fig. 3d and Supplementary Fig. 21). https://doi.org/10.1038/s41467-023-43600-9 Fig. 1 \| Overview of the Redeconve algorithm and benchmark analysis. between observed and reconstructed expression proﬁles per spot based on six ST datasets. N = 4039, 2426, 428, 36550, 2987 and 39431 spots for human lymph nodes, human breast cancer, PDAC (pancreatic ductal adenocarcinoma), human testis, mouse brain and mouse cerebellum respaectively. Spots were sorted by an ascending order of the cosine similarities. e Pearson correlation of cell abundances between Redeconve and the cell counts per spot based on a mouse brain dataset. between observed and reconstructed expression proﬁles per spot based on six ST datasets. N = 4039, 2426, 428, 36550, 2987 and 39431 spots for human lymph nodes, human breast cancer, PDAC (pancreatic ductal adenocarcinoma), human testis, mouse brain and mouse cerebellum respaectively. Spots were sorted by an ascending order of the cosine similarities. e Pearson correlation of cell abundances between Redeconve and the cell counts per spot based on a mouse brain dataset. The ground truth cell counts per spot was obtained by nucleus counting of cell segmentation image12. f computational efﬁciency of different deconvolution-based and mapping-based algorithms on a human lymph nodes dataset. Source data of 1c- e are provided as a Source Data ﬁle. a overview of Redeconve workﬂow for deconvoluting spatial transcriptomics data. Redeconve requires sc/snRNA-seq data together with spatial transcriptomics data as input and performs deconvolution by solving a regularized non-negative least regression model with the aims to estimate cellular composition across spots at single-cell resolution. b heatmap illustrated median spot-level Spearman’s corre- lation of cell type proportions among different algorithms on a human breast cancer dataset. c Sankey diagram demonstrated the cell-type and single-cell reso- lutions of Redeconve results on human breast cancer and mouse cerebellum datasets, respectively. The bar height of cell types or single cells refer to their estimated abundance after deconvolution. d line chart of cosine similarities The ground truth cell counts per spot was obtained by nucleus counting of cell segmentation image12. f computational efﬁciency of different deconvolution-based and mapping-based algorithms on a human lymph nodes dataset. Source data of 1c- e are provided as a Source Data ﬁle. between ST observation and reconstructed proﬁles by different algo- rithms across all spots (See Methods for details). https://doi.org/10.1038/s41467-023-43600-9 Histological analysis based on H&E staining identiﬁed four tissue regions: pancreatic, cancer, duct epithelium, and stroma24 (Fig. 4a). Redeconve, CARD, and DestVI successfully distinguished the four types of tissue regions, consistent with histological analysis (Supplementary Fig. 24,). Meanwhile, cell2location, NovoSpaRc and Tangram failed in several conditions (Fig. 4d and Supplementary Fig. 24). Further inspection into a speciﬁc spot in the upper cancer region (Fig. 4d, the upper zoomed-in piechart) shows that deconvolution-based methods (Redeconve, cell2location, DestVI and CARD) are able to detect ﬁbro- blast, which is known to be abundant in pancreatic cancer24,27,28, while mapping methods (Tangram and NovoSpaRc) fail in this task. Then we examined the detailed characteristics of tumor- inﬁltrating T cells based on these results, which is important to understand the tumor immune microenvironment of pancreatic can- cers. The results of cell2location, NovoSpaRc, Tangram and DestVI reported T cells in almost all spots (Fig. 5a), inconsistent with the nature of PDAC as cold tumors; Meanwhile, Redeconve and CARD clearly suggested the sparsity of tumor-inﬁltrating T cells in pancreatic cancer, consistent with the spatial distribution of T cell-related genes (CD3, IL32 and TMSB4X, Fig. 5a, Supplementary Figs. 25–27). As CARD is limited by the cell-type resolution, it is difﬁcult to provide more detailed insights, but Redeconve analysis enables deeper investigation. We identiﬁed three T cells in the reference scRNA-seq data that appeared in multiple ST spots, indexed as “T.cell.8”, “T.cell.11” and “T.cell.35” separately (Fig. 5b). By examining their expression proﬁles in the reference scRNA-seq, we identiﬁed T cell 11 as regulatory T cell (CD4+ FOXP3+) and 8 and 35 as CD8+ cytotoxic T cells. For fair com- parison, we further divided T cells in the scRNA-seq reference data into three groups, i.e., cytotoxic, helper and regulatory T cells and used these three T cell types together with other cell types as reference to re-run other deconvolution algorithms (Supplementary Fig. 28). Con- sistent with the spatial distribution of CD8 and FOXP3, the result of Redeconve is the most reasonable (Supplementary Figs. 25 and 27). According to the Redeconve deconvolution results, almost all the T cells within cancer region were similar to regulatory T cell 11, and T cell states similar to 8 and 35 only appeared outside or at the edge of the cancer region (Fig. 5b, c), consistent with the immune suppressive status of the cancer region of pancreatic tumors24,29. Evaluating the impact of cell-type resolution on deconvolution by simulation This suggests that Redeconve is capable of handling large-scale scRNA- seq data more effectively and can use ﬁner-grained clusters to increase accuracy instead of becoming confused. Furthermore, simulation (Supplementary Figs. 16–18). When examining the relationship between accuracy and number of clusters in single-cell reference, Redeconve showed an increase in accuracy when the number of clusters grows, while cell2location experienced a sharp drop (Fig. 2d). This suggests that Redeconve is capable of handling large-scale scRNA- seq data more effectively and can use ﬁner-grained clusters to increase accuracy instead of becoming confused. Furthermore, simulation Evaluating the estimating accuracy of cell-type proportion by 10x Genomics Xenium data as ground truth Evaluating the estimating accuracy of cell-type proportion by 10x Genomics Xenium data as ground truth Evaluating the estimating accuracy of cell-type proportion by 10x Genomics Xenium data as ground truth Single-cell ST platforms, such as MERFISH3, Xenium4 and CoxMx5, are commercially emerging as a powerful tool for the high-resolution Single-cell ST platforms, such as MERFISH3, Xenium4 and CoxMx5, are commercially emerging as a powerful tool for the high-resolution Nature Communications\| (2023) 14:7930 3 Article https://doi.org/10.1038/s41467-023-43600-9 Single-cell resolution by Redeconve enables identiﬁcation of pancreatic cancer-clone-speciﬁc T cell inﬁltration a cosine similarity between true and reconstructed spatial expression proﬁles based on Redeconve, cell2location and DestVI with 1000 single cells as input. Each dot represents a spot of the ST data. b the number of different cell states within each spot estimated by the perplexity of cell state composition per spot for results of Redeconve, cell2location and DestVIwith 1000 single cells as input (See Methods for details). c workﬂow of generating simulation data. ScRNA-seq data were aggregated to a pseudo-bulk, which was then used for deconvolution analysis and the results were used for downstream analyses in (d). d cosine similarity between true and reconstructed spatial expression proﬁles vs. number of clusters on simulated pseudo-bulk. PDAC, pancreatic ductal adenocarcinoma. Source data of 2a, b and d are provided as a Source Data ﬁle. 2 \| P f b h ki ith i l ll i t d i l t d f R d ll2l i d D VI i h 1000 i l ll i (S M Fig. 2 \| Performance benchmarking with single-cell inputs and simulated datasets. Redeconve, cell2location and DestVI are currently the only three deconvolution-based tools with the ability to handle thousands of cell states. a cosine similarity between true and reconstructed spatial expression proﬁles based on Redeconve, cell2location and DestVI with 1000 single cells as input. Each dot represents a spot of the ST data. b the number of different cell states within each spot estimated by the perplexity of cell state composition per spot for results Fig. 2 \| Performance benchmarking with single-cell inputs and simulated Performance benchmarking with single-cell inputs and simu of Redeconve, cell2location and DestVIwith 1000 single cells as input (See Methods for details). c workﬂow of generating simulation data. ScRNA-seq data were aggregated to a pseudo-bulk, which was then used for deconvolution analysis and the results were used for downstream analyses in (d). d cosine similarity between true and reconstructed spatial expression proﬁles vs. number of clusters on simulated pseudo-bulk. PDAC, pancreatic ductal adenocarcinoma. Source data of 2a, b and d are provided as a Source Data ﬁle. datasets. Redeconve, cell2location and DestVI are currently the only three deconvolution-based tools with the ability to handle thousands of cell states. a cosine similarity between true and reconstructed spatial expression proﬁles based on Redeconve, cell2location and DestVI with 1000 single cells as input. Each dot represents a spot of the ST data. Single-cell resolution by Redeconve enables identiﬁcation of pancreatic cancer-clone-speciﬁc T cell inﬁltration To demonstrate the power of deconvolution at single-cell resolution on solving practical biological problems, we further investigated the Redeconve results of the human pancreatic ST dataset24. The ST is from the original ST platform, and scRNA-seq data from the same individual were obtained through InDrop. Redeconve with single cells as reference outperformed other methods regarding the reconstruc- tion accuracy for almost all the spots (Fig. 4b, c and Supplementary Fig. 7). Using cell types as reference and varying the cell-type resolu- tion from 20 to 318 clusters, Redeconve still resulted in stable superior performance compared with other methods (with the same inputs) (Supplementary Fig. 22), suggesting the advantage of Redeconve by excluding the interference of single-cell reference vs cell-type refer- ence, although Redeconve is the only algorithm designed to take single cells as reference as we demonstrated in the previous sections. Benchmark regarding individual cell types again showed the super- iority of Redeconve. We identiﬁed marker genes for each cell type (Supplementary Table 2), and calculated the expression consistency We further conducted co-localization analysis of these three T cell states with the resting cell states by calculating the Pearson correlation coefﬁcient of abundance across all spots based on the Redeconve results (Fig. 5d). The results suggested that the regulatory T cell state similar to T cell 11 mainly co-localized with macrophages similar to Nature Communications\| (2023) 14:7930 4 https://doi.org/10.1038/s41467-023-43600-9 Article g. 2 \| Performance benchmarking with single-cell inputs and simulated tasets. Redeconve, cell2location and DestVI are currently the only three convolution-based tools with the ability to handle thousands of cell states. osine similarity between true and reconstructed spatial expression proﬁles sed on Redeconve, cell2location and DestVI with 1000 single cells as input. Each t represents a spot of the ST data. b the number of different cell states within of Redeconve, cell2location and DestVIwith 1000 single cells as input (See Meth for details). c workﬂow of generating simulation data. ScRNA-seq data were aggregated to a pseudo-bulk, which was then used for deconvolution analysis the results were used for downstream analyses in (d). d cosine similarity betw true and reconstructed spatial expression proﬁles vs. number of clusters on simulated pseudo-bulk. PDAC, pancreatic ductal adenocarcinoma. Source data Fig. 2 \| Performance benchmarking with single-cell inputs and simulated datasets. Redeconve, cell2location and DestVI are currently the only three deconvolution-based tools with the ability to handle thousands of cell states. Single-cell resolution by Redeconve enables identiﬁcation of pancreatic cancer-clone-speciﬁc T cell inﬁltration b the number of different cell states within each spot estimated by the perplexity of cell state composition per spot for results Nature Communications\| (2023) 14:7930 5 Fig. 3 \| Benchmarking Redeconve performance on a human breast cancer Xenium dataset. a Left: Overlapped Xenium cells and Visium spots were illustrated on H&E image. Right: the overlapped region was employed for benchmarking Redeconve performance by introducing different single-cell references to predict expression proﬁles, cell type proportions, and cell abundances. b line chart of cosine similarities of cell type proportions between ground truths and algorithm- based predictions per spot. N = 3906 spots for the dataset and spots were sorted by an ascending order of the cosine similarities. c Heatmap illustrating the pairwise Pearson’s correlation of cell abundances among the ground truth, Redeconve, cell2location and Tangram based on various single cell references. d violin and box plot of cosine similarities between observed and reconstructed expression proﬁles for Redeconve and alternative approaches with different single cell references (3’, 5’ and scFFPE-seq). The number of independent single cells in the references are 5527, 13,808 and 28,180 respectively. The center line and the bounds of box refer to median, Q1 and Q3 of scores and the whisker equal to 1.5(Q3–Q1). The minimum and maximum scores refer to Q1-whisker and Q3+whisker. GT, ground truth. scFFPE-seq, single-cell Formalin Fixed Parafﬁn Embedded sequencing. Source data of 3b-d are provided as a Source Data ﬁle. Display items in this ﬁgure were manually generated in Inkscape by the authors. Article https://doi.org/10.1038/s41467-023-43600-9 https://doi.org/10.1038/s41467-023-43600-9 Article plot of cosine similarities between observed and reconstructed expression proﬁles for Redeconve and alternative approaches with different single cell references (3’, 5’ and scFFPE-seq). The number of independent single cells in the references are 5527, 13,808 and 28,180 respectively. The center line and the bounds of box refer to median, Q1 and Q3 of scores and the whisker equal to 1.5(Q3–Q1). The minimum and maximum scores refer to Q1-whisker and Q3+whisker. GT, ground truth. scFFPE-seq, single-cell Formalin Fixed Parafﬁn Embedded sequencing. Source data of 3b-d are provided as a Source Data ﬁle. Display items in this ﬁgure were manually generated in Inkscape by the authors. Fig. 3 \| Benchmarking Redeconve performance on a human breast cancer Xenium dataset. a Left: Overlapped Xenium cells and Visium spots were illustrated on H&E image. Single-cell resolution by Redeconve enables identiﬁcation of pancreatic cancer-clone-speciﬁc T cell inﬁltration 4 \| Single-cell deconvolution of a human PDAC (pancreatic ductal adeno- carcinoma) ST dataset. a four regions were annotated by histological analysis of the original paper: pancreatic, ductal, cancer and stroma regions24. b spatial dis- tribution of the cosine similarity between true and reconstructed expression proﬁles per spot by different computational methods. c pie charts displaying the spatial distribution of the estimated cell type proportion per spot by different computational methods. RBC red blood cell. mDC myeloid dendritic cell. pDC plasmacytoid dendritic cell. Source data are provided as a Source Data ﬁle. T cells. We found that interferon-induced genes (IFIT1 and IFI44L, for example) and HLA-related genes (HLA-A, HLA-B and HLA-C) were all up- regulated in cancer clone B (Fig. 5f), and correspondingly T cell state 8, which is colocalized with cancer clone B, had high expression of HMGB2, HLA-B and HLA-C (Fig. 5f), indicating well-stimulated T cell response33,34. In contrast, T cell state 35 was HMGB2-negative, HLA-low and TMBS10-positive and co-localized with more A-type macrophages, indicating a less efﬁcacy state33,34. Therefore, with accurate deconvo- lution at the single-cell resolution, Redeconve can reveal detailed cell- cell interaction at single-cell level and enables discoveries revealing the underlying mechanisms of tumor immunity. T cells. We found that interferon-induced genes (IFIT1 and IFI44L, for example) and HLA-related genes (HLA-A, HLA-B and HLA-C) were all up- regulated in cancer clone B (Fig. 5f), and correspondingly T cell state 8, which is colocalized with cancer clone B, had high expression of HMGB2, HLA-B and HLA-C (Fig. 5f), indicating well-stimulated T cell response33,34. In contrast, T cell state 35 was HMGB2-negative, HLA-low and TMBS10-positive and co-localized with more A-type macrophages, indicating a less efﬁcacy state33,34. Therefore, with accurate deconvo- lution at the single-cell resolution, Redeconve can reveal detailed cell- cell interaction at single-cell level and enables discoveries revealing the underlying mechanisms of tumor immunity. macrophages B. 6, 8, and 16 together with duct cells of two different states. Interestingly, T cell 8 and 35 were mainly co-localized with cancer cells, indicating dispersed cancer cells outside the cancer region. Although provided scRNA-seq reference with higher T cell resolution (cytotoxic/helper/regulatory T cells), such co-localization was not observed by other methods (Supplementary Fig. 29). Furthermore, these two T cell states were separately co-localized with different cancer clones, with T cell state 8 co-localized with cancer clone B and 35 with cancer clone A. Single-cell resolution by Redeconve enables identiﬁcation of pancreatic cancer-clone-speciﬁc T cell inﬁltration Right: the overlapped region was employed for benchmarking Redeconve performance by introducing different single-cell references to predict expression proﬁles, cell type proportions, and cell abundances. b line chart of cosine similarities of cell type proportions between ground truths and algorithm- based predictions per spot. N = 3906 spots for the dataset and spots were sorted by an ascending order of the cosine similarities. c Heatmap illustrating the pairwise Pearson’s correlation of cell abundances among the ground truth, Redeconve, cell2location and Tangram based on various single cell references. d violin and box Fig. 3 \| Benchmarking Redeconve performance on a human breast cancer Fig. 3 \| Benchmarking Redeconve performance on a human breast cancer Xenium dataset. a Left: Overlapped Xenium cells and Visium spots were illustrated on H&E image. Right: the overlapped region was employed for benchmarking Redeconve performance by introducing different single-cell references to predict expression proﬁles, cell type proportions, and cell abundances. b line chart of cosine similarities of cell type proportions between ground truths and algorithm- based predictions per spot. N = 3906 spots for the dataset and spots were sorted by an ascending order of the cosine similarities. c Heatmap illustrating the pairwise Pearson’s correlation of cell abundances among the ground truth, Redeconve, cell2location and Tangram based on various single cell references. d violin and box Nature Communications\| (2023) 14:7930 6 6 Article https://doi.org/10.1038/s41467-023-43600-9 acrophages B. 6, 8, and 16 together with duct cells of two different T cells. We found that interferon-induced genes (IFIT1 and IFI44L, for g. 4 \| Single-cell deconvolution of a human PDAC (pancreatic ductal adeno- rcinoma) ST dataset. a four regions were annotated by histological analysis of e original paper: pancreatic, ductal, cancer and stroma regions24. b spatial dis- bution of the cosine similarity between true and reconstructed expression proﬁles per spot by different computational methods. c pie charts displaying the spatial distribution of the estimated cell type proportion per spot by different computational methods. RBC red blood cell. mDC myeloid dendritic cell. pDC plasmacytoid dendritic cell. Source data are provided as a Source Data ﬁle. ig. 4 \| Single-cell deconvolution of a human PDAC (pancreatic ductal adeno- i ) ST d t t f i d b hi l i l l i f proﬁles per spot by different computational methods. c pie charts displaying i l di ib i f h i d ll i b diff Fig. Single-cell resolution by Redeconve enables identiﬁcation of pancreatic cancer-clone-speciﬁc T cell inﬁltration Differential gene expression ana- lysis based on the reference scRNA-seq data further indicated the differences between these two pairs of T cells and cancer cells (Fig. 5e, f). It is revealed previously that TM4SF1+ cancer cells denoted late- stage while S1004A+ cancer cells (clone B) denoted early-stage30–32. Our analysis identiﬁed the co-existence of TM4SF1+ cancer cells (clone A) and S1004A+ cancer cells (clone B) with different CD8+ T cells, which is important to understand the interactions between cancer and Nature Communications\| (2023) 14:7930 Redeconve sheds novel insights into the regulatory mechanisms underlying antibody class switch As we expected, IGHA and IGHG were the most differentially- expressed genes; Genes associated with T cells (TRAC, TRBC2, CD3D, CD8A for example) were more up-regulated in IgA+ spots, conﬁrming the existence of such interaction. Since lymph node is one of the organs that generate IgA+ plasma cells, the IgA+ spots might be the potential induction sits for IgA+ plasma cells, and CD8+ T cells may play an important role in such process (Fig. 6d). Further co-localization analysis provides more insights (Fig. 6e). We found co-localization of (Fig. 6a). CellTrek failed to analyze some of the spots. Furthermore, compared with cell-type deconvolution, Redeconve identiﬁed 159 different cell states from 17 cell types (Supplementary Fig. 2). 12 dif- ferent B plasma cell states were identiﬁed in the ST data, which can be further divided into 3 groups (IgA + , IgG+ and negative) based on the expression of IGHA and IGHG genes. Interestingly, we found that IgA+ and IgG+ B plasma cells are spatially mapped to spots in different regions with little overlap, which means that we could deﬁne IgA+ and IgG+ spots based on the abundance of those B plasma cells (Fig. 6b). Next, we took one spot in each of the two regions for detailed inspection at the single-cell resolution. The cell proportion of the two spots shows that CD8+ T cells account for a large proportion in the IgA+ spot, suggesting latent interactions between CD8+ T cells and IgA+ B plasma cells (Fig. 6c). To conﬁrm the universality of such phenom- enon, we conducted differential gene expression analysis between IgA + and IgG+ spots to identify up-regulated and down-regulated genes (Fig. 6d). As we expected, IGHA and IGHG were the most differentially- expressed genes; Genes associated with T cells (TRAC, TRBC2, CD3D, CD8A for example) were more up-regulated in IgA+ spots, conﬁrming the existence of such interaction. Since lymph node is one of the organs that generate IgA+ plasma cells, the IgA+ spots might be the potential induction sits for IgA+ plasma cells, and CD8+ T cells may play an important role in such process (Fig. 6d). Further co-localization analysis provides more insights (Fig. 6e). We found co-localization of methods on this dataset. In terms of cosine similarity-based recon- struction accuracy, Redeconve achieved mean similarities of 0.868 and signiﬁcantly outperformed other methods (Fig. 1d). Redeconve sheds novel insights into the regulatory mechanisms underlying antibody class switch Redeconve were further applied to analyze an ST data of human sec- ondary lymphoid organs12. We again compared Redeconve with other Nature Communications\| (2023) 14:7930 7 https://doi.org/10.1038/s41467-023-43600-9 Article ig. 5 \| Cancer-clone-speciﬁc CD8 + T cell inﬁltration revealed by Redeconve in human pancreatic cancer. a abundance of T cells per spot estimated by different methods. b single-cell identity of inﬁltrated T cells revealed by Redeconve. The hree T cells are indexed as “T.cells.8”, “T.cells.11”, “T.cells.35” separately. c single- ell identity of different cancer clone cells revealed by Redeconve, together with heir abundance difference. d co-localization of the three T cell states with other ellular states. Nodes represent single cells and edges represent co-localization Pearson correlation of cell abundance >0.4). Cancer clone-speciﬁc CD8 + T cell nﬁltration was revealed. e dot plot displaying characteristics genes among the hree T cell states with different spatial preference with cancer clones A and B. f volcano plot displaying differentially expressed genes between the two cancer clones. The blue and red points refer to up-regulated genes in clones A and B-enriched spots, respectively. Vertical dashed line shows the cutoff of log fold change (±0.3). Horizontal dashed line shows the threshold of -lg p (1.301). T cell response-related genes including interferon-stimulating genes and human leuko- cyte antigens were up-regulated in clone B-enriched cells. The two-side exact test was applied in edgeR for the statistical test and the p-values were calculated without adjustments. Treg, T regulatory. Source data are provided as a Source Data ﬁle. Fig. 5 \| Cancer-clone-speciﬁc CD8 + T cell inﬁltration revealed by Redeconve in Fig. 5 \| Cancer-clone-speciﬁc CD8 + T cell inﬁltration revealed by Redeconve in human pancreatic cancer. a abundance of T cells per spot estimated by different methods. b single-cell identity of inﬁltrated T cells revealed by Redeconve. The three T cells are indexed as “T.cells.8”, “T.cells.11”, “T.cells.35” separately. c single- cell identity of different cancer clone cells revealed by Redeconve, together with their abundance difference. d co-localization of the three T cell states with other cellular states. Nodes represent single cells and edges represent co-localization (Pearson correlation of cell abundance >0.4). Cancer clone-speciﬁc CD8 + T cell inﬁltration was revealed. e dot plot displaying characteristics genes among the three T cell states with different spatial preference with cancer clones A and B. Fig. 5 \| Cancer-clone-speciﬁc CD8 + T cell inﬁltration revealed by Redeconve in human pancreatic cancer. Redeconve sheds novel insights into the regulatory mechanisms underlying antibody class switch a abundance of T cells per spot estimated by different methods. b single-cell identity of inﬁltrated T cells revealed by Redeconve. The three T cells are indexed as “T.cells.8”, “T.cells.11”, “T.cells.35” separately. c single- cell identity of different cancer clone cells revealed by Redeconve, together with their abundance difference. d co-localization of the three T cell states with other cellular states. Nodes represent single cells and edges represent co-localization (Pearson correlation of cell abundance >0.4). Cancer clone-speciﬁc CD8 + T cell inﬁltration was revealed. e dot plot displaying characteristics genes among the three T cell states with different spatial preference with cancer clones A and B. f volcano plot displaying differentially expressed genes between the two cancer clones. The blue and red points refer to up-regulated genes in clones A and B-enriched spots, respectively. Vertical dashed line shows the cutoff of log fold change (±0.3). Horizontal dashed line shows the threshold of -lg p (1.301). T cell response-related genes including interferon-stimulating genes and human leuko- cyte antigens were up-regulated in clone B-enriched cells. The two-side exact test was applied in edgeR for the statistical test and the p-values were calculated without adjustments. Treg, T regulatory. Source data are provided as a Source Data ﬁle. (Fig. 6a). CellTrek failed to analyze some of the spots. Furthermore, compared with cell-type deconvolution, Redeconve identiﬁed 159 different cell states from 17 cell types (Supplementary Fig. 2). 12 dif- ferent B plasma cell states were identiﬁed in the ST data, which can be further divided into 3 groups (IgA + , IgG+ and negative) based on the expression of IGHA and IGHG genes. Interestingly, we found that IgA+ and IgG+ B plasma cells are spatially mapped to spots in different regions with little overlap, which means that we could deﬁne IgA+ and IgG+ spots based on the abundance of those B plasma cells (Fig. 6b). Next, we took one spot in each of the two regions for detailed inspection at the single-cell resolution. The cell proportion of the two spots shows that CD8+ T cells account for a large proportion in the IgA+ spot, suggesting latent interactions between CD8+ T cells and IgA+ B plasma cells (Fig. 6c). To conﬁrm the universality of such phenom- enon, we conducted differential gene expression analysis between IgA + and IgG+ spots to identify up-regulated and down-regulated genes (Fig. 6d). Nature Communications\| (2023) 14:7930 Redeconve sheds novel insights into the regulatory mechanisms underlying antibody class switch 6 \| Single-cell deconvolution of a human secondary lymphoid organ ST dataset by Redeconve revealed differences between IgA+ and IgG+ spots regarding cellular composition. a pie chart displaying the spatial distribution of exact test was applied in edgeR for the statistical test and the p-values were cal- culated without adjustments. e co-localization network of IgA+ and IgG+ B plasma cells within the ST data. Nodes represent single cells and edges represent co- located single cells (Pearson correlation of cell abundance >0.2). Abbreviations: GC germinal center, DZ dark zone, LZ light zone, prePB preplasmablast, mem memory, cDC classical dendritic cell, Endo endothelial, FDC follicular dendritic cell, ILC innate lymphoid cell, NK natural killer, NKT natural killer T, TfH T follicular helper, Treg T regulatory, VSMC vascular smooth muscle cell. Source data of 6a-d are provided as a Source Data ﬁle. exact test was applied in edgeR for the statistical test and the p-values were cal- culated without adjustments. e co-localization network of IgA+ and IgG+ B plasma cells within the ST data. Nodes represent single cells and edges represent co- located single cells (Pearson correlation of cell abundance >0.2). Abbreviations: GC germinal center, DZ dark zone, LZ light zone, prePB preplasmablast, mem memory, cDC classical dendritic cell, Endo endothelial, FDC follicular dendritic cell, ILC innate lymphoid cell, NK natural killer, NKT natural killer T, TfH T follicular helper, Treg T regulatory, VSMC vascular smooth muscle cell. Source data of 6a-d are provided as a Source Data ﬁle. Fig. 6 \| Single-cell deconvolution of a human secondary lymphoid organ ST dataset by Redeconve revealed differences between IgA+ and IgG+ spots regarding cellular composition. a pie chart displaying the spatial distribution of the estimated cell type proportion by different methods. b spatial distribution of IgA+ and IgG+ B plasma cells revealed by Redeconve. c comparison of the cell proportion of two selected spots (the IgA+ and IgG+ spots in Fig. 6a with green squares). d volcano plots showing the differential gene expression between IgA+ and IgG+ spots. The red and blue point refer to up-regulated genes in IgG+ and IgA+ spots respectively. Vertical dashed line shows the cutoff of log fold change (±0.3). Horizontal dashed line shows the threshold of -lg(p), namely 1.301. The two-side Fig. 6 \| Single-cell deconvolution of a human secondary lymphoid organ ST dataset by Redeconve revealed differences between IgA+ and IgG+ spots regarding cellular composition. Redeconve sheds novel insights into the regulatory mechanisms underlying antibody class switch Redeconve achieved high reconstruction accuracy for almost all spots, while, as for other methods, low similarities regions were obvious (Supple- mentary Fig. 30). We further checked the sparsity of the results by calculating L0-norm. L0-norm of Redeconve has a reasonable dis- tribution between 4 and 32, indicating that only dozens of cell states appear in one spot. In contrast, other methods except CellTrek demonstrated results that almost all cell types appeared in every spot. CellTrek, a mapping-based algorithm, reached low level of L0-norm by generating many “zero-cell” spots, of which Redeconve successfully reconstructed the cellular composition (Supplementary Fig. 31). We further characterized the spatial heterogeneity at single cell resolution to explore the potential regulators of antibody class switch based on this human lymph node data. During the antibody matura- tion, an activated B cell can change its antibody production from IgM to either IgA, IgG, or IgE depending on the functional requirements, which is termed as class switching35. However, the detailed regulators underlying antibody class switching is unclear. Consistent with pre- vious examples, Redeconve outperformed other methods in recon- structing the ST gene expression proﬁles for almost all spots (Fig. 1d). Spatial pie chart showed that Redeconve produced obvious regional division, while other methods showed blurred or even no boundaries Nature Communications\| (2023) 14:7930 8 https://doi.org/10.1038/s41467-023-43600-9 Article g. 6 \| Single-cell deconvolution of a human secondary lymphoid organ ST taset by Redeconve revealed differences between IgA+ and IgG+ spots garding cellular composition. a pie chart displaying the spatial distribution of e estimated cell type proportion by different methods. b spatial distribution of A+ and IgG+ B plasma cells revealed by Redeconve. c comparison of the cell oportion of two selected spots (the IgA+ and IgG+ spots in Fig. 6a with green uares) d volcano plots showing the differential gene expression between IgA+ exact test was applied in edgeR for the statistical test and the p-values were cal- culated without adjustments. e co-localization network of IgA+ and IgG+ B plasma cells within the ST data. Nodes represent single cells and edges represent co- located single cells (Pearson correlation of cell abundance >0.2). Abbreviations: GC germinal center, DZ dark zone, LZ light zone, prePB preplasmablast, mem memory cDC classical dendritic cell, Endo endothelial, FDC follicular dendritic cell, ILC innate lymphoid cell NK natural killer NKT natural killer T TfH T follicular helper rticle https://doi.org/10.1038/s41467-023-43600-9 Fig. Discussion Integrative analysis of disassociated single-cell and in situ ST data is pivotal to construct a comprehensive map of the cellular composition and interactomes of tissues. However, because of technological lim- itations, current computational methods for integrative analysis of single-cell and ST data are limited to the cell type resolution. To deep mine the biomedical information hidden in the single-cell and ST data, here we present Redeconve, a single-cell resolution deconvolution algorithm for integrative analysis of ST data with sc/snRNA-seq data as reference based on a quadratic programming model with regulariza- tion of cell-cell similarity, which enables building of comprehensive spatial maps at single-cell resolution for diverse tissues. LðβÞ : = P J j = 1 yj P I i = 1 xijβi 2 + c P i1≠i2 Ri1,i2ðβi1 βi2Þ2 s:t: βi ≥0 for i = 1, 2, . . . , I ð1Þ ð1Þ s:t: βi ≥0 for i = 1, 2, . . . , I Here i = 1, 2, . . . , I denotes cells and j = 1,2, . . . ,J denotes genes. The ﬁrst term is the traditional Least Square (LS) term and the second term is a regularization term, c is a hyperparameter tuning the weight between the two terms. We will later explain the regularization term in details. Note that this is a typical quadratic programming problem, so we can rewrite our goal as: We performed stringent evaluation on multiple datasets from a diverse set of ST platforms. The results suggested superiority of Redeconve compared with the state-of-the-art deconvolution-based and mapping-based algorithms in terms of resolution, accuracy, sparsity, robustness, and computational speed. Such improvement from cell-type to single-cell resolution unlocks novel biological dis- coveries as exempliﬁed by applications in human pancreatic cancer and lymph node samples. minβ 1 2 βTGβ dTβ s:t: aTβ ≥b ð2Þ ð2Þ Where G is the Hessian matrix, dT = ð2P jyjx1j, . . . , 2P jyjxIjÞ, and aT, b are separately aT = 1 0 0 0 1 0 .. . .. . .. . .. . 0 0 1 0 B B B B @ 1 C C C C A , b = 0 0 .. . Discussion 0 0 B B B B @ 1 C C C C A ð3Þ While Redeconve enables deconvolution at single-cell resolution and thus will be a powerful tool for biomedical discoveries, matching between scRNA-seq and ST data appears to be an important factor determining the quality of deconvolution analysis as shown by our evaluation on different tissues (Fig. 1d). Therefore, construction and selection of reference scRNA-seq data according to the speciﬁc ST data conﬁguration will be critical in future applications. ð3Þ So we can efﬁciently solve this problem with the solve.QP function in R package “quadprog”. Although Redeconve demonstrates superior computational efﬁ- cacy compared with the state-of-the-art deconvolution algorithms, the single-cell resolution may require extensive computational cost for resolving thousands of cellular states, especially when the cellular throughput of scRNA-seq technologies increases exponentially. Because of the computational complexity of quadratic programming, Redeconve can currently resolve thousands of cellular states based on a standard machine. An enhanced version based on algorithmic inno- vation or hardware acceleration is needed to handle scRNA-seq data- sets of tens of thousands of cellular states. The regularization term. In sc/snRNA-seq data, the collinearity among cells is serious: cells of the same cell type have very similar expression proﬁles. This problem would lead to instability of coefﬁcients and reduction of efﬁciency when directly doing linear regression. To solve this collinearity problem, we further include a regularization term into the loss function. By add this term, we aim at stabilizing the coefﬁ- cients while having minor effect on the residuals. In the regularization term cP i1≠i2Ri1,i2ðβi1 βi2Þ2, Ri1,i2 is a measure of similarity between cell i1 and i2, which is Deconvolution at single-cell resolution unlocked by Redeconve may also beneﬁt the imputation of ST data with the aid of the rich information in scRNA-seq data. Redeconve has implemented a func- tion to reconstruct the gene expression proﬁles of individual spots based on the single-cell deconvolution results based on a parsimony principle. The imputed ST data may be more informative to dissect the cellular states of speciﬁc tissues. Ri1,i2 = ri1, i2, ri1,i2 > 0 0, ri1,i2 ≤0 ( ð4Þ ð4Þ Where ri1,i2 is the Pearson correlation coefﬁcient between cell i1 and i2. Namely, when the Pearson correlation coefﬁcient is greater than zero, Ri1,i2 is equal to the Pearson correlation coefﬁcient; otherwise Ri1,i2 is zero. Methods IgA+ plasma cells with CD8+ cytotoxic T cells, consistent with previous observation that CD8+ cytotoxic T cells can help the formation of IgA+ plasma cells36,37. Furthermore, co-location of IgG+ plasma cells and macrophages was identiﬁed (Fig. 6e), indicating the roles of macro- phages during the genesis of IgG+ plasma cells38,39. Hence, deconvo- lution at single cell resolution by Redeconve gains additional insights that may be helpful for uncovering previously opaque biological question. Algorithm Model overview. In general, we apply an improved linear regression model to deconvolute ST data at single-cell resolution. Given a single- cell (or single-nucleus) expression matrix X with dimensions ngenes × ncells and a ST expression matrix Y with dimensions ngenes × nspots as input, Redeconve returns a matrix β with dimensions ncells × nspots indicating the estimated number of each cell in each spot. The goal of our model is to optimize the following loss function for each spot separately: https://doi.org/10.1038/s41467-023-43600-9 https://doi.org/10.1038/s41467-023-43600-9 Redeconve sheds novel insights into the regulatory mechanisms underlying antibody class switch a pie chart displaying the spatial distribution of the estimated cell type proportion by different methods. b spatial distribution of IgA+ and IgG+ B plasma cells revealed by Redeconve. c comparison of the cell proportion of two selected spots (the IgA+ and IgG+ spots in Fig. 6a with green squares). d volcano plots showing the differential gene expression between IgA+ and IgG+ spots. The red and blue point refer to up-regulated genes in IgG+ and IgA+ spots respectively. Vertical dashed line shows the cutoff of log fold change (±0.3). Horizontal dashed line shows the threshold of -lg(p), namely 1.301. The two-side Nature Communications\| (2023) 14:7930 9 9 Article Nature Communications\| (2023) 14:7930 Real datasets for benchmarking 1. We ﬁrst calculate a hyperparameter cd according to the formula in mode “default”, and set up a series of hyperparameter c1, c2, c3, c4, c5 as 0:01cd, 0:1cd, cd, 10cd, 100cd; 1. We ﬁrst calculate a hyperparameter cd according to the formula in mode “default”, and set up a series of hyperparameter c1, c2, c3, c4, c5 as 0:01cd, 0:1cd, cd, 10cd, 100cd; PDAC. ST data of a human pancreatic ductal adenocarcinomas (PDAC-A) with 438 spots and sample-matched scRNA-seq data (InDrop) with 1926 single cells across 20 cell types were integrated by Moncada et al., and an intersection of 19,736 genes was used in our study. The annotation of four main structural regions based on his- tological analysis by Moncada et al. was used during our analysis to depict the spatial characteristics of the ST data. 2. Then we run deconvolution with these hyperparameters sepa- rately, and calculate the residual εi for each ci; 2. Then we run deconvolution with these hyperparameters sepa- rately, and calculate the residual εi for each ci; i i 3. We further calculate: 3. We further calculate: 3. We further calculate: 3. We further calculate: di = Δε Δc = εi + 1 εi ci + 1 ci ð6Þ ð6Þ Human lymph node. Human lymph node Visium data were down- loaded from the 10x Genomics website (https://www.10xgenomics. com/resources/datasets/human-lymph-node-1-standard-1-1-0), which includes a total number of 4035 spots. ScRNA-seq data were collected from Kleshchevnikov et al, of which 73,260 cells across 34 cell types were collected. Since this scRNA-seq dataset captured a wide spectrum of immune cell states spanning lymph nodes, tonsils and spleen, we used it as reference to reveal the phenotypic diversity of immune cells when deconvoluting at single cell resolution. 4. We checkthese di, then choose ci that maximizes di asthe optimal hyperparameter (This indicates: if the parameter continues to increase, the residual will increase signiﬁcantly). Namely, we choose ci that satisﬁes: 4. We checkthese di, then choose ci that maximizes di asthe optimal hyperparameter (This indicates: if the parameter continues to increase, the residual will increase signiﬁcantly). Namely, we choose ci that satisﬁes: 4. We checkthese di, then choose ci that maximizes di asthe optimal hyperparameter (This indicates: if the parameter continues to increase, the residual will increase signiﬁcantly). Article We add a pseudo-count of 0.5 to the “zeros” in sc/snRNA-seq data, and normalize sc/snRNA-seq data to TPM (transcripts per million). Preprocessing operations are not needed for ST data. 3. Normalization of reference. We add a pseudo-count of 0.5 to the “zeros” in sc/snRNA-seq data, and normalize sc/snRNA-seq data to TPM (transcripts per million). Preprocessing operations are not needed for ST data. cells In mode “autoselection”, we apply the following method to determine the optimal hyperparameter: Real datasets for benchmarking Namely, we choose ci that satisﬁes: maxi21,2,,Idi = εi + 1 εi ci + 1 ci ð7Þ ð7Þ By this procedure, we can get the hyperparameter that maximizes the power of regularization term while having minor effect on the LS term. Mouse cerebellum. The DropViz scRNA-seq dataset were generated by Saunders A. et al. and were collected by Cable D. M. et al. along with the annotations of the cells. The Slide-seq mouse cerebellum data were collected by Cable D. M. et al. using the Slide-seq v2 protocol11. Both of these datasets were downloaded from https://singlecell. broadinstitute.org/single_cell/study/SCP948/robust-decomposition- of-cell-type-mixtures-in-spatial-transcriptomics#study-download. We use examples to illustrate the effect of hyperparameters on the results. We applied Redeconve to the human lymph node dataset with a series of different hyperparameters from 0 to 1e08, then cal- culated the deconvolution residuals (RMSE_normal) to evaluate the effect of hyperparameter (Supplementary Fig. 32). The results showed that an optimal hyperparameter can enhance the deconvolution pre- cision in addition to avoiding co-linearity caused by closely similar cell states. Also, the hyperparameter would also affect the number of cell states selected in the result. A bigger hyperparameter would lead to more cell states selected (Supplementary Fig. 33). We set the hyper- parameter as 0 and 1e04 separately on the PDAC dataset. With a hyperparameter of 1e04, more T cells were detected than a hyper- parameter of zero in the PDAC dataset (Supplementary Fig. 34). Con- sidering the distribution of CD3+ cells (Shown in Supplementary Figs. 25–27), this example clearly illustrates how the hyperparameter enables biological discovery. Human breast cancer. Human Breast Cancer Visium data related to the Wu et al. study40 was available at https://zenodo.org/record/ 4739739#.Ys0v6jdBy3D. Sample ‘CID4290’ that includes 2426 in tissue spots was used for deconvolution. ScRNA-seq data that includes 100,064 single cells with annotations (Access number: GSE176078, the NCBI GEO database) served as reference to do deconvolution analysis. Human testis. The processed Human Testis Slide-seq dataset was download from https://www.dropbox.com/s/q5djhy006dq1yhw/ Human.7z?dl=0 and sample ‘Puck5’ with 36,591 spots was used for evaluation in this study41. The reference scRNA-seq data that includes 6490 single cells was obtained from the NCBI GEO database with access number GSE112013, and the corresponding annotations were available in the supplementary information Table S1 by Guo et al. Article https://doi.org/10.1038/s41467-023-43600-9 2. Gene ﬁltering. Deconvoluting with tens of thousands of genes is time-consuming or even misleading, so we select highly variable genes before deconvolution for computational efﬁcacy. Filtering criteria include the following three standards: (1) These genes appear in both sc/snRNA-seq data and ST; (2) The variance of these genes in sc/snRNA-seq data must be larger than a threshold (default is 0.025); (3) The average counts per spot must be bigger than a threshold (default is 0.003). This ﬁnally results in ~8000 genes for deconvolution. Redeconve allows deconvolution with- out gene ﬁltering with higher computational cost. 2. Gene ﬁltering. Deconvoluting with tens of thousands of genes is time-consuming or even misleading, so we select highly variable genes before deconvolution for computational efﬁcacy. Filtering criteria include the following three standards: (1) These genes appear in both sc/snRNA-seq data and ST; (2) The variance of these genes in sc/snRNA-seq data must be larger than a threshold (default is 0.025); (3) The average counts per spot must be bigger than a threshold (default is 0.003). This ﬁnally results in ~8000 genes for deconvolution. Redeconve allows deconvolution with- out gene ﬁltering with higher computational cost. 2. Gene ﬁltering. Deconvoluting with tens of thousands of genes is time-consuming or even misleading, so we select highly variable genes before deconvolution for computational efﬁcacy. Filtering criteria include the following three standards: (1) These genes appear in both sc/snRNA-seq data and ST; (2) The variance of these genes in sc/snRNA-seq data must be larger than a threshold (default is 0.025); (3) The average counts per spot must be bigger than a threshold (default is 0.003). This ﬁnally results in ~8000 genes for deconvolution. Redeconve allows deconvolution with- out gene ﬁltering with higher computational cost. 3. “autoselection”: automatically calculate and select the optimal hyperparameter. 3. “autoselection”: automatically calculate and select the optimal hyperparameter. In mode “default”, we use the following formula to set the hyperparameter: c = c0 ngenes=n2 cells ð5Þ ð5Þ Where c0 is a predetermined constant and is set to 105. The idea of this formula is: (1) the LS term is approximately proportional to ngenes, so as ngenes increases c should synchronously increase; (2) the regularization term is approximately proportional to the square of ncells, so as ncells increases c should decrease by n2 cells. 3. Normalization of reference. Discussion So, we manually bring the coefﬁcients of cells whose expression proﬁle is similar closer. By doing this, we can guarantee the robustness and precision of our result. Where ri1,i2 is the Pearson correlation coefﬁcient between cell i1 and i2. Namely, when the Pearson correlation coefﬁcient is greater than zero, Ri1,i2 is equal to the Pearson correlation coefﬁcient; otherwise Ri1,i2 is zero. So, we manually bring the coefﬁcients of cells whose expression proﬁle is similar closer. By doing this, we can guarantee the robustness and precision of our result. In summary, we present an algorithm named as Redeconve for conducting deconvolution-based analysis of scRNA-seq and ST data at single-cell resolution. The usage of Redeconve is expected to help mapping the cellular architecture at ﬁne granularity across diverse biomedical situations including tumor, immune, development, neu- rology, and other health and disease conditions. Applications to human pancreatic cancer and lymph nodes showed the potential of Redeconve to bring completely novel insights due to the single-cell resolution unlocked and the superior technical metrics of Redeconve compared to the current state-of-the-art algorithms. We expect Redeconve will be a useful tool to advance the application of scRNA- seq and ST technologies in diverse research disciplines. Determination of the hyperparameter. A key point of this model is how to select the hyperparameter: an extremely small hyperparameter will make the regularization term ineffective, while an extremely large one will greatly affect the ﬁtting residuals. An ideal hyperparameter should be as large as possible while affecting the ﬁtting residual as little as possible. Here we offer 3 ways to set the hyperparameter: 1. “default”: use the default hyperparameter we set according to the number of cells and genes; 1. “default”: use the default hyperparameter we set according to the number of cells and genes; Nature Communications\| (2023) 14:7930 10 Article Comparing Redeconve with alternative methods When β is uniformly distributed (namely βi is a constant, 1 I, for all i), we can know by simple calculation that the perplexity equals to the number of states I. This means that perplexity can reveal the number of states when the distribution is uniform. For other distributions, perplexity can also approximately represent the number of states. “Number of states” in the setting of single-cell deconvolution refers to “number of cell states (or types)”. Namely, the perplexity of each spot can approximately represent the number of cell states/types occurred in this spot. By calculating perplexity on simulated and real datasets, we have veriﬁed that perplexity showed good consistency with number of non-zero cell types/states in the result, but poor consistency with absolute cell abundance (Supplementary Fig. 35 and Supplementary Table 1). Calculating performance metrics. To demonstrate superior perfor- mance of Redeconve, we ﬁrstly estimated predicted expression pro- ﬁles for spatial spots. For all datasets, spot-wise cosine similarities, Pearson’s correlations and RMSEs between observed and predicted spot-by-gene expression matrix were calculated. In order to compute these metrics based on the output of each algorithm, we calculated the predicted expression matrix through two ways: (1) for Redeconve, NovoSpaRc, CellTrek and Tangram, we multiplied spot-by-cell abun- dance or proportion matrix by the cell-by-gene sc/snRNA expression matrix; (2) for cell2location, DestVI and CARD, we multiplied the cell- type abundance or proportion matrix by the reference cell-type expression matrix, where the reference was generated through aver- aging sc/snRNA expression data according to cell types. When calcu- lating RMSEs, the total number of UMIs for each spot in both observed and predicted expression proﬁle was normalized to ngenes. We then estimated sparsity of the results through calculating cell-type pro- portion matrices of all programs and comparing the results according to cell-type information entropy and L0 norm. The L0-norm represents number of cell types present at each spot (nonzero values). We also evaluated the performance of cell abundance estimation by Pearson’s correlation between results of individual methods (Redeconve, Cell-type level benchmark based on the PDAC dataset. Marker genes were ﬁrst identiﬁed for each cell types (Supplementary Table 2). Then, for each cell type, similarities of marker genes expression between ST observation and reconstructed proﬁles by different algo- rithms across all spots were calculated. Ranks of cosine similarities of individual cell types were used as metrics to summarize the overall performance. Comparing Redeconve with alternative methods p g We compared Redeconve with recently developed deconvolution- based methods (cell2location, DestVI13 and CARD10) as well as mapping-based methods (NovoSpaRc, CellTrek8 and Tangram7). Criteria of selecting alternative methods. In considering which methods to include for the comparison, we required methods that (1) are speciﬁcally designed for end-to-end estimating the abundance/ proportion of cells or cell types using scRNA-seq and ST data as input; (2) demonstrate superior performance in the corresponding publica- tions and third-party evaluation papers; and (3) are peer reviewed with a publicly available software implementation before Dec 2022. Information entropy and perplexity. We calculate Information entropy H and perplexity P for each spot separately by the following formula: Parameter setting. Prediction results for the 6 datasets were obtained by running the corresponding programs of the algorithms aforemen- tioned based on the default settings except some special considera- tions: (1) 1000 cells were randomly selected in NovoSpacRc to avoid large number of total cells; (2) 1000 stratiﬁed samples of cells were used for Redeconve in almost all the datasets except PDAC where we used total 1926 cells; (3) minCountGene and minCountSpot of the createCARDObject function were set to 0 to prevent unexpected gene or spot ﬁltering in CARD. The output of each method was either a cell- by-spot matrix represented absolute abundance (Redeconve, Tan- gram) or proportion (NovoSpaRc) of single cells existing at each spot or estimated cell-type abundance (cell2location) or proportion (DestVI, CARD) matrix except CellTrek, of which the outcome was predicted spatial coordinates for individual cells. Hence, for CellTrek, we obtained cell-by-spot abundance matrix by assigning single cells to speciﬁc spots according to whether the spot area designed by ST platforms covered the predicted coordinates. We only evaluated CellTrek on the two 10x Genomics Visium-based datasets (human lymph node and human breast cancer) because of running errors on other ST datasets in our computational environment. H = X i βi log2 ðβiÞ ð8Þ P = 2H ð9Þ H = X i βi log2 ðβiÞ ð8Þ ð8Þ P = 2H ð9Þ ð9Þ where i = 1, 2, . . . , I denotes different cell states. βi were normalized in advance so that their sum equaled to 1 (i.e., they denote proportion rather than absolute abundance). Data preprocessing To run the deconvolution, the following data preprocessing steps are necessary. Note that some steps are alternative according to users’ needs. 1. Get the expression proﬁles of cell type/Sampling of single cells. If a cell-type deconvolution is to be run, we will estimate the expression proﬁle xij of cell type i and gene j as the average expression of gene j across all cells within cell type i. If a single-cell deconvolution is to be run and the number of single cells is overwhelming, we will take stratiﬁed samples of cells by cell type to get a rational number of cells. Mouse brain. 10x Visium and snRNA-seq data (includes annotation) were available in the ArrayExpress database with accession numbers E- MTAB-11114 and E-MTAB-11115, respectively12. Sample ‘ST8059048’ containing 2987 spots was used for evaluation in this study, and all 40,532 single cells across 59 cell types served as reference. In addition, the corresponding data of nuclei counts estimated by histological image segmentation based on deep learning s was downloaded from Nature Communications\| (2023) 14:7930 11 Article https://doi.org/10.1038/s41467-023-43600-9 cell2location, CellTrek and Tangram) and the cell numbers estimated by histological image segmentation based on deep learning for the mouse brain dataset. Finally, computational efﬁciencies were esti- mated through comparing total time spent by each algorithm on a computer with Intel(R) Xeon(R) Platinum 8253 CPU, where we set the maximum number of cores to 96. In addition, we tested the run time of these programs on a single NVIDIA A40 card if GPU acceleration sup- ported (cell2location, DestVI, NovoSpaRc, and Tangram). https://github.com/vitkl/cell2location_paper/blob/master/notebooks/ selected_results/mouse_visium_snrna/segmentation/144600.csv. Nature Communications\| (2023) 14:7930 https://github.com/vitkl/cell2location_paper/blob/master/notebooks/ selected_results/mouse_visium_snrna/segmentation/144600.csv. Human breast cancer xenium. The Human Breast Cancer Xenium dataset is available at https://www.10xgenomics.com/products/ xenium-in-situ/preview-dataset-human-breast. A single FFPE tissue block was analyzed by scFFPE-seq, Visium and Xenium. In addition, 3’ and 5’ gene expression data from dissociated tumor cells is also available4. Assessment at single cell resolution. Cell-by-spot abundance matrix is required for comparison among deconvolution-based methods at single-cell resolution. We, therefore, applied Redeconve with 1000 single cells sampled from the reference scRNA-seq data for the two ST datasets (PDAC and human lymph node) and assigned every single cell a unique cell type since cell2location, DestVI and CARD only support cell-type deconvolution. The result matrices of Redeconve, cell2location and DestVI (no result was available for CARD because of running errors) was obtained according to the corresponding default settings. Cosine similarity, information entropy, perplexity and run- time efﬁciencies were evaluated as mentioned above. Comparing Redeconve with alternative methods In addition, linear regression and statistical test were used to show relationship between cell type abundances and perfor- mance metrics. Cell abundance of ST spots on PDAC dataset. To generate ground truth of cell abundance for each ST spot, we ﬁrst registered H&E and ﬂuorescent images using Adobe Photoshop CC. Such registration enabled the determination of spatial coordinates for ST spots. After that, Cellpose13 was applied through squidpy14 to detect cell nuclei from the H&E image. Finally, we counted the absolute number of nuclei within each spot and referred to these values as cell abundance. Nature Communications\| (2023) 14:7930 12 https://doi.org/10.1038/s41467-023-43600-9 Article Generating and analyzing simulation datasets ﬁrst compared Redeconve with existing tools as described in the aforementioned sections. Then, to study the distribution of T cells, we distinguished from NK cells T cells by the expression of CD3D, CD3E or CD3G in the scRNA-seq data. We further picked out those T cells that frequently appeared in the ST spots (T cells 8, 11, and 35). To study the spatial colocalization of these T cells with other cells, we calculated the Pearson’s correlation of cell abundance across spatial spots, and gen- erated a colocalization network of single cell resolution using those cell pairs whose Pearson correlation were greater than 0.4 with the R package igraph43. ﬁrst compared Redeconve with existing tools as described in the aforementioned sections. Then, to study the distribution of T cells, we distinguished from NK cells T cells by the expression of CD3D, CD3E or CD3G in the scRNA-seq data. We further picked out those T cells that frequently appeared in the ST spots (T cells 8, 11, and 35). To study the spatial colocalization of these T cells with other cells, we calculated the Pearson’s correlation of cell abundance across spatial spots, and gen- erated a colocalization network of single cell resolution using those cell pairs whose Pearson correlation were greater than 0.4 with the R package igraph43. We used 3 scRNA-seq data to generate simulation data separately: PDAC, human lymph node and human testis. Prior to analysis, all scRNA-seq data were down-sampled to around 1000 cells, with the exception of PDAC which contained a total of 1926 cells. To generate a pseudo-bulk for subsequent deconvolution, all single-cells were aggregated together and assigned an abundance value of 1. Data availability y All relevant data supporting the key ﬁndings of this study are available within the article and its Supplementary Information ﬁles. The PDAC data used in this study are available in the Gene Expression Omnibus database under accession code GSE111672. The processed human lymph nodes Visium data are available at 10x Genomics website [https://www.10xgenomics.com/resources/datasets/human-lymph- node-1-standard-1-1-0]. The processed human lymph nodes scRNA-seq data are available from Kleshchevnikov et al. [https://cell2location.cog. sanger.ac.uk/browser.html]. The mouse cerebellum data used in this study are available in the Single Cell Portal database under accession code SCP948 [https://singlecell.broadinstitute.org/single_cell/study/ SCP948/robust-decomposition-of-cell-type-mixtures-in-spatial- transcriptomics#study-download]. The processed human breast can- cer Visium data are available at zenodo [https://zenodo.org/record/ 4739739#.Ys0v6jdBy3D]. The processed human breast cancer scRNA- seq data used in this study are available in the Gene Expression Omnibus database under accession code GSE176078. The processed human testis Slide-seq data are available at dropbox [https://www. dropbox.com/s/q5djhy006dq1yhw/Human.7z?dl=0]. The processed human testis scRNA-seq data used in this study are available in the Gene Expression Omnibus database under accession code GSE112013. The processed mouse brain Visium data used in this study are available in the ArrayExpress database under accession code E-MTAB-11114. The processed mouse brain snRNA-seq data used in this study are available in the ArrayExpress database under accession code E-MTAB-11115. The processed Visium, 3’ scRNA-seq, 5’ scRNA-seq and scFFPE-seq for human breast cancer Xenium dataset are available at 10x Genomics website [https://www.10xgenomics.com/products/xenium-in-situ/ preview-dataset-human-breast]. Source data are provided with this paper. All relevant data supporting the key ﬁndings of this study are available within the article and its Supplementary Information ﬁles. The PDAC data used in this study are available in the Gene Expression Omnibus database under accession code GSE111672. The processed human lymph nodes Visium data are available at 10x Genomics website [https://www.10xgenomics.com/resources/datasets/human-lymph- node-1-standard-1-1-0]. The processed human lymph nodes scRNA-seq data are available from Kleshchevnikov et al. [https://cell2location.cog. sanger.ac.uk/browser.html]. The mouse cerebellum data used in this study are available in the Single Cell Portal database under accession code SCP948 [https://singlecell.broadinstitute.org/single_cell/study/ SCP948/robust-decomposition-of-cell-type-mixtures-in-spatial- i i # d d l d] h d h b Based on the generated ground truths, we computed spot-wise cosine similarities between predicted and ground truth cell type pro- portions for Redeconve and alternative methods. In this approach, we chose scFFPE-seq data as reference for the deconvolution. In addition, Pearson’s correlation was applied to measure the performance of cell abundance estimation for Redeconve, cell2location and Tangram. Data availability Finally, a selection of distinct single-cell references (including 5’, 3’, and scFFPE-seq) were applied for the purpose of assessing robustness of the computational algorithms. transcriptomics#study-download]. The processed human breast can- cer Visium data are available at zenodo [https://zenodo.org/record/ 4739739#.Ys0v6jdBy3D]. The processed human breast cancer scRNA- seq data used in this study are available in the Gene Expression Omnibus database under accession code GSE176078. The processed human testis Slide-seq data are available at dropbox [https://www. dropbox.com/s/q5djhy006dq1yhw/Human.7z?dl=0]. The processed human testis scRNA-seq data used in this study are available in the Gene Expression Omnibus database under accession code GSE112013. The processed mouse brain Visium data used in this study are available in the ArrayExpress database under accession code E-MTAB-11114. The processed mouse brain snRNA-seq data used in this study are available in the ArrayExpress database under accession code E-MTAB-11115. The processed Visium, 3’ scRNA-seq, 5’ scRNA-seq and scFFPE-seq for human breast cancer Xenium dataset are available at 10x Genomics website [https://www.10xgenomics.com/products/xenium-in-situ/ preview-dataset-human-breast]. Source data are provided with this paper. Downstream analyses after Redeconve deconvolution Human lymph node. We ﬁrstly ran Redeconve on default setting to obtain deconvolution result at single-cell resolution. Then, we inves- tigated the spatial distribution of plasma cells after grouping these plasma cells into IgA + , IgG+ and others based on the expression of IGHA1, IGHG1, IGHG3 and IGHG4. IgA+ and IgG+ spots were determined by the following three steps: (1) identifying the top 50% spots with the highest abundance of IgA+ and IgG+ plasma cell enriched, which were named as spot sets A and G; (2) identifying the difference sets between A and G, and naming as AD and GD; (3) selecting spots from AD and GD with the top 1% IgA+ and IgG+ plasma abundance, which were assumed to be IgA+ and IgG+ spots respectively. EdgeR42 was applied to perform differential gene expression analysis and identiﬁed signiﬁcantly dif- ferential genes between IgA+ and IgG+ spots. Then, we calculated Pearson’s correlation coefﬁcient among single cell states in the refer- ence across IgA+ and IgG+ spots and took single cells as nodes and correlated cells (Pearson > 0.2) as edges to generate the cell-cell co- location network. Reporting summary h f Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article. Comparing Redeconve with alternative methods To perform deconvolution, we clustered the scRNA-seq reference with 5 different resolutions using FindCluster() function in Seurat package. Together with directly using all single-cells as input, this results in 6 groups of references. Then the differently annotated references were used for deconvolution by Redeconve and cell2location and the results were used to compare with ground-truth, calculate cosine similarity and perplexity (Fig. 2c, d and Supplementary Figs. 16–18). Statistics and reproducibility For all datasets except for PDAC, we down sampled the sc/snRNA-seq reference to around 1000 cells. Stratiﬁed sampling was performed when cell types are available, otherwise simple random sampling was performed. The exact number of chosen cells for each dataset are as follows: human breast cancer: 1001, human lymph nodes: 1000, human testis: 999, Mouse Brain: 1003, Mouse cerebellum: 1003, human breast cancer Xenium (scFFPE): 1001, human breast cancer Xenium (3’): 998, human breast cancer Xenium (5’): 1002. The seed was set to 2233. All other parts of this study do not involve randomization. The Investigators were not blinded to allocation during experiments and outcome assessment. Benchmarking on human breast cancer Xenium dataset Benchmarking on human breast cancer Xenium dataset The Human Breast Cancer Xenium dataset contains scRNA-seq, Visium and Xenium data for a single FFPE tissue block. By mapping Xenium cells to Visium spots, it becomes possible to generate ground truth data regarding cell abundances and cell type proportions. To achieve this, we chose Replicate 1 of Xenium data to align spatial locations of Xenium cell centers to corresponding H&E images through translation and rotation. After that, a key-point registration approach was employed to align H&E images in Xenium and Visium data based on 155 manually identiﬁed landmark features on commonly shared micro- structures. Then, FindHomography() function in cv2 package with RANSAC method was applied to transform Xenium to Visium coordi- nates. Hence, the ground truths of cell abundance were generated through counting the transformed cell centers located within each Visium spot. To further generate ground truths of cell type proportion for Visium spots, we labeled each cluster in scFFPE-seq and Xenium data with a corresponding cell type designation (Supplementary Table. 3–4). The proportions of various types of Xenium cells in Visium spots were considered as ground truth cell type proportions. References 24. Moncada, R. et al. 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Zenodo, https://doi.org/10.5281/zenodo.8384152 (2023). https://doi.org/10.1038/s41467-023-43600-9 https://doi.org/10.1038/s41467-023-43600-9 Code availability The codes used to generate the ﬁgures in this paper is available at https://codeocean.com/capsule/1351962/tree/v1. The package is avail- able on GitHub with detailed documentation at https://github.com/ ZxZhou4150/Redeconve, https://doi.org/10.5281/zenodo.838415221. PDAC. We ran Redeconve with all the 1926 single cells as reference, and all the parameters were kept default. For downstream analyses, we Nature Communications\| (2023) 14:7930 13 Article https://doi.org/10.1038/s41467-023-43600-9 Reprints and permissions information is available at http://www.nature.com/reprints X.R. conceived this study, designed the algorithm, supervised the ana- lysis, and wrote the manuscript. Z.X.Z developed the software, con- ducted the data analysis, and wrote the manuscript. Y.Z. conducted the data analysis and wrote the manuscript. Z.M.Z provided valuable dis- cussion on the data analysis and wrote the manuscript. Publisher’s note Springer Nature remains neutral with regard to jur- isdictional claims in published maps and institutional afﬁliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/ licenses/by/4.0/. Competing interests p g The authors declare no competing interests. Acknowledgements 22. Stringer, C., Wang, T., Michaelos, M. & Pachitariu, M. Cellpose: a generalist algorithm for cellular segmentation. Nat. Methods 18, 100–106 (2021). This work was supported by Changping Laboratory, the National Natural Science Foundation of China (32022016 X.R., 92159305 X.R., and 31991171X.R.), National Key R&D Program of China (2020YFE0202200 X.R. and 2022YFC3400904 X.R.). 23. Palla, G. et al. Squidpy: a scalable framework for spatial omics analysis. Nat. Methods 19, 171–178 (2022). 14 Nature Communications\| (2023) 14:7930 Article https://doi.org/10.1038/s41467-023-43600-9 Author contributions Author contributions Reprints and permissions information is available at http://www.nature.com/reprints Additional information Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41467-023-43600-9. Supplementary information The o supplementary material available at Correspondence and requests for materials should be addressed to Xianwen Ren Correspondence and requests for materials should be addressed to Xianwen Ren. Peer review information Nature Communications thanks Ken Chen, Jing Su and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. A peer review ﬁle is available. © The Author(s) 2023 Nature Communications\| (2023) 14:7930 15
https://openalex.org/W3120220360	https://research.manchester.ac.uk/files/194102642/fpsyt_11_589545.pdf	English	null	Factors Associated With Psychological Distress in Health-Care Workers During an Infectious Disease Outbreak: A Rapid Systematic Review of the Evidence	Frontiers in psychiatry	2,021	cc-by	21,530	The University of Manchester Researc The University of Manchester Researc The University of Manchester Research Published in: Frontiers in Psychiatry Citing this paper Please note that where the full-text provided on Manchester Research Explorer is the Author Accepted Manuscript or Proof version this may differ from the final Published version. If citing, it is advised that you check and use the publisher's definitive version. 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Oct. 2024 SYSTEMATIC REVIEW published: 28 January 2021 doi: 10.3389/fpsyt.2020.589545 Factors Associated With Psychological Distress in Health-Care Workers During an Infectious Disease Outbreak: A Rapid Systematic Review of the Evidence Fuschia M. Sirois 1* and Janine Owens 2 Fuschia M. Sirois 1* and Janine Owens 2 1 Department of Psychology, University of Shefﬁeld, Shefﬁeld, United Kingdom, 2 School of Clinical Dentistry, University of Shefﬁeld, Shefﬁeld, United Kingdom Objective: Health-care workers (HCW) are at risk for psychological distress during an infectious disease outbreak, such as the coronavirus pandemic, due to the demands of dealing with a public health emergency. This rapid systematic review examined the factors associated with psychological distress among HCW during an outbreak. Method: We systematically reviewed literature on the factors associated with psychological distress (demographic characteristics, occupational, social, psychological, and infection-related factors) in HCW during an outbreak (COVID-19, SARS, MERS, H1N1, H7N9, and Ebola). Four electronic databases were searched (2000 to 15 November 2020) for relevant peer-reviewed research according to a pre-registered protocol. A narrative synthesis was conducted to identify ﬁxed, modiﬁable, and infection-related factors linked to distress and psychiatric morbidity. Keywords: COVID-19, health-care workers, psychological distress, risk factors, resilience, anxiety, stress, depression Edited by: Roger Mcintyre, University of Toronto, Canada Reviewed by: Lorena García-Fernández, Miguel Hernández University of Elche, Spain Felicity Southworth, Public Health England, United Kingdom C d Edited by: Roger Mcintyre, University of Toronto, Canada Reviewed by: Lorena García-Fernández, Miguel Hernández University of Elche, Spain Felicity Southworth, Public Health England, United Kingdom Results: From the 4,621 records identiﬁed, 138 with data from 143,246 HCW in 139 studies were included. All but two studies were cross-sectional. The majority of the studies were conducted during COVID-19 (k = 107, N = 34,334) and SARS (k = 21, N = 18,096). Consistent evidence indicated that being female, a nurse, experiencing stigma, maladaptive coping, having contact or risk of contact with infected patients, and experiencing quarantine, were risk factors for psychological distress among HCW. Personal and organizational social support, perceiving control, positive work attitudes, sufﬁcient information about the outbreak and proper protection, training, and resources, were associated with less psychological distress. Correspondence: Fuschia M. Sirois f.sirois@shefﬁeld.ac.uk Specialty section: This article was submitted to Mood and Anxiety Disorders, a section of the journal Frontiers in Psychiatry Received: 30 July 2020 Accepted: 15 December 2020 Published: 28 January 2021 Conclusions: This review highlights the key factors to the identify HCW who are most at risk for psychological distress during an outbreak and modifying factors to reduce distress and improve resilience. Recommendations are that HCW at risk for increased distress receive early interventions and ongoing monitoring because there is evidence that HCW distress can persist for up to 3 years after an outbreak. Further research needs to track the associations of risk and resilience factors with distress over time and the extent to which certain factors are inter-related and contribute to sustained or transient distress. INTRODUCTION The review focused not only on the COVID-19 pandemic, but also on other related coronavirus and inﬂuenza outbreaks (SARS, H1N1, H7N9, MERS, and Ebola), to expand the potential evidence base and to increase the potential for the ﬁndings to be generalizable across any future infectious disease outbreaks. Under normal circumstances, work-related psychological distress in HCW is associated with several short and long- term adverse outcomes. Psychological distress is linked to adverse occupational outcomes including include decreased quality of patient care (5), irritability with colleagues (6), cognitive impairments that negatively impact patient care (7), and intentions to leave one’s job (8). HCW who experience psychological distress are also at risk of experiencing adverse personal outcomes including substance misuse (6), and suicide (9). In the context of an infectious disease outbreak, such consequences may amplify and heighten psychological distress. HCW who reported elevated levels of psychological distress during the COVID-19 outbreak also experienced sleep disturbances (10), poorer physical health (11), and a greater number of physical symptoms, including headaches (12). Similarly, HCW during the SARS outbreak disclosed a greater number of somatic symptoms and sleep problems (13), substance misuse and more days oﬀ work (14). This review also introduced a conceptual framework for understanding and classifying the factors that contributed to risk or provided resilience for psychological distress. Based on our early scan of the literature, we grouped factors into three conceptual categories: (1) ﬁxed or unchangeable factors, (2) potentially modiﬁable factors, and (3) factors related to infection exposure. Fixed factors were viewed as identifying HCW who might be most vulnerable or resilient to distress and, if the former, require extra support and treatment. Socio- demographic factors and other factors related to work role and experience were included in this category. In contrast, modiﬁable factors were viewed as identifying potential targets for interventions to reduce risk and increase resilience. Social and psychological factors, such as social support, stigma, and psychological resources such as coping styles and personality were included in the modiﬁable category. Lastly, infection- related factors were those that can directly inform hospital procedures and operating policy regarding ways to address and mitigate risk. Factors related to infection exposure and risk of exposure, and the provision of training, resources, and personal protective equipment (PPE) were included in this category. INTRODUCTION HCW who worked in high risk and low risk areas initially, 1 year later the stress of the high-risk HCW was signiﬁcantly increased, and was higher than the stress reported by the low-risk HCW (16). This increased level of stress was also associated with higher levels of depression, anxiety, and post- traumatic stress, indicating a pervasive and sustained negative impact of working during an outbreak on mental health. These ﬁndings underscore the importance of understanding the factors that contribute to risk and resilience for psychological distress in HCW. Several outbreaks of viral diseases have posed signiﬁcant public health threats since 2000. These include SARS, H1N1, H7N9, MERS, EBOLA, and more recently, COVID-19 (see Supplementary Table 1). Such outbreaks place a serious strain on the health-care systems that try to contain and manage them, including health-care workers (HCW) who are at increased risk for nosocomial infections (1). In addition to the threat to their own physical health, HCW can experience psychological distress as a collateral cost of the risk of infection and the demands of dealing with a public health emergency (2). HCW serve a vital role in treating and managing infected individuals during an infectious disease outbreak such as coronavirus. There is an urgent need to understand the factors that create or heighten risk for distress for HCW and aﬀect their immediate and long-term mental health during the COVID- 19 pandemic and other similar outbreaks, as well as those that are protective and may reduce psychological distress. Such knowledge is important for identifying HCW most at risk, and informing strategies and treatments needed to support HCW resilience during and after an outbreak. Psychological distress refers to a state of emotional suﬀering, resulting from being exposed to a stressful event that poses a threat to one’s physical or mental health (3). Inability to cope eﬀectively with the stressor results in psychological distress that can manifest as a range of adverse mental health and psychiatric outcomes including depression, anxiety, acute stress, post-traumatic stress, burnout, and psychiatric morbidity. Although psychological distress is often viewed as a transient state that negatively impacts day-to-day and social functioning, it can persist and have longer-term negative eﬀects on mental health (4). This rapid review synthesized the evidence on the factors associated with psychological distress among health-care workers (HCW) during an infectious disease outbreak. Citation: Sirois FM and Owens J (2021) Factors Associated With Psychological Distress in Health-Care Workers During an Infectious Disease Outbreak: A Rapid Systematic Review of the Evidence. Front. Psychiatry 11:589545. doi: 10.3389/fpsyt.2020.589545 January 2021 \| Volume 11 \| Article 589545 Frontiers in Psychiatry \| www.frontiersin.org 1 Distress in Health-Care Workers Sirois and Owens Frontiers in Psychiatry \| www.frontiersin.org Data Synthesis and Analysis y y We conceptually organized the factors in this Review identiﬁed as contributing to or mitigating psychological distress into three broad categories: (1) ﬁxed or unchangeable factors (sociodemographic and occupational factors), (2) potentially modiﬁable factors (social and psychological factors), and (3) factors related to infection exposure. Evidence was synthesized according to these conceptual categories, with non-signiﬁcant and contrary ﬁndings noted in addition to signiﬁcant ﬁndings to provide a more complete picture of the weight of the evidence for each factor. The balance of evidence for each factor was further presented graphically. We assigned factors within each conceptual category as reﬂecting either risk or resilience for psychological distress according to logic and theory (e.g., maladaptive coping as risk, adaptive coping as resilience). Factors that could be interpreted as either risk or resilience (e.g., sex, age) were assigned according to how they had been framed in the majority of the research that examined these factors. INTRODUCTION Apart from the immediate and short-term impacts on HCW mental health, there is limited but concerning evidence, that working during an infectious outbreak can have lasting and detrimental psychological eﬀects for HCW. In a study of HCW who worked during the SARS outbreak in China, 10 percent experienced high levels of post-traumatic stress (PTS) symptoms when surveyed 3 years later (15). Similarly, HCW who treated patients during the SARS outbreak in Canada reported signiﬁcantly higher levels of burnout, psychological distress, and post-traumatic stress compared to HCW in other hospitals that did not treat SARS patients when surveyed 13– 26 months after the SARS outbreak (14). Lastly, a study of HCW in Hong Kong during the SARS outbreak found that although the levels of perceived stress did not diﬀer between The key questions addressed by this review were: 1) What are the risk factors for psychological distress among HCW during an infectious outbreak? 2) What are the factors associated with reduced risk for psychological distress among HCW during an infectious outbreak? January 2021 \| Volume 11 \| Article 589545 2 Distress in Health-Care Workers Sirois and Owens Frontiers in Psychiatry \| www.frontiersin.org Study Selection and Data Extraction y We used a predeﬁned search strategy (see full details on PROSPERO, https://www.crd.york.ac.uk/PROSPERO/; registration ID: CRD42020178185). Studies were included in this Review if they were empirical research; published or accepted for publication in peer-reviewed journals; written in English; included participants who were HCW who worked in a hospital environment during a major infectious outbreak (COVID19, SARS, MERS, H1N1, H7N9, Ebola); had a sample size of >80, and included data on factors associated with psychological distress during an outbreak. One investigator screened citations for potential full-text review, and a second investigator conducted the full-text review of each study for inclusion. Exclusions were veriﬁed by the other investigator, and disagreements resolved through discussion. Data was extracted by one investigator, entered into a table, and veriﬁed by a second investigator. For studies that included tests for multiple measures of psychological distress, we included the study as reporting a signiﬁcant association with a particular factor if at least one of the measures of distress were signiﬁcant. RESULTS The search yielded 4621 records, with 138 papers reporting 139 studies (Total N = 143,246 HCW) that met inclusion criteria for this Review. Figure 1 presents the complete screening process. Characteristics of the studies are in Table 1. The average sample size was 1,030 (range 82–21,199). The studies included HCW working across 34 countries during COVID-19 (k = 107, N = 120,711), SARS (k = 21, N = 18,096), MERS (k = 7, N = 1,567), H1N1 (k = 2, N = 2,094), Ebola (k = 1, N = 143), and H7N9 (k = 1, N = 102), outbreaks. The rates of psychological distress in HCW varied depending on how distress was measured (Table 1). Figures 2, 3 provide a graphical overview of the weight of the evidence per factor. Data Sources and Searches The search strategy for this pre-registered rapid review involved searching Medline, PsycInfo, Web of Science, and the ﬁrst 10 pages of Google Scholar, as well as hand searching references. Search terms included a combination of terms related to health- care workers (e.g., “physicians,” “nurses”), and distress (e.g., “stress,” “anxiety”). The full search term list is available on PROSPERO (CRD42020178185). We conducted searches in a rolling manner, starting on April 6, 2020, then with updates on June 7, July 2, July 10, July 30, 2020, and November 15, 2020 to capture and integrate the most up-to-date evidence given the ongoing COVID-19 pandemic and the associated rapid release of research. METHODS (19) as being most relevant for the current study, an approach advocated by Quintana (20). Two authors independently rated the quality of the studies using the 11 questions to assess the quality of the study procedures, sampling, and the measures. The assessment yielded a total score that categorized studies as having low (<5), moderate (5–7), or high (8–10) quality. Inter- rater agreement was calculated and assessed using Cohen’s Kappa coeﬃcient (21). Discrepancies were resolved through discussion. In addition to the formal quality assessment, we only included studies that reported ﬁndings for a sample size of >80, which allows enough power to detect a medium eﬀect size with an alpha of 0.05 (21, 22). Evidence was summarized using a rapid, systematic review approach because of the urgent need to support the mental health of HCW during and after the ongoing novel coronavirus pandemic. Rapid Reviews are a form of systematic review that provide an expedient and useful means of synthesizing the available evidence during times of health crises to inform evidence-based decision making for health policy and practice (17, 18). To accomplish this, rapid reviews take a streamlined approach to systematically reviewing evidence. Modiﬁed methods in the current review included: (1) search limited to English language studies; (2) gray literature limited to one search source; (3) no formal critical appraisal of the research. Evidence was summarized using a rapid, systematic review approach because of the urgent need to support the mental health of HCW during and after the ongoing novel coronavirus pandemic. Rapid Reviews are a form of systematic review that provide an expedient and useful means of synthesizing the available evidence during times of health crises to inform evidence-based decision making for health policy and practice (17, 18). To accomplish this, rapid reviews take a streamlined approach to systematically reviewing evidence. Modiﬁed methods in the current review included: (1) search limited to English language studies; (2) gray literature limited to one search source; (3) no formal critical appraisal of the research. Methodological Quality Although rapid reviews do not always include a formal assessment of study quality and risk for bias (18), a lack of a quality assessment can have important implications for the utility of the results (17). Accordingly, we evaluated the methodological quality of the studies in the review using a tool adapted for the current study. The assessment tool included eleven questions chosen from the Appraisal tool for Cross Sectional Studies, AXIS The quality of the studies ranged from moderate to high, with no studies rated as having low quality. The majority of the 139 studies were rated as having high quality (118; 84.9%), and 21 studies were rated as having a moderate quality (15.1%). Inter- rater agreement was high, 90.65% agreement, Cohen’s Kappa = 0.642 (see Supplementary Table 2). January 2021 \| Volume 11 \| Article 589545 3 Distress in Health-Care Workers Sirois and Owens FIGURE 1 \| PRISMA ﬂow diagram for literature screening. Sociodemographic Factors working during the SARS outbreak who met psychiatric caseness for PTSD were more likely to be younger (144). In a study during the H1N1 outbreak, hospital staﬀwho were in their 20’s had greater anxiety about becoming infected than did older staﬀ(103). During COVID-19, HCW who were younger were more likely to experience higher levels of post-traumatic stress symptoms, depression, anxiety, and acute stress compared to older HCW (23, 26–28, 30, 32, 42, 49, 54, 55, 57, 61, 65, 71, 75, 78, 89, 90, 117, 118, 121, 123, 127, 131, 149, 153, 154). In contrast, eight studies conducted during COVID found that HCW who were older were at greater risk of experiencing higher levels of psychological distress (40, 66, 86, 95, 102, 114, 122, 132). Seventy-two studies examined age as a predictor of psychological distress among HCW during an epidemic (see Table 2). Of these, 39 found that age was a signiﬁcant risk factor for distress. In two studies of HCW during the SARS outbreak, staﬀwho were younger than 33 experienced greater stress, but not greater psychiatric morbidity, compared to older staﬀ(134), and staﬀ under 35 were more likely to report severe depressive symptoms 3 years after the outbreak (92). In another study, medical staﬀ who were between 20 and 30 years old and exposed to patients with H7N9 had elevated post-traumatic stress disorder scores compared to older staﬀ(157). Similarly, general practitioners in January 2021 \| Volume 11 \| Article 589545 Frontiers in Psychiatry \| www.frontiersin.org 4 Distress in Health-Care Workers Sirois and Owens Lastly, 33 studies found that age was not a signiﬁcant predictor of distress in HCW during the SARS, MERS or during the COVID-19 outbreaks (Table 2). (41) were more likely to experience distress while working during the COVID-19 pandemic and the SARS outbreak. Sixteen studies conducted during the COVID-19 outbreak did not ﬁnd that occupational role was a risk factor for distress (Table 2). Ninety studies tested sex as a possible risk factor for distress among HCW during an outbreak (Table 2), with all but 33 ﬁnding that being female was associated with higher risk for psychological distress. Notably, the 57 studies that found that female sex was a signiﬁcant risk factor spanned six diﬀerent infectious diseases (MERS, SARS, COVID-19, H1N1, H7N9, and SARS), suggesting that being a female HCW increases vulnerability for distress more generally when working during an infectious outbreak. Sociodemographic Factors Notably, among the studies 30 studies that did not ﬁnd that being female created signiﬁcant risk for distress, eleven (36.6%) were conducted with nurses and included predominantly female participants (24, 43, 44, 59, 70, 79, 80, 89, 108, 140, 153). Other occupational factors examined included years of work experience, and full-time vs. part-time status. Twelve of the 35 studies found evidence to suggest that less work experience may create risk (Table 2). HCW who had worked for <2 years experienced signiﬁcantly greater stress than those with more work experience in a large sample of HCW during the SARS pandemic (13). In HCW during the SARS outbreak, those with <10 years of experience reported higher levels of psychological distress, but not burnout or post-traumatic stress, 13–26 months after the outbreak (14). HCW who had less clinical experience were also more likely to experience stress during the COVID-19 outbreak (23, 28, 55, 65, 69, 154). Years of clinical experience was not associated with PTSD symptoms, acute stress or anxiety, depression, mental health status, or burnout in 21 other studies (Table 2). Two studies found that less work experience was protective against distress for HCW during COVID-19 (121, 122). Lastly, in one study, part-time worker status was a signiﬁcant predictor of greater emotional distress in HCW during the SARS outbreak (107), whereas another study found no evidence of part-time work status creating risk for distress in HCW during COVID-19 (119). Of the 69 studies that examined marital status as a risk or resilience factor for psychological distress, 19 found evidence to suggest this as a risk factor (Table 2). For example, two studies of HCW during the SARS outbreak found that HCW who were single were 1.4 times more likely to experience psychological distress than married HCW (41), and more likely to have sever depressive symptoms 3 years later (92). Similarly, HCW during the COVID-19 outbreak who were single experienced higher levels of distress than those who were married (54, 57, 66, 69, 111, 122, 126). Conversely, four studies conducted during COVID-19 found that being married was a risk factor for greater distress (66, 75, 89, 94), and two studies found that married HCW with children reported greater stress than single HCW or those who were married without children (72, 83). Social Factors A number of social and interpersonal factors mitigated or contributed to psychological distress. Receiving direct social support from friends, family, colleagues and supervisors was a key protective factor in all of the 19 studies that examined its association with psychological distress (Table 2). For example, in HCW during the COVID-19 outbreak, higher levels of social support were associated with signiﬁcantly lower levels of stress, depression, anxiety, depression and PTSD (28, 31, 38, 62, 70, 78, 88, 90, 94, 102, 139, 156). These ﬁndings were consistent with that of a study of frontline medical staﬀduring the COVID-19 outbreak who reported that a positive attitude from co-workers was important for reducing their distress (39). Analogously, emergency nurses working during MERS outbreak who reported poor support from family and friends experienced higher levels of burnout (81). Similarly, studies of HCW during the SARS outbreak found that higher levels of family support were associated with lower depression and anxiety whereas inadequate support from relatives, lack of gratitude from patients and relatives, and perceiving less of a team spirit at work was associated with higher levels of psychological distress (44, 134). Thirty-three studies examined education levels in association with distress. Only eight studies, six conducted during the COVID-19 pandemic (27, 66, 70, 89, 94, 149, 151), along with studies conducted during the Ebola outbreak (76), and the MERS outbreak (81) found that HCW with higher educational levels reported signiﬁcantly lower psychological distress. Twenty- two studies found that education level was not predictive of psychological distress among HCW working during the MERS or the COVID-19 outbreaks (Table 2). Sociodemographic Factors Forty-seven other studies conducted during the SARS, MERS, and COVID- 19 outbreaks found no associations between HCW marital status and distress (Table 2). Occupational Factors Thirty-four studies examined and found evidence that the HCW occupational role created risk for psychological distress while working during the SARS, H1N1, MERS, and COVID- 19 outbreaks (Table 2). In all but 16 studies, being a nurse was associated with a range of mental health issues, including higher stress, burnout, anxiety, depression, PTSD symptoms, psychiatric morbidity, and psychological distress compared to being a physician or other HCW (see Tables 1, 2). The extent to which nurses experienced greater psychological distress whilst working during an outbreak was estimated in several studies. For example, nurses were 1.2 (83), 1.4 (124), 2.2 (63), and 2.8 (107) times more likely to be at risk for poor mental health. In contrast, ﬁve studies found that physicians (13, 97, 119, 128) and technicians Organizational support was an important factor in buﬀering psychological distress of HCW during an outbreak in all 11 studies that examined this factor. In nurses working during the SARS outbreak in Canada, higher perceived organizational support in the form of receiving positive performance feedback from doctors and co-workers, was associated with lower perceptions of SARS-related threat and reduced feelings of emotional exhaustion (59). Similarly, nurses, physicians, and HCW working during the MERS, COVID-19, and SARS January 2021 \| Volume 11 \| Article 589545 Frontiers in Psychiatry \| www.frontiersin.org 5 Rates of distress %) Risk/resilience factors tested 21.1 (Low stress) 69.4 (Moderate stress) 9.6 (high stress) Age, sex, work experience 65.0 (moderate to severe psychological distress) Social support- professional/organizational NR Sex, exposure to conﬁrmed infected cases. 30.0 (moderate to severe anxiety) Sex, age, HCW type 17.8 (moderate depression) 16.8 (severe depression) 18.1 (extremely severe depression) 17.8 (moderate anxiety) 13.9 (severe anxiety) 22.6 (extremely severe anxiety) 19.7 (moderate stress) 16.6 (severe stress) 7.9 (extremely severe stress Age, sex, marital status, HCW type, higher education level, direct contact with conﬁrmed infected cases 69.0 (depression) 59.8 (anxiety) 55.9 (stress) Sex, age, social support-personal, social support- professional/organizational 27.84 (mild anxiety) 23.90 (moderate anxiety) 9.74 (severe anxiety) Age, Sex 46.5 (anxiety) 30.2 (depression) Age, sex, marital status single vs. married NR Sex, marital status; married with children, social support-personal, direct contact with infected cases, adaptive and maladaptive coping style, positive work attitudes (Continued) n the rapid review. Occupational Factors % female) Infectious disease Study period Psychological distress measures Rates of distress (%) Risk/resilience factors tested ors (25.4) COVID-19 09/04/2020– 14/04/2020 PSS-10 to measure stress 21.1 (Low stress) 69.4 (Moderate stress) 9.6 (high stress) Age, sex, work experience s (78.87) COVID-19 18/01/2020– 20/01/2020 K-6 to measure non-speciﬁc psychological distress 65.0 (moderate to severe psychological distress) Social support- professional/organizational s and (92.70) COVID-19 1/04/2020– 14/04/2020 STAI to measure anxiety NR Sex, exposure to conﬁrmed infected cases. ors and 7.30) COVID-19 NR PSS-10 to measure stress, GAD-7 to measure anxiety 30.0 (moderate to severe anxiety) Sex, age, HCW type (79.10) COVID-19 16/04/2020– 20/04/2020 DASS to measure depressive symptoms, anxiety and stress 17.8 (moderate depression) 16.8 (severe depression) 18.1 (extremely severe depression) 17.8 (moderate anxiety) 13.9 (severe anxiety) 22.6 (extremely severe anxiety) 19.7 (moderate stress) 16.6 (severe stress) 7.9 (extremely severe stress Age, sex, marital status, HCW type, higher education level, direct contact with conﬁrmed infected cases ors, nurses -ancillary 49.8) COVID-19 14/04/2020–24/04/ 2020 DASS-21 to measure stress, depressive symptoms and anxiety 69.0 (depression) 59.8 (anxiety) 55.9 (stress) Sex, age, social support-personal, social support- professional/organizational ors (44.78) COVID-19 Last week of March 2020 GAD-7 to measure anxiety 27.84 (mild anxiety) 23.90 (moderate anxiety) 9.74 (severe anxiety) Age, Sex W (34.20) COVID-19 30/04/2020–25/5/ 2020 HADS to measure anxiety and depressive symptoms, MBI to measure burnout 46.5 (anxiety) 30.2 (depression) Age, sex, marital status single vs. Occupational Factors married (80.3) COVID-19 11/04/2020– 16/04/2020 PSS-10 to measure stress NR Sex, marital status; married with children, social support-personal, direct contact with infected cases, adaptive and maladaptive coping style, positive work attitudes (Continued) d Sample (% female) Infectious disease Study period Psychological distress measures Rates of distress (%) Risk/resilience factors tested 509 doctors and nurses (80.30) COVID-19 1st 2 weeks of April 2020 GAD-7 to measure anxiety, PSS-10 to measure stress 25.9 (moderate to severe anxiety) 56.4 (high stress) Age, sex, marital status, HCW type, exposure to conﬁrmed infected cases 376 doctors and nurses (73.70) COVID-19 5 weeks from the beginning of COVID-19 epidemic in Italy MBI to measure burnout 37.0 (high emotional exhaustion) Sex, HCW type 117 doctors, nurses and allied health professionals (77.00) COVID-19 3/04/2020– 18/04/2020 GAD-7 to measure anxiety, PCL-5 to measure post-traumatic stress disorder 33.0 (anxiety) 17.0 (distress: PTSD) HCW type 580 doctors, nurses and allied health professionals (40.00) COVID-19 26/03/2020– 9/04/2020 Middle of outbreak in Italy GHQ-12 to measure psychological distress 33.5 (psychological distress) Sex, HCW type, marital status: married with children, direct contact with infected cases, perceived control, adaptive copin style 270 HCW (73.7) COVID-19 10/04/2020– 13/04/2020 PDI to measure levels of distress, PHQ-9 to measure depressive symptoms, PTSD-8 to measure post-traumatic stress disorder 16.7 (distress PTSD) Age, sex 386 HCW (86.00) MERS NR Study speciﬁc measure of worry about contracting MERS 33.2 (extremely or very worried) Sex, direct contact with conﬁrme infected cases 1,521 HCW (75.54) COVID-19 NR SCL-90-R to measure psychological distress 14.1 (psychological distress) Age, sex, marital status: married with children, HCW type, Social support-personal, less work experience, adaptive personality traits 534 HCW (68.70) COVID-19 01/2020–03/2020 Study speciﬁc measure of stress NR Social support-personal 208 HCW in the ICU (75.00) COVID-19 8/04/2020– 21/04/2020 Peak of the pandemic HADS to measure anxiety and depressive symptoms, IES-R to measure post-traumatic stress disorder 48.0 (anxiety) 16.0 (depression) 27.0 (distress; PTSD) Sex, age, HCW type, risk of exposure to conﬁrmed cases 661 doctors and nurses (NR) SARS 05/2003 2 months after SARS outbreak IES-R, to measure post-traumatic stress disorder, GHQ-28 to measure distress 27.0 (distress; PTSD) HCW type, marital status, social support-personal, adequate information, positive work attitude (Continue ed e, at ample (% female) Infectious disease Study period Psychological distress measures Rates of distress (%) Risk/resilience factors test 52 doctors (21.70) COVID-19 28/03/2020– 06/04/2020 DASS-21 to measure depressive symptoms, stress and anxiety 34.9 (depression) 39.5 (anxiety) 32.9 (stress) Age, sex, less work experienc risk of being in contact with infected patients 28 nurses (100.00) SARS During mid-May 2003, at the peak of the SARS outbreak. Occupational Factors IES to measure PTSD, SCL-90-R to measure psychological distress 11.0 (distress: PTSD) At risk of being in contact with infected patients 16 nurses (98.30) SARS May 2003 SAS to measure anxiety, SDS to measure depressive symptoms NR Social support-personal, train for dealing with SARS provide 02 HCW (68.63) COVID-19 9/02/2020– 11/02/2020 Peak of pandemic CMBI to measure post-traumatic stress disorder, GAD-7 to measure anxiety, PHQ-9 to measure depressive symptoms 24.5 (moderate-severe anxiety and depression) 16.63 (moderate to severe anxiety) 18.29 (moderate to severe depression) Sex, HCW type, adaptive and maladaptive coping style, ada personality traits 2,956 nurses 95.60) COVID-19 April 2020 MBI GS to measure extent of emotional exhaustion, 24.7 and 23.5 (emotional exhaustion HRW) Sex, exposure to conﬁrmed infected cases 71 HCW (67.83) 94 HRW [74.50], 77 RW [59.70]) COVID-19 NR PCL-C to measure post-traumatic stress disorder, GAD-7 to measure anxiety, PHQ-9 to measure depressive symptoms 28.7 (distress; PTSD:HRW) 13.0 (distress; PTSD:LRW) 63.8 (anxiety: HRW) 45.5 (anxiety: LRW) 19.1 (moderate to severe depression: HRW) 6.5 (moderate to severe depression LRW) Sex, higher education level, H type, direct exposure with conﬁrmed infected cases ,146 HCW (65.10) COVID-19 29/04/2020– 4/06/2020 DASS-21 to measure stress, depressive symptoms and anxiety, IES to measure post-traumatic stress disorder NR Sex ,257 HCW (81.10) SARS 12/05/2003– 27/06/2003 6 weeks during outbreak IES-R to measure post-traumatic stress disorder, CHQ to measure psychiatric morbidity 75.3 (psychiatric morbidity) Sex, marital status, HCW type work experience, exposure to conﬁrmed infected cases (Contin ple (% female) Infectious disease Study period Psychological distress measures Rates of distress (%) Risk/resilience factors teste doctors (25.80) COVID-19 14/05/2020– 31/05/2020 GAD-7 to measure anxiety, IES-R to measure post-traumatic stress disorder, PHQ-2 to measure depressive symptoms, Mini-Z to measure physician burnout 14.7 (emotional burnout) 19.7 (moderate-severe anxiety) 26.3 (distress; PTSD) 16.3 (depression) Age, sex 2 HCW (82.90) COVID-19 4/04/2020– 10/04/2020 Peak of pandemic GAD-7 to measure anxiety, PHQ-9 to measure depressive symptoms, PHQ-15 to measure physical symptoms related to distress 77.10 (emotional burnout) 63.4 (distress) 88.4 (anxiety) 86.1 (depression) Sex, HCW type doctors (47.00) COVID-19 3/04/2020– 11/04/2020 8 days Study speciﬁc measures for anxiety and stress 61.0 (anxiety) Sex, Hospital resources/protection/training f the treatment of infection doctors and es (72.40) COVID-19 19/03/2020– 05/04/2020 PCL-5 to measure PTSD, BDI-II to measure depressive symptoms, STAI to measure anxiety NR Exposure to conﬁrmed infecte cases HCW (78.50) COVID-19 16/04/2020– 13/05/2020 PHQ-9 to measure depressive symptoms, GAD-7 to measure anxiety, IES-R to measure post-traumatic stress disorder, PFI to measure burnout 2126.2 (distress: PTSD) 31.0 (moderate-severe depression) 71.0 (anxiety) 29.0 (distress: PTSD) Adaptive personality traits, les work experience, direct contac with conﬁrmed infected cases HCW (56.80) COVID-19 10/03/2020– 15/03/2020 DASS-21 to measure depressive symptoms, stress and anxiety 64.7 (depression) 51.6 (anxiety) 41.2 (stress) Age, sex, marital status, less w experience, social support- professional/organizational, hospital resources, protection, training, at risk of being in con with infected patients doctors and es (51.90) COVID-19 18/04/2020– 28/04/2020 HADS to measure anxiety and depression 56.3 (depression) 46.7 (anxiety) Age, sex, marital status, less w experience, stigma HCW (50.00) COVID-19 April–May 2020 GAD-7 to measure anxiety, PHQ-9 to measure depressive symptoms, PSS to measure level of perceived stress 76.4 (anxiety) 77.2 (depression) 80.9 (stress) Sex (Contin Sample (% female) Infectious disease Study period Psychological distress measures Rates of distress (%) Risk/resilience factors tested 395 HCW (73.60) COVID-19 March–April 2020 DASS-21 to measure stress, depressive symptoms and anxiety, HARS to measure anxiety, MADRS to measure depressive symptoms 31.4 (moderate-severe anxiety) 12.1 (moderate-severe depression) 14.5 (moderate-severe stress) Age, sex, marital status, direct contact with conﬁrmed infected cases ,050 doctors 71.50) COVID-19 May 2020 1 month DASS-21 to measure stress, depressive symptoms and anxiety 31.0 (depression) 29.7 (anxiety) 23.5 (stress) Perceived control, adaptive coping styles 333 nurses (94.59) SARS 03/2004–05/2004 Study speciﬁc measures on worry about contracting SARS, MBI GS to assess extent of emotional exhaustion NR Social support- professional/organizational, direct contact with infected cases, time spent in quarantine 781 HCW (NR) COVID-19 29/03/2020– 05/04/2020 1 week during the peak of the outbreak HAM-A to measure anxiety, BDI to measure depressive symptoms, ASDI to measure stress NR Work experience, Adequate information, Hospital resources, protection, training ,059 HCW (72.70) COVID-19 5/06/2020– 25/06/2020 GADS to measure anxiety and depression 81.0 (depression) 76.5 (anxiety) Age, sex, HCW type, direct contact with conﬁrmed infected cases 330 HCW (62.60) COVID-19 16/04/2020– 11/05/2020 STAI to measure anxiety, DASS-21 to measure stress, depressive symptoms and anxiety, IES-6 to measure post-traumatic stress disorder, MBI to measure burnout 71.2 (anxiety) 26.8 (depression) 34.3 (stress) 36.7 (distress; PTSD) Sex, HCW type, social support-personal, direct contact with conﬁrmed infected cases 469 HCW (68.40) H1N1 1/09/2009– 30/09/2009 At the beginning of the second wave of the pandemic GHQ-28 to measure psychological distress, study speciﬁc measure of worry about H1N1 27.5 (mild to severe psychological distress) 56.7 (worry) HCW type, stigma, adequate information, positive work attitudes 93 physicians 32.10) SARS During the SARS outbreak in 2003 Study speciﬁc question about new distressing psychological symptoms 18.1 (new distressing symptoms) Direct contact with conﬁrmed infected cases ,124 HCW (36.10) COVID-19 30/03/2020– 2/04/2020 4 days HADS to measure anxiety and depression 37.2 (anxiety) 31.4 (depression) Age, sex, marital status, higher education level, HCW type, less experience, direct contact with infected cases, hospital resources, protection, training 21,199 nurses 98.60) COVID-19 7/02/2020– 10/02/2020 SAS to measure anxiety, SDS to measure depressive symptoms 3.9 (moderate anxiety) 0.8 (severe anxiety) 6.9 (moderate depression) 1.3 (severe depression) Sex, age, marital status, direct contact with infected cases, at risk of being in contact with infected patients, (Continued) disease measures (%) 151 doctors (56.30) COVID-19 30/04/2020– 16/05/2020 GAD-7 to measure anxiety 14.6 (moderate anxiety) 3.3. Occupational Factors (severe anxiety) Sex, direct contact with conﬁrmed cases 82 HCW (56.09) SARS 5/04/03–5/05/03 During height of outbreak Study speciﬁc measures of worry about contracting SARS NR Perceived control 97 HCW (82.50) SARS Sample 2 08/2003 CIES–R to measure post-traumatic stress disorder NR Perceived control 688 HCW (85.00) COVID-19 15/05/2020– 10/06/2020 DASS-21 to measure stress, depressive symptoms and anxiety 25.0 (psychological distress) Sex, marital status, less experience, direct contact with conﬁrmed infected cases 4,692 nurses (96.90) COVID-19 8/02/2020– 14/02/2020 2 weeks after the authority in Wuhan suspended all public transport on 23/01/2020 PHQ-9 to measure depressive symptoms, GAD-7 to measure anxiety 9.4 (depressive symptoms) 8.1 (anxiety) Marital status, higher education level, social support-personal, social support-professional and organizational, perceived risk 200 HCW (80.00) COVID-19 March 2020–May 2020 DASS-21 to measure stress, depressive symptoms and anxiety NR Age, sex, marital status, higher education level, 2,014 nurses (87.10) COVID-19 13/02/2020- 24/02/2020 At the peak of the outbreak MBI-HSS to measure burnout, SAS to measure anxiety, SDS to measure depressive symptoms 60.5 (emotional exhaustion) 14.3 (anxiety) 10.7 (depression) Age, sex, marital status, social support-personal, higher education level, less work experience, social support-personal, perceived control, adaptive personality traits, at risk of being in contact with infected patients, hospital resources, protection, training 587 mixture of radiology staff (52.00) COVID-19 7/02/2020– 9/02/2020 CPSS to measure stress, CSAS to measure anxiety NR Sex, marital status 720 HCW (NR) COVID-19 21/04/2020 for 3 weeks Survey was open during the state of Indiana’s peak day of COVID-19 cases on 26/04/2020 Grit-S to measure perceived grit NR Adaptive coping style, hospital resources, protection, training 512 anaesthesiologists (44.30) COVID-19 12/05/2020– 22/05/2020 GAD-7 to measure anxiety 74.2 (anxiety) Age, sex, marital status, less work experience, direct contact with infected cases, hospital protection (PPE) for treatment of infected cases (Continued) Sample (% female) Infectious disease Study period Psychological distress measures Rates of distress (%) Risk/resilience factors tested 143 medical staff and students (49.50) Ebola (EVD) 13/02/2015– 19/03/2015 During Ebola outbreak SCL-90-R to measure psychological symptoms NR Educational level 253 HCW (83.00) COVID-19 NR IES-R to measure post-traumatic stress disorder NR Sex, HCW type 456 doctors and Nurses (70.60) COVID-19 01/02/2020– 14/02/2020 IES-R to measure post-traumatic stress disorder, GAD-7 to measure anxiety, PHQ-9 to measure depressive symptoms 37.5 (psychological distress) 31.6 (anxiety) 29.6 (depression) Sex, age, level of education, HCW type, direct contact with infected cases, risk of contact with infected cases, stigma, social support-personal, time spent in quarantine 147 nurses (NR) MERS 1/10/2015– 30/11/2015 Shortly after the MERS epidemic ended IES-RK to measure post-traumatic stress disorder, GHQ-12 to measure mental health, study speciﬁc measure of stress 57.1 (distress: PTSD) Social support- Professional/organizational 380 nurses (84.21) COVID-19 NR CAPS to measure post-traumatic stress disorder, NR Social support-organizational/ professional 223 ED nurses (93.50) MERS 20/07/2015– 31/07/2015. Occupational Factors 2 months after the outbreak of MERS during uncontrolled disease period OLBI to assess MERS-related burnout NR Age, sex, marital status, level of education, work experience, direc contact with infected cases, socia support-personal, hospital resources, protection, training 112 nurses (88.30) MERS 30/06/2015– 10/07/2015 IES to measure post-traumatic stress disorder, MBI-HSS to measure burnout. Occupational Factors 50.0 (distress: PTSD) Age, sex, marital status, higher level of education, less work experience 7,614 HCW (82.00) SARS 05/2003–07/2003 Toward the tail end of the pandemic IES to measure post-traumatic stress disorder; single item to measure perceived stress at work 56.0 (stress) HCW type, marital status, Stigma exposure to SARS 1,257 HCW (76.70) COVID-19 29/01/20–3/02/20 During pandemic PHQ-9 to measure depression, GAD-7 to measure anxiety, CIES-R to measure post-traumatic stress disorder 50.4 (depression) 44.6 (anxiety) 71.5 (distress: PTSD) Sex, HCW type, direct contact with conﬁrmed infected cases (Continued Sample (% female) Infectious disease Study period Psychological distress measures Rates of distress (%) Risk/resilience factors tested 359 HCW (81.90) MERS 05/32015–12/2015 During the outbreak IES-R to measure post-traumatic stress 51.0 (distress: PTSD) Sex, age, HCW type, at risk of being in contact with infected patients, time spent in quarantine 90 nurses (72.20) COVID-19 11/03/2020– 18/03/2020 At the time of the survey, nurses had worked in Wuhan for at least 32 days CPSS to measure psychological distress, PCL-C to measure post-traumatic stress disorder 5.6 (distress: PTSD) Sex, age, marital status, level of education, less work experience 908 HCW (75.55) COVID-19 3/02/2020- 24/02/2020 Survey began 10 days after state of emergency declared on 23/01/2020 SAS to measure anxiety, SDS to measure depressive symptoms 24.34 (anxiety) 32.93 (depression) Less work experience, direct contact with conﬁrmed infected cases 225 reserve medics (72.0) COVID-19 4/04/2020– 6/04/2020 IES-R to measure post-traumatic stress disorder, DASS-21 to measure depressive symptoms, stress and anxiety 46.7 (depression) 35.6 (anxiety) 16.0 (stress) 31.6 (distress: PTSD) Sex, age social support-professional/ organizational 356 nurses (86.2) COVID-19 01/2020–03/2020 PSS-10 to measure stress, PCL-5 to measure post-traumatic stress disorder NR Age, marital status, level of education, les work experience, job role, direct contact with infected cases, adaptive personality traits 1,092 nurses (99.51) COVID-19 02/2020 SSAR to measure stress NR Age, sex, marital status, level of education, social support-personal, perceived control 114 HCW (79.80) COVID-19 02/2020 HADS to measure anxiety and depression NR Adaptive and maladaptive coping styles, adaptive personality traits 549 HCW (75.2) SARS In 2006, 3 years after Beijing’s SARS outbreak CES-D to measure depressive symptoms 22.8 (moderate or severe depression) Sex, age, marital status, altruistic perspective toward work, exposure to infection, being quarantined 512 HCW (79.96) COVID-19 10/02/20–20/02/20 During pandemic SAS to measure anxiety 12.5 (mild to severe anxiety) Sex, age, marital status, level of education, HCW type, direct contact with conﬁrmed infected cases 1,090 HCW (80.20) COVID-19 24/02/2020– 9/03/2020 PSS-10 to measure stress, GAD-7 to measure anxiety, PHQ-9 to measure depressive symptoms 13.3 (anxiety) 18.4 (depression) 23.9 (anxiety and depression) Age, sex, marital status, HCW type, level of education, less experience, social support-personal (Continued ted vel o h mple (% female) Infectious disease Study period Psychological distress measures Rates of distress (%) Risk/resilience factors test 31 doctors and ses (85.52) COVID-19 17/02-2020– 23/02/2020 DASS-21 to measure stress, depressive symptoms and anxiety 14.81 (depression) 18.3 (anxiety) 9.98 (stress) Sex, age, HCW type, role, lev education, direct contact with conﬁrmed infected cases HCW (58.27) SARS 07/2003–03/2004 CHQ to assess psychiatric morbidity 17.3 (psychiatric morbidity) Neuroticism 42 HCW (77.90) COVID-19 25/02/2020– 26/02/2020 HAM-A to measure anxiety, HAM-D to measure depressive symptoms NR Direct contact with conﬁrmed infected cases HCW (70.10) COVID-19 27/03/2020– 30/04/2020 GADS to measure anxiety and depression 16.6 (anxiety) 20.3 (depression) Age, sex, exposure to conﬁrm infected cases doctors, nurses, allied health fessionals (54.80) COVID-19 29/03/2020– 15/04/2020 Study speciﬁc measure of stress 74.0 (stress) Age, sex, HCW type, marital status; married with children, contact with infected cases, s support-organizational, hospi resources, protection, training HCW (79.00) COVID-19 6/04/2020– 19/04/2020 Middle of lockdown in Spain and at peak of pandemic MBI to measure burnout Age, sex, HCW type, hospita resources (PPE) for treatment infection HCW (NR) COVID-19 24/03/2020– 13/05/2020 Phase 1 of Italian COVID-19 emergency GHQ-12 to measure psychological distress 21.26 (psychological distress) Age, less experience, perceiv control nurses (76.70) COVID-19 3/02/2020– 11/02/2020 GHQ-12 to measure psychological distress, IES-R to measure post-traumatic stress disorder 25.1 (psychological distress) Sex, age, level of education, marital status, less experience adaptive and maladaptive cop styles, stigma, social support-personal, hospital resources, protection, training 25 HCW (75.60) H1N1 16/03/2009– 31/07/2009 Approximately 1 month after the peak of outbreak IES to measure post-traumatic stress disorder, study speciﬁc measures on stress NR Age, sex, HCW type, at risk o being in contact with infected patients 57 HCW (74.60) SARS 12/05/2003– 20/06/2003 During the outbreak IES to measure psychological stress NR Direct contact with infected c stigma (Conti ample (% female) Infectious disease Study period Psychological distress measures Rates of distress (%) Risk/resilience factors tested 7 HCW (87.80) SARS 23/10/2004– 30/09/2005 13–26 months after outbreak IES to measure post-traumatic stress disorder, K10 to measure non-speciﬁc psychological distress, MBI-EE to measure burnout NR Work experience, stigma, maladaptive coping styles, maladaptive personality traits, direct contact with infected cases time spent in quarantine = 176 = 184 HCW 3.25, T1; 64.50, ) SARS T1: 15/04/2003– 15/05/2003. Occupational Factors During the peak period of hospital admissions for SARS. T2: 2004 PSS-10 to measure stress, DASS-21 to measure stress, depressive symptoms and anxiety, IES-R to measure post-traumatic stress disorder NR At risk of being in contact with infected patients 0 nurses (89.00) COVID-19 22/02/2020 SAS to measure subjective anxiety, SOS to measure stress NR Sex, marital status, level of education, perceived control, direct contact with conﬁrmed infected cases 106 doctors (49.0) COVID-19 19/03/2020– 22/03/2020 Whilst conﬁrmed cases were rising Study speciﬁc measures of stress NR Marital status, hospital training fo treatment of infection, adaptive personality traits 0 HCW (78.80) SARS 10/04/2003– 22/04/2003 Conducted during the peak of the initial phase of the SARS outbreak GHQ-12 to measure psychological distress 29.0 (distress) HCW type, part-time work status 7 nurses (100.00) MERS 30/08/2015– 21/09/2015 Conducted during MERS epidemic PSS to measure level of perceived stress, SF-36 MH to measure mental health status NR Marital status, work experience, stigma, adaptive personality traits 003 HCW (77.10) COVID-19 2/04/2020– 10/04/2020 Whilst cases were increasing PHQ-9 to measure depressive symptoms, GAD-7 to measure anxiety NR HCW type, stigma, direct contact with infected cases, time spent in quarantine doctors and rses (64.60) SARS 1/11/2003– 14/11/2003 6 months after the end of the outbreak IES to measure post-traumatic stress disorder, GHQ-28 to measure psychiatric morbidity 18.8 (psychiatric morbidity), 17.7 (distress: PTSD) HCW type, maladaptive coping styles 4 doctors (44.53) COVID-19 03/04/2020– 10/04/2020 PSS-10 to measure stress 85.6 (moderate and high stress) Age, sex, marital status (Continue 87 HCW (87.80) SARS 23/10/2004– 30/09/2005 13–26 months after outbreak IES to measure post-traumatic stress disorder, K10 to measure non-speciﬁc psychological distress, MBI-EE to measure burnout NR Work experience, stigma, maladaptive coping styles, maladaptive personality traits, direct contact with infected cases time spent in quarantine = 176 2 = 184 HCW 3.25, T1; 64.50, 2) SARS T1: 15/04/2003– 15/05/2003. During the peak period of hospital admissions for SARS. Occupational Factors T2: 2004 PSS-10 to measure stress, DASS-21 to measure stress, depressive symptoms and anxiety, IES-R to measure post-traumatic stress disorder NR At risk of being in contact with infected patients 00 nurses (89.00) COVID-19 22/02/2020 SAS to measure subjective anxiety, SOS to measure stress NR Sex, marital status, level of education, perceived control, direct contact with conﬁrmed infected cases 106 doctors (49.0) COVID-19 19/03/2020– 22/03/2020 Whilst conﬁrmed cases were rising Study speciﬁc measures of stress NR Marital status, hospital training fo treatment of infection, adaptive personality traits 0 HCW (78.80) SARS 10/04/2003– 22/04/2003 Conducted during the peak of the initial phase of the SARS outbreak GHQ-12 to measure psychological distress 29.0 (distress) HCW type, part-time work status 87 nurses (100.00) MERS 30/08/2015– 21/09/2015 Conducted during MERS epidemic PSS to measure level of perceived stress, SF-36 MH to measure mental health status NR Marital status, work experience, stigma, adaptive personality traits 003 HCW (77.10) COVID-19 2/04/2020– 10/04/2020 Whilst cases were increasing PHQ-9 to measure depressive symptoms, GAD-7 to measure anxiety NR HCW type, stigma, direct contac with infected cases, time spent in quarantine 6 doctors and rses (64.60) SARS 1/11/2003– 14/11/2003 6 months after the end of the outbreak IES to measure post-traumatic stress disorder, GHQ-28 to measure psychiatric morbidity 18.8 (psychiatric morbidity), 17.7 (distress: PTSD) HCW type, maladaptive coping styles 84 doctors (44.53) COVID-19 03/04/2020– 10/04/2020 PSS-10 to measure stress 85.6 (moderate and high stress) Age, sex, marital status (Continue Sample (% female) Infectious disease Study period Psychological distress measures Rates of distress (%) Risk/resilience factors tested 1,926 HCW (NR) SARS 05/2003–06/2003 Diagnosis of the ﬁrst case of SARS occurred on 12/03/2003. Occupational Factors Hong Kong declared SARS-free on 23/06/2003 STAI to measure anxiety, MBI-EE to measure emotional burnout NR HCW type, contact with conﬁrmed infected cases 441 nurses (95.20) COVID-19 7/04/2020– 12/04/2020 GAD-7 to measure anxiety, PHQ-9 to measure depressive symptoms 38.8 (anxiety) 37.4 (depression) Age, sex, marital status, level of education, less work experience, risk of contact with infected cases, hospital resources, protection, training 347 HCW (90.80) COVID-19 14/04.20202– 25/04/2020 GAD-7 to measure anxiety, Mini Z to measure burnout, IES to measure distress, PHQ-2 to measure depressive symptoms 69.5 (anxiety) 84.1 (mild distress) 22.8 (depression) Age, HCW role 2,285 HCW (69.06) COVID-19 16/02/2020– 23/02/2020 Early stage of COVID-19 pandemic GAD-7 to measure anxiety, PHQ-9 to measure depressive symptoms 46.0 (anxiety) 44.4 (depression) Sex, at risk of being in contact with infected patients 1,407 HCW (71.50) COVID-19 11/05/2020– 31/05/2020 GHQ-28 to measure distress, SASR to measure perceived anxiety 24.7 (acute stress) Sex, age, HCW type, Hospital resources, protection, training, Social support – professional/organizational, adequate information 3,109 HCW (NR) COVID-19 09/04/2020– 19/04/2020 10 days during the outbreak Study speciﬁc measure of stress NR Age 1,379 HCW (77.20) COVID-19 27/03/2020– 31/03/2020 Days immediately preceding the peak 77.2 of the COVID-19 outbreak in Italy GAD-7 to measure anxiety, PSS to assess perceived stress, PHQ-9 to measure depressive symptoms, GPS to assess post-traumatic stress symptoms (PTSS) 49.4 (distress: PTSD) Sex, age, HCW type, colleagues being infected, quarantined, deceased 506 doctors and nurses (76.70) COVID-19 30/03/2020– 16/04/2020 PSS-14 to measure stress NR Sex, marital status, HCW type, part-time work, direct contact with conﬁrmed infected cases 135 HCW (39.30) COVID-19 6/04/2020– 11/04/2020 GAD-7 to measure anxiety, PHQ-9 to measure depressive symptoms NR Sex, marital status, HCW type, hospital resources, protection, training (Continued) Sample (% female) Infectious disease Study period Psychological distress measures Rates of distress (%) Risk/resilience factors tested 939 HCW (66.00) COVID-19 23/04/2020– 23/05/2020 GAD-7 to measure anxiety, IES-R to measure post-traumatic stress disorder, PHQ-9 to measure depressive symptoms 77.6 (depression) 60.2 (anxiety) 76.4 (psychological distress) Sex, age, HCW type, less work experience, risk of contact with infected cases 123 nurses (74.00) COVID-19 10/04/2020– 20/04/2020 STAI to measure anxiety 46.3 (anxiety) Sex, age, marital status, less work experience, direct contact with conﬁrmed infected cases 448 nurses (73.00) COVID-19 NR SAS to measure anxiety, BSI-18 to measure psychological distress 64.0 (acute stress) 41.0 (signiﬁcant psychological distress) Sex, age, perceived control 657 HCW (70.90) COVID-19 09/04/2020– 24/04/2020 GAD-2 to measure anxiety, PHQ-2 to measure depressive symptoms, PC-PTSD to measure acute stress 57.0 (acute stress) 48.0 (depression) 33.0 (anxiety) HCW type 863 HCW (70.70) COVID-19 23/02/2020– 5/03/2020 IES-6 to measure post-traumatic stress disorder, DASS-21 to measure stress, depressive symptoms and anxiety 13.6 (depression) 13.9 (anxiety) 8.6 (stress) Sex, age, marital status, level of education, HCW type, direct contact with infected cases, time spent in quarantine 153 HCW hospital staff (74.30) MERS 25/08/2015– 14/09/2015 Approximately 1 month after the end of the outbreak on 28/07/2015 IES-RK to measure post-traumatic stress disorder 18.6 (distress: PTSD) Loss of control and perceived risk adaptive coping styles and ability 14,825 doctors and nurses (64.30) COVID-19 28/02/2020– 18/03/2020 CES-D to measure depression, PCL-5 to measure post-traumatic stress disorder (PTSD) 25.2 (depression) 9.1 (PTSD) Age, sex, marital status, HCW type, less work experience, social support-personal 1,800 HCW Phase 1: 223 (79.50) Phase 2: 1577 (89.50) COVID-19 1st week of self-isolation Phase 1: 30/03/2020– 5/04/2020 Phase 2: 4/05/2020– 10/05/2020 PSM-25 to measure anxiety and distress NR Marital status, HCW type, direct contact with conﬁrmed infected cases 201 HCW Group 1: 118 (65.60) Group 2: 83 (66.30) COVID-19 NR GAD-7 to measure anxiety, SDS to measure depressive symptoms NR Direct contact with conﬁrmed infected cases (Continued Sample (% female) Infectious disease Study period Psychological distress measures Rates of distress (%) Risk/resilience factors tested 248 HCW (87.02) SARS 16/06/2003– 9/07/2003 IES-R to measure post-traumatic stress disorder NR Age, sex, marital status, work experience, adequate information, at risk of being in contact with infected patients 536 HCW (69.00) COVID-19 2/03/2020– 6/03/2020 PHQ-9 to measure depressive symptoms, GAD-7 to measure anxiety, NR Age, sex, marital status, colleagues being infected/quarantined, direct contact with conﬁrmed infected cases 442 HCW (84.30) COVID-19 31/01/2020– 4/02/2020 IES to measure post-traumatic stress disorder NR Age, sex, marital status, HCW type, less work experience, at risk of being in contact with infected patients, direct contact with infected cases, time spent in quarantine 122 HCW (64.40) COVID-19 04/2020–05/2020 HADS to measure anxiety and depression, PSS to measure perceived stress 35.6 (anxiety) 27.9 (depression) 72.8 (moderate stress) Sex, HCW type, direct contact with infected cases, hospital resources, protection, training 652 front-line Hospital HCW (79.00) SARS 06/2003–08/2003 GHQ-12 to measure psychological distress, Study speciﬁc measure for job-related stress 56.7 (psychological distress) 68.0 (stress) HCW type, age, sex, social support-personal, direct contact with infected cases, hospital resources, protection, training 3,075 HCW (71.50) COVID-19 29/05/2020– 24/06/2020 OLBI to measure burnout, HADS to measure anxiety and depression NR Sex, HCW type, level of education, positive work attitudes 102 HCW (66.70) H7N9 01/2015 and 05/2016 PCL-C to measure post-traumatic stress disorder 20.6 (distress: PTSD) Age, sex, HCW type, direct contact with infected cases, hospital resources, protection, training 798 HCW (39.60) COVID-19 20/05/2020– 20/06/2020 GAD-7 to measure anxiety NR Direct contact with conﬁrmed infected cases 452 HCW (66.20) COVID-19 20/05/2020– 10/06/2020 HADS to measure anxiety and depression NR Stigma 150 nurses (80.00) COVID-19 5/2020 Last 2 weeks PSS to measure perceived stress 50.3 (stress) Age, sex, less experience, social support-personal, direct contact with conﬁrmed infected cases 100 nurses (100.00) COVID-19 07/02/2020– 25/02/2020 In the initial stage of the outbreak when there was a shortage of nurses GAD-7 to measure anxiety, PHQ-9 to measure depressive symptoms 40.0 (anxiety) 46.0 (depression) Age, marital status, level of education, less work experience 113 doctors (46.90) COVID-19 1/04/2020– 14/04/2020 GAD-7 to measure anxiety, Beck Inventory to measure anxiety and depressive symptoms NR Sex, age, marital status, direct contact with conﬁrmed infected cases (Continued) Sample (% female) Infectious disease Study period Psychological distress measures Rates of distress (%) Risk/resilience factors tested 210 HCW (57.10) COVID-19 NR STSS to measure work related stress, study speciﬁc measure (Emergency Stress Questionnaire) of stress NR Age, sex, HCW type, adaptive coping styles, adequate information 100 doctors (44.00) COVID-19 04/2020–05/2020 Before the peak of the pandemic Beck Depression Inventory to measure anxiety and depression, 14.0 (moderate anxiety) 15.0 (moderate depression) 2.0 (severe depression) Sex, direct contact with conﬁrmed infected cases 721 doctors (38.80) SARS 05/2003 2 months after the ﬁrst case of SARS was reported in Singapore GHQ-28 to measure psychological distress, IES-R to measure post-traumatic stress disorder 14.1 (psychological distress) Age, stigma, direct contact with conﬁrmed infected cases 202 nurses (87.60) COVID-19 02/2020–03/2020 PCL-C to measure PTSD 16.8 (distress: PTSD) Sex, marital status, level of education, adaptive coping styles and adaptability, maladaptive coping styles, positive work attitudes 1,045 HCW (85.80) COVID-19 02/02/2020– 03/02/2020 HADS to measure anxiety and depression, PSS-14 to measure perceived stress 13.6 (moderate to severe depression) 20.0 (moderate to severe anxiety) Sex, HCW type, level of education, less experience, direct contact with infected cases, risk of being in contact with infected cases 350 HCW (46.60) COVID-19 10/04/2020– 25/04/2020 GAD-7 to measure anxiety, PHQ-9 to measure depressive symptoms, PSS-10 to measure distress 17.7 (moderate and severe anxiety) 11.4 (severe depression) 3.7 (high levels of stress) Sex 466 ED nurses and doctors (65.70) SARS 24/06/2003– 24/07/2003 Study speciﬁc measures on distress caused by SARS NR HCW type, loss of control and perceived risk 180 HCW treating patients with COVID-19 (71.70) COVID-19 01/2020–02/2020 SASR to measure perceived stress, SAS to measure anxiety NR Social support-personal, perceived control 309 HCW (97.40) COVID-19 7/02/2020– 21/02/2020 SAS to measure anxiety, SDS to measure depression, 28.5 (anxiety) 56.0 (depression) Age, marital status, level of education, HCW type, direct contact with conﬁrmed infected cases 223 Nurses (97.30) COVID-19 16/02/2020– 25/02/2020 GAD-7 to measure anxiety, PHQ-9 to measure depressive symptoms 40.8 (anxiety) 26.4 (depression) Age, sex, level of education, less work experience, role type, direct contact with infected cases„ perceived control (Continued) tudy period Psychological distress measures Rates of distress (%) Risk/resilience factors teste /02/2020– 0/02/2020 GHQ-12 to measure psychological distress 61.1 (psychological distress) Age, sex, marital status, level o education, HCW type, less wo experience, direct contact with infected cases, risk of contact infected cases 1/02/2020– 5/02/2020 uring the early ages of the andemic PCL-5 to measure post-traumatic stress symptoms (PTSS) 3.8 (distress: PTSD) Sex, education level, HCW typ direct contact with conﬁrmed infected cases 0/05/2020– 3/06/2020 weeks MBI-HSS to measure burnout, Beck Depression Inventory to measure depression, PSS to measure perceived stress 31.8 (depression) Age, level of education, marita status, less work experience, direct contact with infected ca adaptive personality traits 4/2020 DASS-21 to measure depressive symptoms, stress and anxiety 37.2 (mild-severe stress) 59.0 (depression) 42.6 (anxiety) Age, sex, marital status, level o education, less work experienc 9/02/2020– 6/03/2020 weeks after the utbreak in Wuhan SCL-90-R to measure psychological symptoms, PHQ-4 to measure anxiety and depressive symptoms NR Sex, at risk of being in contact infected patients /06/2020– 3/06/2020 PCL-C to measure post-traumatic stress disorder, HADS to measure depression and anxiety 41.87 (anxiety) 27.61 (depression) Sex, age, marital status, level o education, HCW type, direct contact with conﬁrmed infecte cases, time spent in quarantin social support-personal N1; MERS, Middle East respiratory syndrome; SARS, severe acute respiratory syndrome; ED, Emergency Departme s in COVID wards; LRW, low-risk workers in non-COVID wards. Sample (% female) Infectious disease S ,002 HCW (85.20) COVID-19 1/ 20 377 HCW (61.50) COVID-19 0 05 D st pa 377 midwives and urses (NR) COVID-19 30 13 2 540 HCW (45.60) COVID-19 04 927 HCW (64.96) COVID-19 19 06 8 ou 678 HCW (85.05) COVID-19 6/ 13 ARS-CoV-2); H1N1, inﬂuenza A virus subtype H1 spital; NR, not reported; HRW, high-risk workers BDI, Beck Depression Inventory; BDI-II, Beck De alth Questionnaire; CMBI, Chinese version of M ety and Stress Scale 42-item; DASS-21, Depress Scale; GHQ-12, General Health Questionnaire 1 milton Anxiety Scale; HAM-D, Hamilton Depress r Post-Traumatic Stress Disorder; IES-RK, Impac Rating Scale; MBI, Maslach Burnout Inventory; rvey; Mini-Z, Z Clinician Questionnaire (for “Zero ritraumatic Distress Inventory; PHQ-2, Patient H ; PC-PTSD, Primary Care Post-Traumatic Stress SD-8, Post-Traumatic Stress Disorder 8-item; SA Chinese version; SF-36 MH, Short Form Survey Occupational Factors pression Inventory 1996 revision; BSI-18, Brief Symptom Inventory; CAPS, Clinician Administered PTSD Scale; CES aslach Burnout Inventory; CIES-R, Chinese Impact of Event Scale—Revised; CPSS, Chinese Perceived Stress Sca sion; Anxiety and Stress Scale 21-item; GAD-2, Generalized Anxiety Disorder Scale 2-item; GAD-7, Generalized Anxi 2-item; GHQ-28, General Health Questionnaire 28-item; GPS, Global Psychotrauma Screen; Grit-S, Short Grit Sca sion Scale; HARS, Hamilton Rating Scale for Anxiety; IES, Impact of Events Scale; IES-6, Impact of Event Scale for Po t of Event Scale revised Korean version; K-10, Kessler Psychological Distress Scale 10-item; K-6, Kessler Psycholog MBI-EE, emotional exhaustion scale of the Maslach Burnout Inventory; MBI GS, Maslach Burnout Inventory—Gene ” Burnout); OLBI, Oldenburg Burnout Inventory; PCL-5; Post-traumatic Stress Disorder Check List (for DSM 5); PCL Health Questionnaire 2-item; PHQ-4, Patient Health Questionnaire 4-item; PHQ-9, 9-item Patient Health Questionna Disorder Screen for DSMIV; PSS, Perceived Stress Scale; PSS-10, Perceived Stress Scale 10-item; PSS-14, Perceiv AS, Self-Rating Anxiety Scale; SASR, Stanford Acute Stress Reaction scale; SCL-90-R, Symptoms Checklist 90-item mental health component; SOS, Stress Overload Scale; STAI, State-Trait Anxiety Inventory; STSS, Secondary Trauma ed with risk and resilience for psychological distress in health-care workers. Evidence for resilience Non-signiﬁcant ﬁndings 23), Al Mahyijari et al. (26), 28), Azoulay et al. (30), erjee et al. (42), Civantos et al. et al. (55), Erquicia et al. (57), t al. (65), al. (71), Jain et al. (75), Juan et al. (90), Liu et al. (92), ero et al. (117), Rossi et al. ahrour and Dardas (123), (131), Tam et al. (134), Tang 4), Xing et al. (149), Yörük al. (154) Caillet et al. (40), Han et al. (66), Leng et al. (86), Liu et al. (95), Master et al. (102), Prasad et al. (114), Saricam (122), Sun et al. (132) Arshad et al. (29), Blekas et al. (36), Cai et al. (38), Chen et al. (47), Chen et al. (45), Chew et al. (48), Chong et al. (13), Dobson et al. (53), Elkholy et al. (56), Hu et al. (72), Kim and Choi (81), Kim et al. (82), Lee et al. (85), Li et al. (88), Liu et al. (93), Liu et al. (94), Magnavita et al. (98), Maraqa et al. (99), Martínez-López et al. (100), Marton et al. (101), Podder et al. (111), Pouralizadeh et al. (113), Rodriguez-Menéndez et al. (116), Si et al. (125), Styra et al. (130), Tselebis et al. (139), Tu et al. Alan Cai e Mosh (132) Güle Babo (35), Elhad Hoss et al. (83), 90), Liu et al. (92), l. (117), Rossi et a and Dardas (123), Tam et al. (134), Ta g et al. (149), Yörük 4) n et al. (27), Arafa ay et al. (30), Babo ettinsoli et al. (35), 7), Caillet et al. (40) Civantos et al. (49 1), Elbay et al. (54) 7), Giardino et al. ( Han et al. (66), Ha einzadeh-Shanjani al. (73), Jain et al. t al. (84), Lee et al Magnavita et al. (9 (111) P li d Occupational Factors (140), Uyaroglu et al. (141), Vagni et al. (142), Xiong et al. (150), Yao et al. (151), Yin et al. (152), Zhang et al. (156) 23), Alan et al. (27), Arafa , Azoulay et al. (30), Babore 32), Bettinsoli et al. (35), t al. (37), Caillet et al. (40), l. (13), Civantos et al. (49), t al. (51), Elbay et al. (54), et al. (57), Giardino et al. (61), . (65), Han et al. (66), Hasan Hosseinzadeh-Shanjani ang et al. (73), Jain et al. (75), ), Lai et al. (84), Lee et al. l. (93), Magnavita et al. (98), er et al (111) Pouralizadeh Song et al. (127), Liu et al. (95), Veeraraghavan and Srinivasan (143) Aksoy and Koçak (25), Al Mahyijari et al. (26), Barello et al. (33), Cai et al. (38), Chatterjee et al. (42), Chen et al. (47), Chen et al. (45), Chew et al. (48), Elhadi et al. (55), Elkholy et al. (56), Kim and Choi (81), Kim et al. (82), Lai et al. (84), Leng et al. (86), Li et al. (88), Liu et al. (92), Liu et al. (94), Maraqa et al. (99), Martínez-López et al. (100), Master et al. (102), Mo et al. (105), Que et al. (115), Saricam (122), Shahrour and Dardas (123), Si et al. (125), Styra et al. (130), Sun et al. (132), Tan et al. (135), Wang et al. (146), Xiong et al. (150) n al. ), al. 3), u al. al. , ai Liu 0), ), al. 9), Cai ng et al. oi (81), Li et al. is et al. 38), Dobson al. (72), Kim Leng et al. ( on et al. (101 ralizadeh et ), Tselebis et ), Xiong et al ández et al. ( 151), Zhang et al. (156 afa et al. (28), (13), Elbay et al. (54), Holton et al. (69), Li ng et al. (127), Yousse 21), Sar ) Babo . (63), Hu l. B S S re et al. (31), Chan al. (92), Tan et al. ( t al. (38), Chen et al (72), Li et al. (89), L Park et al. (108), Y k (41), Goul ang et al. (14 obson et al. 91), Moshev d Güler (153 Maraqa et al. (99), Maunde ebis et al. (139), Veerarag ong et al. (150) . (99), Maunde 139), Veerarag 50) (35), un et (35) un et 1), Bettinsoli 9), Mo et al. ( deh et al. (11 g et al. (149), t al. (32), Bet (47), Chong 65), Hasan et 71), Hu et al. Kim et al. (82) (90), Liu et a 1), Bettinsoli 9), Mo et al. ( deh et al. (11 g et al. (149) t al. (32), Bet (47), Chong 65), Hasan et 71), Hu et al. Kim et al. (82) (90), Liu et a 121), Tang s et al Wang e t al. (1 et al. (1 6), Ho et al. ( (73), L , Soro ussef ndez . (82) qa et rk et 0), Su Wan (153 nt ﬁndin 99), Mas 8), Poura z et al. (1 0), Sun e (140), Uy al. (149), Chen et Shanjan et al. (82 Liu et al. adeh et a al. (145) (152), Y al. (26), B al. (40), C (61), Jua riguez-M l. (132), S (152) 151), Zhang et al. (156 afa et al. (28), (13), Elbay et al. (54), Holton et al. (69), Li ng et al. (127), Yousse Evidence for resilience Non-signiﬁcant ﬁndings Ahmed et al. (24), Arafa et al. (28), Chan and Huak (41), Elbay et al. (54), Fiksenbaum et al. (59), Hong et al. (70), Jung et al. (79), Khattak et al. (80), Li et al. (88), Maraqa et al. (99), Rodriguez-Menéndez et al. (116) Chan and Huak (41), García-Fernández et al. (60), Goulia et al. (63), Maraqa et al. (99), Rodriguez-Menéndez et al. (116), Styra et al. (130), Vagni et al. (142) et al. (63), Juan et al. (78), Koh (102), Maunder et al. (104), k et al. (108), Park et al. (109), al. (116), Teksin et al. (138), Bettinsoli et al. (35), Fauzi et al. (58), Ho et al. (68), Hu et al. (72), Liao et al. (90), Marton et al. (101), Mo et al. (105), Shahrour and Dardas (123), Xiao et al. (10), Xiong et al. (150) t al. (130), Wong et al. (148) Hong et al. (70) Bettinsoli et al. (35), Chen et al. (47), Chen et al. (45), Fauzi et al. (58), Huffman et al. (74), Lin et al. (91), Master et al. (102), Vagni et al. (142), Wang et al. (145) Babore et al. (31), Son et al. (126) et al. (47), Chen et al. (45), Lin (102) Maunder et al (14) Phua . n-Teyssier et al. (120), Vag y et al. (5 65), Hu m and C al. (100 Pouraliza (35), un et (99), Maunde 139), Veerarag 50) Maraqa et al ebis et al. ( ng et al. (15 ng et al. (70) bore et al. (31), Son . (63), Hu l. B S S Juan et al. (78), Koh nder et al. (104), 8), Park et al. (109), Teksin et al. (138), Wong et al. (148) Chen et al. (45), Lin nder et al. (14), Phua Non-signiﬁcant ﬁndings Dobson et al. (53), Liu et al. (94), Sun et al. (132) Juan et al. (78), Lee et al. (85), Park et al. (109), Si et al. (125), Zhang et al. (156) 1), Elbay et al. (54), ta et al. (65), Hu et al. . (75), Kim and Choi López et al. (100), (106) Pouralizadeh Distress in Health-Care Workers Sirois and Owens Sirois and Owens Distress in Health-Care Workers FIGURE 2 \| Findings from the studies that examined ﬁxed (demographic and occupational) and modiﬁable (social and psychological) factors and associations with risk and resilience for psychological distress. FIGURE 2 \| Findings from the studies that examined ﬁxed (demographic and occupational) and modiﬁable (social and psychological) factors and associations with risk and resilience for psychological distress. felt stigmatized, perceived stigma concerning negative public attitudes and disclosing about one’s work, experienced higher levels of depression, anxiety, and psychological distress (55, 78, 102, 108, 109, 116, 138). During the SARS outbreak, HCW who felt people avoided their family because of their job were twice as likely to have elevated levels of post-traumatic stress symptoms (83). Importantly, experiencing stigma and avoidance from others was signiﬁcantly associated with higher levels of post-traumatic stress symptoms during the SARS outbreak (104), and 13–26 months later (14). outbreaks who perceived support from their supervisors and colleagues, experienced better mental health in the form of lower PTSD symptoms, lower distress, and being less likely to develop psychiatric symptoms, respectively (24, 28, 41, 54, 59, 70, 79, 80, 88, 99, 116). Seven studies examined receiving useful information from others (a common form of social support). In one study, HCW who received adequate communication and information about the H1N1 outbreak from their organization were less likely to experience psychiatric symptoms because it helped them cope better, and worry less about the pandemic (63). Frontiers in Psychiatry \| www.frontiersin.org (35), un et Similarly, HCW during the SARS outbreak who had conﬁdence in the information they received from their organization (130), and who received clear communication about directives and how to take precautionary measures (41), experienced reduced psychological distress. HCW working during the COVID- 19 outbreak who felt that they did not receive suﬃcient information, scored signiﬁcantly higher on anxiety and acute stress than those who were satisﬁed with the information provided (60, 99, 116, 142). Psychological Factors y g The psychological factors examined in the studies included adaptive and maladaptive coping responses, beliefs and attitudes, and personality traits. Fourteen studies examined how perceptions of control were associated with distress among HCW (Table 2). In eight studies, higher self-eﬃcacy was associated with lower anxiety, depression, distress, and lower levels of fear about SARS and post-traumatic stress symptoms during the COVID-19 and SARS outbreaks, respectively (10, 35, 68, 72, 90, 105, 123, 150). Conversely, feeling a loss of control was associated with greater distress (148) during the SARS outbreak in Hong Kong. Analogously, appraisals of personal risk were linked to higher levels of PTSD symptoms in HCW during the MERS (126) and SARS (130) outbreaks. Negative social perceptions created risk for poor mental health for HCW in all 12 studies that examined this factor. In nurses during the MERS outbreak, perceived social stigma was associated with higher stress and poorer mental health (108). Similarly, during the COVID-19 pandemic, HCW who January 2021 \| Volume 11 \| Article 589545 25 Sirois and Owens Distress in Health-Care Workers FIGURE 3 \| Findings from the studies that examined factors related to infection exposure and associations with risk and resilience for psychological distress. Distress in Health-Care Workers Sirois and Owens FIGURE 3 \| Findings from the studies that examined factors related to infection exposure and associations with risk and resilience for psychological distress. toward their work reported less stress during the peak of the COVID-19 outbreak (31). Only one study conducted with nurses during COVID-19 did not ﬁnd evidence that risk appraisals were linked to greater distress (70). Seventeen studies examined whether coping styles were associated with HCW distress during an outbreak (Table 2). Emergency physicians and nurses working during the SARS outbreak who used denial, mental disengagement, or venting of emotions to cope were more likely to score higher on psychiatric morbidity (110). Similar results were found in frontline nurses during COVID-19, with use of negative coping associated with higher PTSD and psychological distress (102), and positive coping linked to lower PTSD (145). In HCW during the SARS outbreak, those who used maladaptive coping strategies, such as escape-avoidance, and self-blame coping, reported higher levels Positive attitudes toward one’s work were protective against distress in all six studies that examined this factor. Frontiers in Psychiatry \| www.frontiersin.org Factors Related to Infection Exposure Factors Related to Infection Exposure Fifty-three studies examined the impact of direct contact with infected patients on HCW’s psychological distress. Of these, the majority (65) found that being in direct contact with and/or treating patients infected with COVID-19, SARS, MERS, or H7N9 was a risk factor for psychological distress (Table 2). Only eight studies did not ﬁnd that contact with infected patients increased risk for distress in HCW during the COVID-19, SARS, and MERS outbreaks. Similarly, 24 studies found that risk of contact with infected patients due to working in high- risk areas (e.g., ICU, isolation areas and infection units) was associated with higher levels of anxiety, stress, and post-traumatic stress symptoms than not working in such areas (Table 2). Notably, one study found that HCW in a high-risk unit during SARS reported higher and sustained perceived stress 1 year after the outbreak compared to those in low-risk units, with those in low-risk units reporting a decrease in stress over time, but those in high-risk units experiencing an increase in stress post-outbreak (16). Three studies conducted during COVID- 19 found that risk of contact was not associated with greater distress (53, 94, 132). Spending time in quarantine due to risk of being infected was associated with higher levels of burnout, depression, and psychological distress in HCW during SARS and COVID-19 (14, 59, 92, 132), but was unrelated to post- traumatic stress symptoms and psychological distress in HCW during the MERS outbreak or the COVID-19 outbreak (78, 85, 109, 125, 156). Lastly, one study found that HCW who had colleagues who became infected, had deceased due to infection, or had been quarantined, also experienced higher levels of post- traumatic stress symptoms and acute stress during the COVID- 19 outbreak (118). For the ﬁxed factors, the weight of the evidence indicated that HCW who were female or a nurse were at signiﬁcant risk for psychological distress (Figure 2). Nurses tend to tend to be predominantly female, have higher workloads (104), and have more patient contact than other HCW. Indeed, we found that over 36 percent of the studies that found no signiﬁcant relationship between being female and increased psychological distress involved only nurses. There was also clear and consistent evidence that HCW who had or were at risk for contact with infected patients, were more likely to experience psychological distress (Figure 3). Psychological Factors Higher work satisfaction was associated with less psychological distress among hospital staﬀduring the H1N1 outbreak (63), lower PTSD among nurses (145), and lower rates of burnout among HCW during the COVID-19 outbreak. Similarly, HCW during the SARS outbreak who felt their work had become more important were less likely to develop psychiatric symptoms (41), and those who viewed their work altruistically were less likely to have severe symptoms of depression 3 years later (92). HCW who held a positive attitude January 2021 \| Volume 11 \| Article 589545 26 Distress in Health-Care Workers Sirois and Owens with less psychological distress in all 19 studies that examined this factor (Table 2). Receiving clear infection control guidelines predicted lower psychological morbidity in frontline HCW during SARS (134), and having suﬃcient hospital resources for the treatment of MERS was associated with lower MERS-related burnout (81). After the implementation of a SARS protection training program, HCW experienced signiﬁcant decreases in anxiety and depression 2 weeks and 1 month after the starting the program (44). Similarly, medical staﬀreceiving inadequate training related to managing H7N9 had higher PTSD symptoms than those who received appropriate training (81). During COVID-19, HCW who felt HCW who felt that they did not have adequate information, training, personal protective equipment (PPE), felt unsafe, and perceived lower logistic support, reported higher levels of depression, anxiety, and acute stress symptoms (51, 54, 60, 65, 72, 74, 99, 100, 102, 106, 120, 142). of burnout, psychological distress, and post-traumatic stress when surveyed 13–26 months after the outbreak (14). However, the use of adaptive strategies, such as problem-solving and positive reappraisal, were not associated with any of the distress outcomes. This ﬁnding was consistent with those from studies in which coping ability was not signiﬁcantly associated with PTSD symptoms during the MERS outbreak (126), and problem- solving and turning to religion to cope were not associated with reduced distress during COVID-19 (31). g Twelve studies investigated the role of personality in HCW’s psychological distress (Table 2). During the SARS outbreak, neuroticism was linked to poorer mental health (96), and HCW who had an anxious attachment style reported experiencing higher burnout, psychological distress, and post-traumatic stress 13–26 months after the outbreak (14). Those with an avoidant attachment style reported greater distress, but not burnout or post-traumatic stress. Eight studies examined the role of dispositional resilience. Psychological Factors Among nurses working during the MERS outbreak, higher levels of hardiness were associated with lower stress and better mental health (108), and resilience was associated with lower anxiety, depression, post-traumatic stress symptoms, and burnout among frontline nurses and HCW during COVID-19 (38, 45, 72, 74, 89, 91, 153). DISCUSSION To our knowledge, this rapid systematic review of 139 samples of 143,246 HCW working during an infectious outbreak is the largest and most up to date review of the evidence on the factors that contribute to risk or resilience to psychological distress. In this review we introduced a conceptual framework that categorized the factors contributing to increased and reduced risk of psychological distress among HCW during an infectious disease outbreak into three main categories, including factors that were ﬁxed, modiﬁable, and related to infection exposure. The majority of the studies reviewed examined the role of ﬁxed factors (demographic and occupational), with fewer studies examining how modiﬁable factors (social and psychological) were associated with psychological distress in HCW working during an outbreak. For the ﬁxed factors, the weight of the evidence indicated that HCW who were female or a nurse were at signiﬁcant risk for psychological distress (Figure 2). Nurses tend to tend to be predominantly female, have higher workloads (104), and have more patient contact than other HCW. Indeed, we found that over 36 percent of the studies that found no signiﬁcant relationship between being female and increased psychological distress involved only nurses. To our knowledge, this rapid systematic review of 139 samples of 143,246 HCW working during an infectious outbreak is the largest and most up to date review of the evidence on the factors that contribute to risk or resilience to psychological distress. In this review we introduced a conceptual framework that categorized the factors contributing to increased and reduced risk of psychological distress among HCW during an infectious disease outbreak into three main categories, including factors that were ﬁxed, modiﬁable, and related to infection exposure. The majority of the studies reviewed examined the role of ﬁxed factors (demographic and occupational), with fewer studies examining how modiﬁable factors (social and psychological) were associated with psychological distress in HCW working during an outbreak. Frontiers in Psychiatry \| www.frontiersin.org Limitations and Strengths There are several limitations of this rapid systematic review. Conducting the review during the ongoing outbreak of COVID-19 imposed time constraints. This meant that we only included published peer-reviewed literature and did not search more thoroughly through gray literature or online pre-print repositories. Most study samples were quite large, increasing conﬁdence in the generalisability of the ﬁndings. The evidence reviewed was also consistent in indicating that harmful coping strategies linked to greater distress, and positive coping strategies were protective for distress. Interventions that target harmful coping strategies, such as avoidance and self- blame, that can that may maintain or increase stress, may be worthwhile. Identifying when HCW may be using such strategies and ﬁnding ways to foster more positive approaches for managing stress are important for not only for reducing distress, but also for reducing the risk of other adverse health consequences. For example, HCW who experienced post- traumatic stress during the SARS outbreak and used harmful coping were at greater risk for substance abuse (166). Mental health check-ups are one approach that could help monitor both HCW distress and whether appropriate coping strategies are being used (167). In terms of the evidence base, the majority of the studies were cross-sectional, providing only a snapshot of the factors associated with HCW psychological distress. This limits conclusions about the direction of causality between the factors and distress, especially for those factors that are modiﬁable. Only three studies examined the potential long-term eﬀects of the risk and resilience factors on HCW’s mental health by using follow- up and time-lagged designs (14, 16, 92), providing some support for the assumed contribution of the factors to distress. More research needs to track the associations of risk/resilience factors over time with distress and the extent to which certain factors link to sustained or transient distress. In keeping with evidence that low perceived control is a transdiagnostic vulnerability factor for anxiety (168), perceptions of control were consistently associated with lower distress in the evidence reviewed. Indeed, having a sense of control is a well-known factor for reducing health-related distress (162). Feeling a loss of control may be inevitable during an infectious outbreak, as perceptions of risk are inversely related to perceived control (169). However, interventions focused on increasing a sense of autonomy can be eﬀective for reducing distress in HCW during times of upheaval (170). Factors Related to Infection Exposure Worry about becoming infected is a key stressor for HCW in the context of an outbreak as risk of infection has implications not only for their own health but also for that of their families (83). Evidence also indicated that being in quarantine contributes to distress, perhaps due to being isolated from the team (158), and that vicariously experiencing these risks can be detrimental for HCW mental health (118). Although relatively fewer studies investigated modiﬁable factors (Figure 2), the evidence highlighted key target areas to reduce HCW distress. It is also worth noting that the ﬁndings from the studies examining the role of social and psychological factors were extremely consistent. This lends conﬁdence to the suggestion that these factors are important Provision of adequate training, protection, and other resources to manage and reduce risk of infection was associated January 2021 \| Volume 11 \| Article 589545 Frontiers in Psychiatry \| www.frontiersin.org 27 Distress in Health-Care Workers Sirois and Owens information and provision of needed resources increased a sense of empowerment among ICU nurses (171). targets for intervention to reduce distress and bolster resilience. Stigmatizing attitudes from the public toward HCW were consistently associated with greater distress across the studies reviewed. Although stigma can be eﬀectively reduced through social contact with those who experience stigmatization (159), this approach may not be practical or advisable during an outbreak. Instead, public health campaigns that deliver accurate messages and highlight facts to reduce the fears underlying stigma (160), counteracting the climate of fear cultivated through the media which can promote stigma during an infectious outbreak (161) could assist. Adaptive personality traits consistently linked to better mental health outcomes in HCW working during an outbreak. Dispositional resilience was examined in the majority of the studies reviewed, with hardiness examined in one study. Dispositional resilience can be conceptualized in several diﬀerent ways, including as a personal quality reﬂecting the capacity to cope, or as type of hardiness (172). When conceptualized as the former, resilience involves being ﬂexible to change, managing unpleasant emotions, and not getting discouraged (173). Although personality traits are often viewed as being relatively stable, personality can also be viewed as reﬂecting personal qualities and tendencies that are expressed to a greater or lesser degree, and are therefore amenable to change (174). Factors Related to Infection Exposure From this perspective, approaches that help HCW develop a tendency to use resilient coping skills may help reduce vulnerability to psychological distress during an outbreak. The evidence was unanimous in indicating that perceiving social support was associated with lower distress. Adequate social support is a resilience factor that is well-known to be eﬀective reducing stress across a number of stressful situations (162), and is equally important for reducing stress among HCW (163). This support can come from supervisors and co-workers (164), either formally or informally, through positive performance feedback (59), and positive attitudes, and through peer support groups. Organizational social support may be especially important to ﬁll the gap when personal social support may be sparse because regular social support sources are struggling with their own distress during an outbreak. Such support can also foster positive work attitudes and satisfaction (165), which were associated with lower distress. Frontiers in Psychiatry \| www.frontiersin.org Limitations and Strengths The evidence reviewed suggests that this might be accomplished at the organizational level by providing HCW with the resources needed to manage the risk of infection. For example, providing personal protective equipment (PPE), adequate training, and clear guidelines, information, and protocols for infection control are important, because having such resources is linked to lower distress. This conclusion is consistent with research that found that access to The majority of the studies were conducted during COVID- 19, with relatively fewer studies reporting results from other infectious outbreaks such as SARS, MERS, H1N1, H7N9, and Ebola. On the one hand, this could be viewed as a limitation on the generalisability of the ﬁndings from the predominant outbreak, COVID-19, to other infectious outbreaks. On the other hand, we would argue that the consistency of the ﬁndings for a number of factors including participant sex, being a nurse, all 10 of the social and psychological factors, four of the ﬁve infection exposure factors, demonstrate that ﬁndings are likely to be generalizable across infectious outbreaks for these factors. Although a number of studies investigated ﬁxed factors and infection-related factors, relatively fewer studies examined how modiﬁable factors linked to distress (Figures 2, 3). There is a need for more research focusing on these factors to January 2021 \| Volume 11 \| Article 589545 28 Distress in Health-Care Workers Sirois and Owens provide a more solid evidence base about potential targets for clinical intervention and treatment. A handful of studies used unvalidated measures of psychological distress, raising concerns about whether the ﬁndings would be the same had validated measures been used. For those studies that used validated measures, the ways in which cut-oﬀscores for caseness were calculated, and/or the ways in classiﬁcation of symptoms met thresholds for psychological distress, undoubtedly varied between measurement instruments. This likely introduced some variance into the results. identiﬁed modiﬁable factors that warrant further investigation as possible points of intervention to mitigate distress. Viewing risk and resilience factors from the lens of ﬁxed and modiﬁable factors provides an eﬃcient and useful approach for understanding who is most at risk and how to address that risk during and after an outbreak. Further research focusing on possible interactions among these factors would be useful to gain a better understanding of both the risk proﬁles and key modiﬁable factors, as the evidence reviewed did not consistently examine this area. Limitations and Strengths There is evidence that the psychological distress from working during an outbreak can persist for 2–3 years after the outbreak (14–16). Therefore, monitoring and providing appropriate support should continue beyond the outbreak period to ensure mental health recovery, especially among HCW who are most at risk. Our ﬁndings suggest that particular attention should be paid to female HCW and nurses (regardless of sex), and those who come into contact with infected patients or their environments to ensure that they receive necessary resources and provision of support to manage psychological distress. Proactive approaches at the organizational level can be eﬀective (164) and may be necessary to help reduce the psychological distress of HCW. For example, a study of HCW during the COVID-19 outbreak in China found that mental health resources and services were mainly used by those experiencing mild and subthreshold levels of psychological distress rather than those who experienced more severe distress (11). Addressing the mental health needs of HCW with more severe distress will likely require more proactive means from health-care organizations. Few studies considered potential confounders in the associations with distress, compared found associations in matched non-HCW samples, or the extent to which the factors were predictive of distress outside of an outbreak. As well, the results extracted from the studies reﬂect a mix of bivariate and multivariate associations, as not all studies reported the bivariate only ﬁndings, which would be more comparable for making comparisons. Studies that examined the factors in multivariate analyses often used diﬀerent covariates making it diﬃcult to draw equitable conclusions from the studies. It is therefore diﬃcult to assess the degree to which certain factors may independently predict psychological distress over and above other factors. Collectively, these limitations may have contributed to the equivocal ﬁndings noted for several of the factors reviewed. Several strengths of the Review balance these limitations. Conceptually organizing the factors according to risk or resilience and whether they were ﬁxed or modiﬁable, provided a theoretical framework for identifying who might be at most risk for psychological distress. This facilitates appropriate clinical intervention, and for noting which factors would be suitable targets for potential interventions. We also reported non-signiﬁcant and contrary ﬁndings alongside signiﬁcant ﬁndings to provide a more balanced and critical overview of the evidence. Limitations and Strengths The Review included evidence from across six infectious disease outbreaks, with the majority of the research reporting ﬁndings from coronavirus outbreaks—Severe Acute Respiratory Syndrome Coronavirus-2 (COVID-19), Severe Acute Respiratory Syndrome-related coronavirus (SARS), and Middle East Respiratory Syndrome-related coronavirus (MERS)—that share similarities in their symptom and contagion proﬁles. Consistent evidence for risk and resilience factors was found across these various infectious diseases, suggesting that the ﬁndings from this review may be applicable across diﬀerent outbreaks. This is relevant for understanding the mental health of HCW in future outbreaks. Lastly, conducting a series of search updates ensured integration of the most recent evidence from the ongoing COVID-19 outbreak into the review at the time of submission. There are a number of delivery methods to provide support and help HCW modify risk factors and foster resilience factors. These include telehealth, mobile apps, online toolkits, and peer-support, either in person or virtual (175). Combining diﬀerent approaches may also be eﬀective. For example, social support and perceived control can have an additive eﬀect for reducing stress related to job demands (176). There is also evidence for the eﬀectiveness of interventions for reducing HCW distress when delivered at the person level and organizational level (164), as well as those that target lifestyle practices (177, 178). Evidence from randomized controlled trials suggests that third-wave cognitive behavioral therapeutic approaches, such as mindfulness (178), gratitude (177), and self-compassion (179), are eﬀective for reducing stress and burnout among healthcare professionals, and could be beneﬁcial. In low-resource settings, peer-support is one option that has been shown to be eﬀective for reducing occupational distress in HCW (164). 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Ahmed F, Zhao F, Faraz NA. How and when does inclusive leadership curb psychological distress during a crisis? Evidence from the COVID-19 outbreak. Front Psychol. (2020) 11:1898. doi: 10.3389/fpsyg.2020.01898 11. Implications and Conclusions Whereas, other reviews have documented the extent of distress experienced by HCW during an outbreak (2), the current Review highlights the proﬁles of HCW most at risk for psychological distress and psychiatric morbidity during an outbreak. This January 2021 \| Volume 11 \| Article 589545 Frontiers in Psychiatry \| www.frontiersin.org 29 Distress in Health-Care Workers Sirois and Owens ACKNOWLEDGMENTS An earlier version of this manuscript has been released as a pre-print at medRxiv (181). DATA AVAILABILITY STATEMENT the work. All authors contributed to the article and approved the submitted version. the work. All authors contributed to the article and approved the submitted version. 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Attenuating the impact of job demands: additive and interactive eﬀects of perceived control and social support. J Vocational Behav. (1991) 39:40–53. doi: 10.1016/0001-8791(91)90003-5 Copyright © 2021 Sirois and Owens. 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. 177. Cheng S-T, Tsui PK, Lam JHM. Improving mental health in health care practitioners: randomized controlled trial of a gratitude intervention. J Consult Clin Psychol. (2015) 83:177–88. doi: 10.1037/a0037895 January 2021 \| Volume 11 \| Article 589545 Frontiers in Psychiatry \| www.frontiersin.org 35
https://openalex.org/W4252744765	https://bmcpediatr.biomedcentral.com/track/pdf/10.1186/s12887-021-02551-0	English	null	Clinical Signs Associated With Earlier Diagnosis of Children With Autism Spectrum Disorder	Research Square (Research Square)	2,020	cc-by	10,795	© The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Sicherman et al. BMC Pediatrics (2021) 21:96 https://doi.org/10.1186/s12887-021-02551-0 Sicherman et al. BMC Pediatrics (2021) 21:96 https://doi.org/10.1186/s12887-021-02551-0 Open Access Background: The objective of this study is to gain new insights into the relationship between clinical signs and age at diagnosis. Background: The objective of this study is to gain new insights into the relationship between clinical signs and age at diagnosis. Method: We utilize a new, large, online survey of 1743 parents of children diagnosed with ASD, and use multiple statistical approaches. These include regression analysis, factor analysis, and machine learning (regression tree). Results: We find that clinical signs that most strongly predict early diagnosis are not necessarily specific to autism, but rather those that initiate the process that eventually leads to an ASD diagnosis. Given the high correlations between symptoms, only a few signs are found to be important in predicting early diagnosis. For several clinical signs we find that their presence and intensity are positively correlated with delayed diagnosis (e.g., tantrums and aggression). Even though our data are drawn from parents’ retrospective accounts, we provide evidence that parental recall bias and/or hindsight bias did not play a significant role in shaping our results. Conclusion: In the subset of children without early deficits in communication, diagnosis is delayed, and this might be improved if more attention will be given to clinical signs that are not necessarily considered as ASD symptoms. Our findings also suggest that careful attention should be paid to children showing excessive tantrums or aggression, as these behaviors may interfere with an early ASD diagnoses. Keywords: Autism spectrum disorder, Clinical signs, Symptoms, Early diagnosis, Diagnosis age, Regression trees Clinical signs associated with earlier diagnosis of children with autism Spectrum disorder Nachum Sicherman1* , Jimmy Charite1, Gil Eyal2, Magdalena Janecka3, George Loewenstein4, Kiely Law5, Paul H. Lipkin5, Alison R. Marvin6 and Joseph D. Buxbaum7 * Correspondence: ns38@columbia.edu 1Columbia University, Graduate School of Business, 511 Uris Hall, 3022 Broadway, New York, NY 10027, USA Full list of author information is available at the end of the article Background children affected across different age categories is also essential to guide policies and plan service needs [11]. Given evidence that the timing of interventions can have a large impact on trajectories of children diagnosed with autism spectrum disorder (ASD) [1–7], timing of diag- nosis has assumed increasing significance. Timely diag- nosis not only enables early clinical and educational intervention, but also relieves parental distress [8, 9], which in turn ameliorates secondary impacts of ASD [10]. Obtaining an accurate estimate of the number of There is extensive evidence, from both population and clinical samples, that ASD can be reliably diagnosed be- fore the age of 2 [12–17], yet the Center for Disease Control and Prevention (CDC) estimates that the me- dian age at diagnosis is 3 years 10 months for more se- vere autistic disorders, 4 years 1 month for pervasive developmental disorder – not otherwise specified, and 6 years 2 months for Asperger disorder [12]. Thus, most children who are ultimately diagnosed with ASD are not diagnosed until after the age of 4, despite the fact that parents often express concerns a year or two before this * Correspondence: ns38@columbia.edu 1Columbia University, Graduate School of Business, 511 Uris Hall, 3022 Broadway, New York, NY 10027, USA Full list of author information is available at the end of the article Methods Sample An online survey, approved by the Columbia University Institutional Review Board (IRB), was completed by 1, 815 parents of children who were previously diagnosed with ASD. Potential participants, who were contacted by email, were selected from the Interactive Autism Net- work (IAN) Research Database and Registry at the Ken- nedy Krieger Institute, Baltimore, MD. Numerous studies show that both the prevalence and age of diagnosis are correlated with family socioeconomic status (SES). Children of more educated and wealthier parents are more likely to be diagnosed with ASD, and to be diagnosed earlier. With regard to race and ethnicity, the evidence is mixed. While minority children are less likely to be diagnosed with ASD [19], differences in age of diagnosis across racial and ethnic groups are not signifi- cant after controlling for parents’ wealth and education [11, 19–21]. Other factors shown to be correlated with age of diagnosis are family structure (e.g., the presence of grandparents in the household) [22], birth order, the type of diagnosis (e.g., Asperger being diagnosed later than other conditions), and level of urbanization; children in urban areas are diagnosed significantly earlier [23]. The IAN Research database and research registry was designed to facilitate ASD research efforts by informing participants about studies for which they qualify. All the participants in the database are parents of children whose ASD diagnosis has been clinically validated [29, 30] as well as verified by a review of parent- and profes- sional-provided medical records [31]. To date, IAN Re- search has provided recruitment and/or data services for more than 500 research studies. IAN Research is gov- erned by a Johns Hopkins Medicine IRB (NA_00002750; PI: Dr. Paul H. Lipkin). In the US, the median age at diagnosis is highly vari- able across states [12, 21]. Rates of diagnosis in different states are affected by the availability of resources [23, 24], distribution of clinics, density of doctors, existence of early screening and intervention programs, and strength of parents' organizations. Rates of diagnosis also correlate with the type of welfare regime in the state, and the state’s history of de-institutionalization for intel- lectual disabilities [25]. Across states, however, the age of diagnosis is decreasing over time [12, 23], suggesting continuous improvements in ASD detection. It is difficult to estimate response rate. Sicherman et al. BMC Pediatrics (2021) 21:96 Page 2 of 14 Page 2 of 14 age [17]. Family members and caretakers are typically the first to raise concerns, usually before the second year of age [13, 15, 18]. Methods Sample We estimate that the recruitment email went to approximately 11,300 e-mail addresses (excluding emails of participants re- ported as deceased, bounced emails, and emails excluded per participant request). However, we cannot tell how many emails were actually received and read. Thus, the response rate is at least 16%. Demographic and socio-economic information on par- ents collected in the study included ethnicity, education, household income, and urban/rural residence. Data from the survey was supplemented with information previ- ously collected by IAN on each of the children in our sample. Given a lack of clinical biomarkers for ASD, currently, diagnosis typically relies on both behavioral observation by a qualified clinician and parental report of developmen- tal history and current presentation. Clinical signs are clearly important, given that family members, caretakers, and health-care professionals, who are typically the first to raise concerns, necessarily rely on observations of signs to initiate the processes that eventually lead to diagnosis and treatment. However, only a small number of studies have examined the correlation between the age of diagnosis and the presence and severity of various specific signs, ei- ther separately or jointly [26, 27]. Several studies have shown that overall severity leads to earlier diagnosis [11], and that specific or broad categories of signs are associ- ated with either earlier or later diagnosis [28]. However, we are not aware of any study that systematically looks at a large set of symptoms and signs and their level of sever- ity, their correlation with one-another, and their connec- tion to age of diagnosis. In cases where more than one child in the family was diagnosed, parents were asked to answer the survey with regard to their first diagnosed child. After deleting cases for which crucial variables were missing (e.g., age of diagnosis), the sample size was reduced to 1743. Descriptive statistics of the sample as well as other vari- ables that were used in some of the analyses are reported in Additional file 1: Appendix Table 1 (Supplemental Material). These variables are also listed in the “Covari- ates” section below. Survey This survey is part of a larger project that addresses vari- ous factors that could affect the age of diagnosis. In this study we focus on a limited number of questions from the survey. Parents were provided with a list of 25 signs and asked to report those exhibited by their child around the time of his/her diagnosis. Most signs were taken from the diagnostic criteria for autism from the Diagnostic and Statistical Manual for Mental Disorders (DSM). Some modifications to the list were made The objective of the research reported in this article is to disentangle the relationship between symptoms, clin- ical signs, and age of diagnosis, utilizing a new, large, on- line survey of parents of children diagnosed with ASD. Our goal is to statistically identify the clinical signs that are most diagnostic of an early age of diagnosis. Page 3 of 14 Sicherman et al. BMC Pediatrics (2021) 21:96 Sicherman et al. BMC Pediatrics (2021) 21:96 Sicherman et al. BMC Pediatrics (2021) 21:96 replacing them with the scores of a limited number of uncorrelated factors. following a pilot study in which parents also listed signs that are not unique to individuals with autism [22]. This prior study found that there were some signs that, while not unique to ASD, were frequently cited by parents, and may have led them to seek professional help and to obtain a diagnosis. Results Sample demographic and symptom characteristics Additional file 1: Appendix Table 1 reports the socio- economic characteristics of the sample and compares them to that of the US population. Similar to other stud- ies, the children in our sample are from wealthier, more educated, and more urban families than the US average. Representation of minority groups, especially Hispanic and Black, is also lower than their share of the US popu- lation. The effects of various socioeconomic variables on the age of diagnosis in our sample are reported in Additional file 1: Appendix Table 2. Subsequently, we used two statistical methods to study the link between symptoms and age of diagnosis: factor analysis and regression trees. Each of these methods takes a different approach to addressing the same prob- lem: identifying the patterns of signs that lead, most reli- ably, to early diagnosis of ASD. Statistical analyses To explore characteristics of the data we examined the median and the distribution of all items on the clinical signs list, as well as the correlation structure between in- dividual signs. Regression trees g Regression trees is a class of machine learning models that make it possible to estimate non-linear relationships in an easily interpretable fashion. The estimation pro- cedure is similar to running numerous Ordinary Least Squares (OLS) regressions for each sign, where the dependent variable is the age of diagnosis and the inde- pendent variable is a dummy variable indicating high vs. low severity of the sign. In each regression we use a different level of severity to construct the cutoff for the high/low dummy variable. Clinical signs whose severity allows for best prediction of early vs late diagnosis were identified in an iterative and hierarchical way. The re- gression tree analysis first identifies, for each sign, the level of severity that best splits the sample between chil- dren who are diagnosed earlier and those diagnosed later. Then, among all signs, the signs that allows for the best separation of the sample into children with early versus late diagnosis is selected as the first node in the tree. Two branches are then created, one for children with early diagnosis and one for children with late diag- nosis. The above procedure is repeated for each branch, creating two new nodes. This process continues at each node of the tree, until no symptom can further split the sample into two groups such that the differences in age of diagnosis between the groups (early and late diagno- sis) are statistically different. In the Results section, we provide more details on the tree construction. For each clinical sign or symptom, the respondent could indicate, using a slider, the level of severity ranging from “none” to “severe.” Other intermediate levels were labeled (at equal intervals) as “minor,” “moderate,” and “serious.” Respondents could position the slider anywhere along the scale. Responses were translated to a numerical value on a 0–10 scale. A zero response indicates that a child did not display that par- ticular symptom. The list of the symptoms that parents were asked to assess is presented in Table 1. Covariates In addition to the detailed description of clinical signs, we control in some statistical analyses for socio- economic variables such as ethnicity, parents’ education, family income, and location (urban vs. rural). Other co- variates that we include in some analyses are year of birth, to account for the overall increase in diagnosis over time, and a dummy variable for a diagnosis of Asperger, to account for the fact that such children tend to be diagnosed later. Given the retrospective nature of the study, we also use the time elapsed between the time of diagnosis and the date of survey to check for potential biases (see the Study Limitations section and App. B). Factor analysis The percent of children that, at time of diagnosis, dis- played each of the signs listed in the survey, as well as (for those who reported them) their median values of severity, are presented in Table 1. Additional file 1: Appendix Fig. 1 depicts the value distributions of each sign where it is shown that the modal responses for most signs are either zero (indicating that a child did not exhibit the symptom) or the highest severity level [10]. However, there are sufficient numbers of intermediate Factor analysis is designed to distill multiple variables into smaller sets of factors representing key sources of unique – i.e., non-redundant – variance. For example, a large number of questions designed to measure person- ality could be distilled, using factor analysis, into mea- sures of a much smaller number of specific traits, such as introversion and impulsivity. This approach allowed us to avoid the problems associated with estimating the effects of multiple signs that are highly correlated by Page 4 of 14 Sicherman et al. BMC Pediatrics (2021) 21:96 Table 1 Reported Clinical Signs & Symptoms and Level of Severity Clinical Signs & Symptom Obs. Level of Severity on a 0–10 scale Mean Median Std. % Non Ze Social and Communication Delayed speech 1609 6.18 7.40 3.58 0.83 Delayed response to name 1609 4.66 4.90 3.51 0.77 Poor eye contact 1609 6.07 6.30 2.99 0.91 Lack of gestures (e.g., pointing, nodding or shaking head) 1609 5.23 5.30 3.54 0.82 Difficulty understanding gestures 1609 5.08 5.20 3.38 0.83 Preference to play alone or play with objects rather than with others 1609 7.10 7.80 2.90 0.93 More focus on objects than people 1609 6.79 7.50 2.96 0.92 Failure to initiate or respond to social interactions 1609 6.94 7.70 2.80 0.94 Difficulties in initiating and/or maintaining relationships and friendships 1609 7.48 8.30 2.81 0.94 Restricted and Repetitive behaviors Played with toys or objects in an unusual way (e.g. repetitive play, lining up toys) 1609 6.19 6.60 3.02 0.92 Need for sameness (e.g. difficulties with changes in routine) 1330 6.30 6.90 3.01 0.76 Unusual motor mannerisms (e.g. hand flapping, spinning) 1330 5.29 5.40 3.41 0.71 Unusual interest in specific objects or toys (e.g. high in intensity or focus) 1330 6.45 7.10 3.00 0.75 Sensory Symptoms Unusual interest in sensory aspects of the environment (e.g. Factor analysis excessive smelling of objects or people, fascination with lights or movement) 1609 4.77 4.90 3.16 0.86 Sensory hyperreactivity (e.g. excessive or adverse response to specific sounds, lights, touch, smell or tastes) 1609 6.16 6.50 3.01 0.93 Sensory hyporeactivity (e.g. insensitivity or indifference to sensory pain or temperature, slow response to sensory stimuli in the environment) 1609 4.81 5.00 3.39 0.83 Aggression Temper tantrums 1609 4.85 5.00 3.20 0.85 Aggression toward self 1609 2.48 1.30 3.01 0.60 Aggression toward others 1609 2.65 1.80 3.01 0.64 Regression (At some point in time, did x’s behavior regress (deteriorate) in any of the following ways?) Loss of skills 1609 2.31 0.10 3.12 0.50 Loss of language (words only) 1609 2.68 0.10 3.60 0.49 Loss of language (phrases) 1609 2.16 0.00 3.46 0.41 Less social engagement 1609 2.96 1.20 3.49 0.56 Loss of motor skills 1609 1.18 0.00 2.36 0.35 Loss of daily living skills 1609 1.56 0.00 2.80 0.38 The smaller number of observations for three of the clinical signs was the result of a technical problem that resulted in a loss of data Table 1 Reported Clinical Signs & Symptoms and Level of Severity Clinical Signs & Symptom severity values reported to render severity level a potentially useful input into statistical analyses. diagnosis (in months). For example, the number −2 indi- cates a decrease of 2 months in the age of diagnosis. Each horizontal line describes the 95% confidence inter- val for the effect, and the dot in the center displays the point estimate (see Additional file 1: Appendix A for more details). Factor analysis Factor analysis is most suitable when the correlation be- tween variables of interest is relatively high. A visual examination of the correlations across signs (Table 2) shows a relatively large number of correlations with values of 0.3 and higher (35.7% of the pairwise correla- tions). Using the Kaiser-Meyer-Olkin (KMO) measure of sampling adequacy, the standard test of whether a data set is appropriate for factor analysis, yielded a value of 0.91, suggesting a high degree of suitability for factor analysis [32]. Interestingly, signs associated with aggression as well as “need for sameness” and sensory hyperreactivity are positively correlated with the age of diagnosis; children exhibiting these symptoms are diagnosed later, on average. Using varimax rotation, we identified 5 distinct factors. Table 3 reports the mapping of the 25 signs to these five factors and the labels we chose for these factors. Three of those factors mapped well onto a triad of ASD diag- nostic impairments (social interaction, communication, and restrictive/repetitive behaviors). Two additional fac- tors represented items relevant to developmental regres- sion and aggressive behaviors. A limitation of the univariate analysis is that the exist- ence of some signs may be correlated with the existence of others, so that individual signs provide redundant clues about a child’s condition. We use two methodolo- gies to estimate the joint effects of various symptoms on the age of diagnosis: factor analysis and regression trees. A seemingly natural approach to dealing with this problem would be to estimate the effect of one sign con- trolling for the effect of each of the others in a multiple regression. This approach proved infeasible due to mul- ticolinearity. The high correlations between many of the signs (see Table 2) increases the variance of the coeffi- cient estimates and makes the coefficient estimates un- stable and difficult to interpret. We, therefore, use factor analysis and regression trees to avoid such problems. The estimation results using a multiple regression are reported in Additional file 1: Appendix A. The next step in our analysis was to use the weights from the factor analysis to generate factor scores for each child and each factor. The factor scores are then included as independent variables in a regression model in which, as before, the dependent variable is age of diagnosis. Therefore, instead of including all 25 symptoms in one re- gression, we use the 5 factor scores. Correlations between severity and age of diagnosis (Univariate analysis) Before presenting results using factor analysis and regression trees, we report some basic correlations between each sign and the age of diagnosis. Figure 1 displays graphically, for each sign, the average effect of a one unit increase in reported severity on the age of For most signs, higher severity was predictive of an earlier age of diagnosis (the lines are on the left side of the panel, indicating a negative relationship between severity and age of diagnosis). Page 5 of 14 Sicherman et al. BMC Pediatrics (2021) 21:96 Sicherman et al. BMC Pediatrics Fig. 1 “Correlation Between Clinical Signs & Symptoms Severity and Age of Diagnosis” Note: “Negative” months indicate earlier diagnosis” Delayed speech, delay in response to own name, and lack of gesture had the strongest effects. Most regressions of skills were associated with an earlier age of diagnosis, except for loss of motor and daily living skills, whose ef- fects were non-significant. However, given that regressive symptoms were less frequently reported by parents, their usefulness for early diagnosis might be limited. Regression trees We conducted a regression tree analysis using a package called RPART [33]. As described earlier, the first step in constructing the tree is to find, for each sign, the level of severity (on the 0–10 scale) that best split the sample into two groups, those who are diagnosed earlier and those who are diagnosed later. The best cutoff point is produced by the RPART package using “Gini Index” and is, intuitively, similar to choosing a cutoff point that pro- duces the highest explained variation (R2) for each sign. Developmental regression and restricted-and-repetitive- behaviors (RRBs) were also predictive of earlier age at diagnosis, although much less predictive than communi- cation difficulties. Presence of aggressive behaviors, on the other hand, was associated with a delayed diagnosis. Add- ing to the regression socio-economic indicators, the child’s year of birth, and an indicator for an Asperger diagnosis (Table 4, Column 2), did not much affect the results. The results of this first step are presented in Table 5. For example, for “delayed speech” the level of severity of 5.75 best divides the sample by age of diagnosis. For children with severity levels of 5.75 or below, the mean age of diag- nosis is 63.7 months (median = 58), while for children with levels of severity above 5.75 the mean age of diagnosis is 34.9 (median = 30). The extremely low p-values indicate that, with almost certainty, the population mean age of diagnosis for children above the split is different (and lower) than those with severity level below the split. To test the hypothesis that individual signs play an im- portant role in predicting age of diagnosis beyond what is captured by the overall severity of the child condition, we constructed a measure of overall severity by counting the number of signs that the parents reported with a positive level of severity. The results, reported in Table 4, column 3, show that, on average, a one unit increase in the number of signs reduced the age of diagnosis by almost one month. However, when the regression also includes the five factors representing the effects of the individual signs (Table 4, columns 4 and 5), the effect of overall severity becomes insignificant. Factor analysis This avoids the prob- lem of multicollinearity, since factors are, by construction, uncorrelated with one-another, and, by reducing the num- ber of independent variables, increases statistical power. Sicherman et al. BMC Pediatrics ( Sicherman et al. BMC Pediatrics (2021) 21:96 Page 6 of 14 (2021) 21:96 Table 2 Pairwise Correlations Between Clinical Signs & Symptoms (those highlighted in red are significant at the .05 level) Table 2 Pairwise Correlations Between Clinical Signs & Symptoms (those highlighted in red are significant at the .05 level) Table 2 Pairwise Correlations Between Clinical Signs & Symptoms (those highlighted in red are significant at the .05 level) The results of this regression are reported in Table 4, Column 1. The factor representing communication diffi- culties was the strongest predictor of age of diagnosis, with higher levels of severity associated with significantly lower age of diagnosis. Unfortunately, one of the draw- backs of factor analysis is that there is no interpretation to the values of the estimated coefficients. Regression trees At the bottom of the table are signs for which no level of severity could separate the sample into two groups where the age of diagnosis of one group was significantly different from that of the other. In creating the tree (Fig. 2) we work from top down, first picking the sign for the top of the tree that best di- vides the sample between children who are diagnosed Sicherman et al. BMC Pediatrics (2021) 21:96 Page 7 of 14 Table 3 Factor Loadings, 5 Factors, Orthogonal Varimax Rotation, loading>.3 Variable Factor 1 Factor 2 Factor 3 Factor 4 Factor 5 Regressive Autism Social Awkwardness Communication difficulties Sensory reactivity and need for sameness Aggressive Behavior 1 Delayed speech 0.6610 2 Delayed response to name 0.7492 3 Poor eye contact 0.4324 0.4654 4 Lack of gestures 0.7728 5 Difficulty understanding gestures 0.3613 0.6916 6 Played alone 0.7254 7 Too focused on objects 0.7400 8 Poor social skills 0.6906 0.3184 9 Difficulty initating relationships 0.6150 10 Unusual play with toys 0.4290 0.3545 11 Need for sameness 0.4949 0.3918 12 Unusual motor mannerisms 0.5210 13 Unusual interest in objects 0.6638 14 Sensory hyperreactivity 0.4932 0.3374 15 Sensory hyporeactivity 0.3659 16 Unusual sensory interest 0.5741 17 Temper tantrums 0.7523 18 Aggression toward self 0.6514 19 Aggression toward others 0.7224 20 Loss of skills 0.8197 21 Loss of language (words only) 0.8146 22 Loss of language (phrases) 0.8222 23 Less social engagement 0.7676 24 Loss of motor skills 0.6562 25 Loss of daily living skills 0.6496 See Table 1 for the full labels of symptoms Table 3 Factor Loadings, 5 Factors, Orthogonal Varimax Rotation, loading>.3 See Table 1 for the full labels of symptoms earlier and those who are diagnosed later, based on the criteria discussed above. This is “delayed speech,” which, with a cutoff level of 5.8 (all cutoffs numbers are rounded in the figure) splits our sample into two groups: 62% (n = 740) with a severity level of 5.8 or above have an average age of diagnosis of 35 months, and 38% of the sample (n = 463), with a severity level below 5.8, have an average age of diagnosis of 64 months. earlier and those who are diagnosed later, based on the criteria discussed above. Regression trees BMC Pediatrics (2021) 21:96 Page 8 of 14 k “ l ” b d h d ff l n age of diagnosis using 5 factor Varimax Rotation Model (2) (3) (4) (5) −2.01* −1.77 −1.64 (0.65) (0.79) (0.78) −0.90 0.88 −0.69 (0.72) (0.76) (0.76) −13.5* −15.3* −13.2* (0.74) (0.77) (0.79) −2.55* −1.61 −2.34* (0.74) (0.80) (0.77) 6.25* 7.81* 6.46* −0.73 (0.76) (0.76) −4.04 −4.07 (1.60) −1.6 0.52 0.48 (1.78) (1.78) −3.65 −3.61 (2.45) (2.45) 1.49 1.42 (2.97) (2.97) 3.20 3.24 (1.97) (1.97) 3.02 3.06 (3.46) (3.46) −1.45 −1.48 (1.54) (1.54) −2.76 −2.76 (1.79) (1.79) 10.9* 10.8* (1.84) (1.85) −0.75* −0.75* (0.10) (0.10) −0.97* −0.31 −0.18 (0.14) (0.21) (0.21) 1549* 64.3* 52.7* 1553* (202) (2.63) (4.12) (202) 1330 1669 1359 1330 0.387 0.030 0.328 0.387 delayed for a variety of reasons and the delay can take many forms, for example, an ability to receive and “autistic aloneness.” Combined with difficulties in initiat- ing relationships we get the “Asperger” type and a later Table 4 Regression Models for the effects of factor scores on age of diagnosis using 5 factor Varimax Rotation Model (1) (2) (3) (4) (5) Factor 1: Regressive Autism −2.43* −2.01* −1.77 −1.64 (0.66) (0.65) (0.79) (0.78) Factor 2: Social Awkwardness 0.53 −0.90 0.88 −0.69 (0.73) (0.72) (0.76) (0.76) Factor 3: Communication Difficulties −15.7* −13.5* −15.3* −13.2* (0.70) (0.74) (0.77) (0.79) Factor 4: Sensory Reactivity & Need for Sameness −1.97* −2.55* −1.61 −2.34* (0.76) (0.74) (0.80) (0.77) Factor 5: Aggressive Behavior 7.46* 6.25* 7.81* 6.46* (0.73) −0.73 (0.76) (0.76) Both Parents Graduated College −4.04 −4.07 (1.60) −1.6 Only Mother Graduated College 0.52 0.48 (1.78) (1.78) Only Father Graduated College −3.65 −3.61 (2.45) (2.45) Black 1.49 1.42 (2.97) (2.97) Hispanic/Latino 3.20 3.24 (1.97) (1.97) Other 3.02 3.06 (3.46) (3.46) Family Income > $100,000 −1.45 −1.48 (1.54) (1.54) Lived in Urban Area −2.76 −2.76 (1.79) (1.79) Asperger Diagnosis 10.9* 10.8* (1.84) (1.85) Year of Birth −0.75* −0.75* (0.10) (0.10) Number of Symptoms Reported −0.97* −0.31 −0.18 (0.14) (0.21) (0.21) Constant 46.7* 1549* 64.3* 52.7* 1553* (0.62) (202) (2.63) (4.12) (202) Observations 1359 1330 1669 1359 1330 R-squared 0.327 0.387 0.030 0.328 0.387 Each column reports the estimated coefficients of one regression Standard errors in parentheses * p < 0.01, ** p < 0.05, * p < 0.1 * p < 0.01, p < 0.05, * p < 0.1 “autistic aloneness.” Combined with difficulties in initiat- ing relationships we get the “Asperger” type and a later diagnosis (node 8). Regression trees This is “delayed speech,” which, with a cutoff level of 5.8 (all cutoffs numbers are rounded in the figure) splits our sample into two groups: 62% (n = 740) with a severity level of 5.8 or above have an average age of diagnosis of 35 months, and 38% of the sample (n = 463), with a severity level below 5.8, have an average age of diagnosis of 64 months. severity level of 5.7 or above have an average age of diag- nosis of 32 months, and 20% of the sample (n = 240), with a severity level below 5.7, have an average age of diagnosis of 42 months. As the graph shows, these two groups, both relatively large, cannot be further divided. Going back up the tree, of children with relatively low severity of delayed speech (node 3), the symptom that best divides this sample is “delayed response to name.” Notice that here the cutoff level is quite low (0.75), sug- gesting that among children with no (or low level of) de- layed speech, any level of delayed response to name is important in predicting the age of diagnosis. The cutoff level of 0.75 splits the subsample into two groups (nodes 6 and 7): 22% (n = 264) with a severity level of 0.75 or above have an average age of diagnosis of 55 months (node 6), and 17% of the sample (n = 199), with a sever- ity level below 0.75, have an average age of diagnosis of 75 months (node 7). Again, the rationale for such a split seems intuitive, at least after the fact. Speech could be Next, for each of these sub-groups, we again split the sample, using the same criteria. We repeat this process until we cannot split the sample into two sub-groups such that the difference in the age of diagnosis is statisti- cally significant. From Fig. 2, we can see that when we limit the sample to the children with high severity of delayed speech (node 2, where severity level is ≥5.8), the only remaining sign that further splits this sub-sample is “lack of ges- tures” where the cutoff level of 5.7 splits our sample into two groups (nodes 4 and 5): 42% (n = 500) with a Sicherman et al. Regression trees Children with the higher level of severity have a lower age of diagnosis. The 6% of our sample (n = 67) with a severity level of 5.6 or greater have an average age of diagnosis of 68 months, and 9% of the sample (n = 107), with a severity level below 5.6, have an average age of diagnosis of 86 months. Notice that this sub-group has the highest age of diagnosis so far. Looking at this specific subgroup we see that once again “delayed speech” is the sign that best splits it. Note that we have here a small sub-group of children with relatively low levels of delayed speech. However, when limiting the sample to this sub-group, those with relatively more delayed speech are diagnosed earlier. A cutoff level of 3.8 divides the group into our last two subgroups, represented by nodes 12 and 14. Children with delayed speech levels of 3.8 or above are diagnosed at an average age of 56 months, and children with sever- ity level below 3.8 are diagnosed at an average age of 91 months. The groups’ sizes are 15 (1%) and 92 (8%) respectively. level of 6.0 splits our sample into two groups (nodes 8 and 9). Fourteen percent (n = 170) with a severity level of 6 or above have an average age of diagnosis of 61 months, and 8% of the sample (n = 94), with a severity level below 6, have an average age of diagnosis of 45 months. It should be noted that here, children that exhibit the sign at higher severity are actually diagnosed at an older age. Moving up to the sample represented by node 7 (low levels of delayed speech and low levels of delayed re- sponse to own name), the symptom that best split the sample is “need for sameness,” and the cutoff level of 2.3 splits our sample into two groups (nodes 11 and 15). Here again, children who exhibit the sign at higher se- verity are diagnosed at an older age. The 14% (n = 174) with a severity level of 2.3 or above have an average age of diagnosis of 79 months, and the 2% of the sample (n = 25), with a severity level below 2.3, have an average age of diagnosis of 48 months. This group cannot be fur- ther split, most likely due to small sample size. Regression trees delayed for a variety of reasons and the delay can take many forms, for example, an ability to receive and understand speech without the capacity to speak. If the child does not respond to his name, however, the lack of response is likely to be more diagnostic, to be indicative of a lack of attachment or lack of social awareness, g Looking at the sample represented by node 6, we see that the sign that best splits this sample is “difficulties in initiating and/or maintaining relationships.” The cutoff Sicherman et al. BMC Pediatrics (2021) 21:96 Page 9 of 14 Table 5 Optimal Splits and Mean/Median Age at First Diagnosis Above/Below Split Clinical Signs & Symptoms Split Mean Above Mean Below p-value* Median Above Median Below Delayed speech 5.75 34.9 63.7 0.00000 30 58 Delayed response to own name 3.85 35.6 60.4 0.00000 32 52 Poor eye contact 6.15 40.5 52.1 0.00000 33 42 Lack of gestures 6.05 33.6 56.7 0.00000 30 48 Difficulty understanding gestures 3.75 41.1 54.9 0.00000 34 47 Preference to play alone 7.55 42.3 50.7 0.00000 36 40 Too focused on objects 1.05 44.8 61.6 0.00000 36 54 Poor social skills 5.25 43.2 53.9 0.00000 36 42 Unusual play with toys or objects 5.55 41.3 52.7 0.00000 35 42 Need for sameness 2.35 47.4 35.1 0.00000 38 31 Unusual motor mannerisms 3.15 42.6 52.7 0.00000 35 40 Unusual sensory interest 1.65 47.3 35.2 0.00000 38 28 Sensory hyperreactivity 8.55 39.4 47.3 0.00019 33 38 Sensory hyporeactivity 1.75 44.5 51.2 0.00042 36 39 Temper tantrums 2.35 48.2 39.6 0.00000 38 32 Aggression toward self 2.65 51.4 43.3 0.00000 40 36 Aggression toward others 2.15 50.8 41.9 0.00000 41 34 Loss of skills 5.15 39.8 47.5 0.00011 35 38 Loss of language (words only) 5.8 34.8 49.3 0.00000 30 39 Loss of language (phrases) 7.25 35.7 48.0 0.00000 32 38 Difficulty initating on maintaing relationships “n.a.” “n.a.” “n.a.” “n.a.” “n.a.” “n.a.” Unusual interest in objects or toys “n.a.” “n.a.” “n.a.” “n.a.” “n.a.” “n.a.” Less social engagement “n.a.” “n.a.” “n.a.” “n.a.” “n.a.” “n.a.” Loss of motor skills “n.a.” “n.a.” “n.a.” “n.a.” “n.a.” “n.a.” Loss of daily living skills “n.a.” “n.a.” “n.a.” “n.a.” “n.a.” “n.a.” * The p-value is for the difference between the means way.” The cutoff level of 5.6 splits our sample into two groups (nodes 10 and 13). Regression trees Going back to node 11, the sign that best split this sample is “played with toys or objects in an unusual Page 10 of 14 (2021) 21:96 Sicherman et al. BMC Pediatrics (2021) 21:96 Sicherman et al. BMC Pediatrics Fig. 2 “Regression Tree of Age of Diagnosis and Clinical Signs & Symptoms”. Note: Out of the sample of 1743, only 1330 respondents were able to see the full list of clinical signs and symptoms (see footnote to Table 1). Only 1203 of the 1330 also had a valid age of diagnosis reported Fig. 2 “Regression Tree of Age of Diagnosis and Clinical Signs & Symptoms”. Note: Out of the sample of 1743, only 1330 respondents were able to see the full list of clinical signs and symptoms (see footnote to Table 1). Only 1203 of the 1330 also had a valid age of diagnosis reported In sum, the regression tree analysis identifies speech delay, lack of gestures and delayed response to name – all components of factor 3 in the factor analysis - as the key signs leading to early diagnosis (the lighter the node’s color in Fig. 2, the earlier is the diagnosis). an important factor in early diagnosis, findings that cor- roborate and build on previous research. We showed that deficits in early communication are predictive of age at diagnosis over and above overall symptom severity. Im- portantly, these symptoms were observed by parents much earlier than the current median age of diagnosis. This suggests the potential value of designing targeted tools that could enable family members to identify key early symptoms of ASD before they come to the attention of healthcare specialists. Discussion Our study analyzed, using several different analytical ap- proaches, variation in age of diagnosis as a function of a child’s signs and symptoms at time of diagnosis. Lever- aging a unique sample of over 1600 children with an ASD diagnosis, for whom detailed demographic and clinical information was available, allowed us to explore a variety of clinical factors associated with age of diagno- sis, while controlling for key environmental factors iden- tified by previous studies (SES, urban/rural residence and cohort). Deficits in communication as the earliest indicators of ASD Our results indicated that early deficits in communication – particularly responding to one’s name, lack of gestures, and delayed language, −are all strongly associated with an earlier age at diagnosis. Using a machine learning ap- proach, we show that children whose parents reported the presence of high severity of those signs were diagnosed with ASD 33 months earlier compared to children whose The different methodologies we employed yielded over- lapping, but informatively distinct findings. All analyses strongly point towards deficits in early communication as Page 11 of 14 Sicherman et al. BMC Pediatrics (2021) 21:96 better reflect how the disorder manifests differently over time due to both maturational changes, and the transitions in social environments that children typic- ally undergo. The full set of signs required for a for- mal diagnosis are not likely to emerge until later in development; yet by this time an important window of opportunity for early intervention may have been missed. This argument is supported by studies show- ing that clinician judgement, rather than use of stan- dardized diagnostic criteria, was associated with an earlier and more stable diagnosis [44]. It is also in agreement with a tri-level model of autism screening and diagnosis, the first level of which consist of gen- eric developmental surveillance as soon and as often as possible and the second which involves referral to early intervention when necessary. Only at the third level, which would typically follow early intervention at a somewhat later age, would the full and formal diagnostic schedule become relevant. parents either did not observe those sings or observed only their milder presentations. Factors associated with a delayed diagnosis Our results suggest that temper tantrums and aggression as well as “need for sameness” and sensory hyperreactiv- ity are positively correlated with the age of diagnosis; children exhibiting these symptoms are diagnosed later, on average. Note, moreover, that these clinical signs are relatively uncorrelated with all other signs in Table 3 (apart from “need for sameness”). This suggests that this cluster is associated with a particular ASD subtype, the presenta- tion of which is milder, and hence does not become obvious until the child is older and has transitioned into relatively complex and socially demanding settings. Indeed, the diagnostic delay associated with these symptoms may itself be a cause of those symptoms, which emerge as a consequence of non-recognized ASD-related difficulties. Daniels and Mandell (2014) speculated that the later age of diagnosis in Asperger’s Syndrome vs. autism is due to lack of speech delay in the former, making it less evident until children move into the more socially de- manding environment of school. Chawarska and col- leagues provide evidence for such heterogeneity in the early course of ASD, and propose that the lack of such communication problems in early development may mask social difficulties until later in development, con- tributing to a delay in the diagnosis [38, 39]. Similarly, several studies suggested that restrictive/repetitive be- haviors appear to worsen with age [40–43]. Lord (1995) speculates that, since these behaviors are also present in normally developing young children, the worsening may reflect the fact that “parents become better able to recognize the absence of normal development as chil- dren grow older” (page 1377). This improved ability owes, no doubt, to a greater range of experiences and comparisons as children begin to exit the home environ- ment, and as normally developing children respond more appropriately to the demands of new environ- ments. Collectively, this evidence suggests that some of the efforts towards reducing the age at diagnosis should An alternative explanation is that such clinical signs may mask other ASD-associated signs. For example, an aggressive child would be first treated for aggression and not much else and then, over time, additional diagnoses would be considered This in turn indicates that children showing excess temper tantrums and/or aggression should be evaluated for autism, rather than awaiting more obviously ASD-linked signs. Discussion y p The finding that early deficits in communication are predictive of early diagnosis is consistent with earlier studies which found that problems with verbal and non- verbal communication, rather than abnormal social in- teractions or presence of restricted/repetitive behaviors, are key for early detection of ASD [27]. Using, to our knowledge, one of the biggest sample to date, we repli- cated the finding from a number of studies that parental concerns about child’s communication are the earliest signs of an underlying ASD [34–36]. As we noted earlier, the most likely explanation for this finding is that these symptoms index what parents expect of normal develop- ment in the home in the first 2 years of life. If we think of child development not only as a process of biological maturation, but also as a social “career” [25, 37] in the course of which children transition from simpler to more complex social situations, it stands to reason that the factors predictive of early diagnosis would involve the most basic skills of social communication. These signs are likely to trigger a referral to early intervention programs, where such programs exist (recall the finding that early diagnosis is strongly correlated with urban residence). Other signs more strongly indicative of ASD will then become more evident against the background of the type of interactions expected in these programs, in which therapists work one-on-one with children and often involve children in group activities. Given the ex- perience of early intervention workers with many types of disabilities. Study limitations One limitation of our study is that the sample is limited to children who were eventually diagnosed with ASD, and hence does not include information on the preva- lence and severity of clinical signs among children who were not diagnosed with ASD. As a consequence, we could only establish their connection to age at diagnosis, but not their overall usefulness in predicting which Page 12 of 14 Page 12 of 14 Page 12 of 14 Sicherman et al. BMC Pediatrics (2021) 21:96 children will go on to develop ASD. Collecting such in- formation would, however, be difficult. The relevant population to sample for signs could include all children, children identified as being “at risk,” or children with a demographic profile similar to that of children who are diagnosed with ASD. Use of each of these comparison populations would likely yield different results. as signs that occurred earlier but did not reach the thresh- old for parents or other care-givers to delve further or seek help, were not reported, and hence not used in the analysis. In addition, given that the signs and their level of severity are reported by the parents, they are, by definition, subjective and could be biased. We also cannot infer causation between the sign profile and age of diagnosis, and the direction of many of the effects reported in our study remains to be estab- lished. For example, playing alone could cause children to be diagnosed later, perhaps because it reduces the salience of other cues, such as delayed speech. However, it is more likely that playing alone is associated with late diagnosis because it becomes apparent only later, when children transition to environments where they are expected to play with other kids. y y Given the retrospective nature of our study, a potential concern could be that the accuracy and quality of re- sponses could diminish with the time elapsed since the child was diagnosed until the survey date. To deal with this issue, we conducted several statistical analyses to test for such bias (see Addtional file 1: Appendix B). First, we tested, for each sign, whether the probability that it would be recalled by the parent was influenced by the passage of time; it was not. Second, we conducted a similar test, but for the severity of each sign. Again, re- ported severity levels were unrelated to time passed since diagnosis. Study limitations Finally, we included time elapsed since diagnosis as a covariate in our regression analyses. Doing so did not affect our findings. Even when we suspect that a causal chain does exist, identification of the exact pathway of causation cannot be established. It is likely, for example, that delayed speech contributes to early diagnosis not because parents identify it as a cause for concern about ASD, but because it leads to referral to speech experts, who are more attune to the possibility of ASD as well as familiar with other patterns of signs that predict it. Parents’ recall of the type of clinical signs and symp- toms exhibited by their child prior to diagnosis could also be distorted by their awareness, at the time of com- pleting the survey, of their child’s diagnosis. However, our results run contrary to what one would expect if such a bias were present. If parents’ recall was influ- enced by current diagnosis in a fashion consistent with hindsight bias – the tendency to believe that one knew in the past what one knows in the present – then they would be more likely to emphasize symptoms that are specific to autism, i.e., that differentiate autism from other conditions, yet we find the opposite. We caution, however, that the same does not necessarily hold for parents’ recall of the severity of their children’s signs. Consequently, we have less confidence in our results re- garding severity. Finally, similar to most studies of children with ASD, the indices of socioeconomic status were higher in our sample than in the general US population. If this discrep- ancy results, in part, from a greater willingness to partici- pate in surveys on the part of high SES individuals, then this could affect the generalization of our findings to the broad population. However, the general findings of our study should still hold unless the path to diagnosis is systematically different for lower SES families. Conclusion Similarly, in the subset of children without early deficits in communication, diagnosis is delayed, and this might be improved if more attention will be given to clinical signs that are not necessarily considered as ASD symptoms. Consent for publication Not applicable. Consent for publication Not applicable. Consent for publication Not applicable. Abbreviations ASD: Autism Spectrum Disorder; IAN: Interactive Autism Network 3. Anderson DK, Liang JW, Lord C. Predicting young adult outcome among more and less cognitively able individuals with autism spectrum disorders. J Child Psychol Psychiatry Allied Discip. 2014;55(5):485–94. Supplementary Information Th li i i l y The online version contains supplementary material available at https://doi. org/10.1186/s12887-021-02551-0. The online version contains supplementary material available at https://doi. org/10.1186/s12887-021-02551-0. The online version contains supplementary material available at https://doi. org/10.1186/s12887-021-02551-0. Additional file 1: Appendix Table 1. Sample Socio-Economic Charac- teristics. Appendix Figure 1. Distribution of Clinical Signs & Symptoms by Levels of Severity. Appendix Table 2. The Effects of Various Variables on Age of Diagnosis. Appendix A. Regression Analyses. Appendix Table A1. Regressions of First Age of Diagnosis on Symptom Severity. Appendix Figure A1. The Effect of all Clinical Signs & Symptoms (Com- bined) on Age of Diagnosis. Appendix B: Testing for potential recall- bias. Appendix Table B1. Child age at diagnosis, at time of survey, and time elapsed. Appendix Table B2. The effects of Time Elapsed on the Liklihood that a sign is recalled and the reported severity of the symptom. Received: 19 August 2020 Accepted: 10 February 2021 Received: 19 August 2020 Accepted: 10 February 2021 Author details 1Columbia University, Graduate School of Business, 511 Uris Hall, 3022 Broadway, New York, NY 10027, USA. 2Department of Sociology, Columbia University, New York, NY, USA. 3Seaver Autism Center for Research and Treatment, Icahn School of Medicine at Mount Sinai, New York, USA. 4Social and Decision Sciences, Carnegie Mellon University, Pittsburgh, PA, USA. 5Kennedy Krieger Institute and Johns Hopkins School of Medicine, Baltimore, MD, USA. 6Kennedy Krieger Institute and Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA. 7Seaver Autism Center for Research and Treatment, Department of Psychiatry, Friedman Brain Institute, Mindich Institute for Child Health and Development, Icahn School of Medicine at Mount Sinai, New York, NY, USA. Funding l 10. Howlin P. Children with autism and Asperger syndrome: A guide for practitioners and carers: Wiley; 1999. 10. Howlin P. Children with autism and A practitioners and carers: Wiley; 1999. Financial support for this research was provided by a generous grant from the Organization for Autism Research (OAR). OAR did not play any role in the design of the study or collection, analysis, and interpretation of data and in writing the manuscript. 11. Wiggins LD, Baio J, Rice C. Examination of the time between first evaluation and first autism spectrum diagnosis in a population-based sample. J Dev Behav Pediatr. 2006;27(2):79–87. 12. Christensen D, Baio J, Braun K, Bilder D, Charles J, Constantino J, et al. Prevalence and Characteristics of Autism Spectrum Disorder Among Children Aged 8 Years - Autism and Developmental Disabilities Monitoring Network, 11 Sites, United States, 2012 MMWR Surveill Summ. 2016;65(No. SS-3):1023. 12. Christensen D, Baio J, Braun K, Bilder D, Charles J, Constantino J, et al. Prevalence and Characteristics of Autism Spectrum Disorder Among Children Aged 8 Years - Autism and Developmental Disabilities Monitoring Network, 11 Sites, United States, 2012 MMWR Surveill Summ. 2016;65(No. SS-3):1023. Conclusion Utilizing a large survey of parents of children with ASD, this is the first study that systematically looks at a large set of clinical signs and symptoms, their presence and level of severity, the correlations between them, and how they are related to the age of diagnosis. We show that individual signs play an important role in predicting age of diagnosis beyond what is captured by the overall se- verity of the child condition. These limitations arising from potential recall biases occur because of the retrospective nature of the study. Prospective epidemiological research on this question is, however, impractical. Only a small fraction of all chil- dren are diagnosed with ASD; hence an epidemiological sample of parents of children who have not yet been di- agnosed with ASD will contain too few cases of children who will end up with ASD diagnosis later. For this rea- son, we believe that despite the well-known problems with retrospective studies, our approach – especially given the direction of our results and the additional tests we conducted to rule out retrospective bias – is the best way to shed light on this crucial question. Since many signs are highly correlated, we show that most of the variation in age of diagnosis can be captured by a small number of signs. Using regression tree, we can both rank-order the signs and show how they inter- act with each other to provide an earlier diagnosis. While some of our findings are consistent with practi- tioners’ experience, this study provides statistical support for existing intuitions, as well as new insights. A key new insight is that clinical signs not specifically associated with ASD can lead to earlier diagnosis if they bring the child to the attention of professionals who have greater Another limitation is that questions about signs refer to only one point in time: around the time of the diagno- sis. Clinical signs that appear later and might have raised concerns had the child not already been diagnosed, as well Page 13 of 14 Sicherman et al. BMC Pediatrics (2021) 21:96 Sicherman et al. BMC Pediatrics (2021) 21:96 Sicherman et al. BMC Pediatrics (2021) 21:96 experience with, and hence greater skill in diagnosing, ASD. Another key insight is that careful attention should be paid to children showing excessive tantrums or aggression, as these behaviors may interfere with an early ASD diagnoses. Competing interests N h f hi i l No author of this article had any financial or otherwise conflict of interest relating to the article. Acknowledgements We thank Patrick Kennedy, Brenden Eum, and Jacqueline Araya for superb research assistance. We thank all participants for taking part in this study. Useful comments and suggestions were provided by Kamel Jedidi and Oded Netzer. 4. Siu AL, Bibbins-Domingo K, Grossman DC, Baumann LC, Davidson KW, Ebell M, et al. Screening for Autism Spectrum Disorder in Young Children. JAMA. 2016;315(7):691. 5. Brasher S, Stapel-Wax JL. Autism Spectrum Disorder in the Primary Care Setting. Adv Fam Pract Nurs. 2020;2:159–68. 5. Brasher S, Stapel-Wax JL. Autism Spectrum Disorder in the Primary Care Setting. Adv Fam Pract Nurs. 2020;2:159–68. Availability of data and materials h d d d l y The datasets generated and/or analyzed during the current study are not publicly available due to privacy constraints but are available from the corresponding author on reasonable request. 13. Howlin P. Asgharian a. The diagnosis of autism and Asperger syndrome: findings from a survey of 770 families. 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https://openalex.org/W4388792391	https://link.springer.com/content/pdf/10.1007/s40789-023-00650-0.pdf	English	null	Apparent activation energy of mineral in open pit mine based upon the evolution of active functional groups	International Journal of Coal Science & Technology/International journal of coal science & technology	2,023	cc-by	8,731	Abstract This study aimed to investigate the mechanism of mineral spontaneous combustion in an open pit. On the study of coal and mineral mixture in open pit mines, as well as through the specific surface area and Search Engine Marketing (SEM) experi- ments, the specific surface area and aperture characteristics of distribution of open pit coal sample and pit mineral mixture samples were analyzed. Thermal analysis experiments were used to divide the oxidation process was divided into three stages, and the thermal behavior characteristics of experimental samples were characterized. On the basis of the stage division, we explored the transfer law of the key active functional groups of the experimental samples. The apparent activation energy calculation of the key active groups, performed by combining the Achar differential method with the Coats–Redfern integral method, microstructural and oxidation kinetic properties were revealed. The resulted showed that the mixed sample had high ash, the fixed carbon content was reduced, the specific surface area was far lower than the raw coal, the large aperture distribution was slightly higher than the medium hole, the micropore was exceptionally low, the gas adsorption capacity was weaker than the raw coal, the pit coal sample had the exceedingly more active functional groups, easy to react with oxygen, more likely to occur naturally, and its harm was relatively large. The mixed sample contained the highest C–O–C functional group absorbance. The functional groups were mainly influenced by the self-OH content, alkyl side chain, and fatty hydro- carbon in the sample. The main functional groups of the four-like mixture had the highest apparent activation energy, and the two reactions were higher in the low-temperature oxidation phase. Keywords Specific surface area · Aperture distribution characteristics · Thermal behavior characteristics · Oxidation kinetic properties · Gas adsorption capacity Apparent activation energy of mineral in open pit mine based upon the evolution of active functional groups Shipng Lu1 · Jingyu Zhao1,2 · Jiajia Song1 · Jiaming Chang1 · Chi‑Min Shu3 Received: 8 June 2023 / Revised: 24 September 2023 / Accepted: 27 September 2023 © The Author(s) 2023 Received: 8 June 2023 / Revised: 24 September 2023 / Accepted: 27 September 2023 © The Author(s) 2023 * Jingyu Zhao zhaojingyu2014@126.com 1 School of Safety Science and Engineering, Xi’an University of Science and Technology, Xi’an 710054, China RESEARCH RESEARCH RESEARCH 3 Department of Safety, Health, and Environmental Engineering, National Yunlin University of Science and Technology, Douliou 64002, Yunlin, Taiwan (2023) 10:75 (2023) 10:75 International Journal of Coal Science & Technology https://doi.org/10.1007/s40789-023-00650-0 * Jingyu Zhao zhaojingyu2014@126.com 1 School of Safety Science and Engineering, Xi’an University of Science and Technology, Xi’an 710054, China 2 Shaanxi Industrial Process Safety and Emergency Rescue Engineering Technology Research Center, Xi’an 710054, Shaanxi, China 3 Department of Safety, Health, and Environmental Engineering, National Yunlin University of Science and Technology, Douliou 64002, Yunlin, Taiwan 2 Shaanxi Industrial Process Safety and Emergency Rescue Engineering Technology Research Center, Xi’an 710054, Shaanxi, China 1 Introduction underground mining, the productivity of open pit mining can reach up to 3 to 5 times that of underground mining. However, open pit mines usually continue to expand, and the continuous increase of pit minerals leads to an increase in the risk of coal spontaneous combustion (CSC) (Cao et al. 2020; Tang 2020; Tang et al. 2020). CSC is a major problem in coal mine safety production (Deng et al. 2015; Liang et al. 2023; Zhao et al. 2022). In the initial stage of CSC, water evaporation and gas desorption will occur, which cause the gradual increase of coal sample surface temperature, thus prompting the oxygen molecule to react deeply with coal through the coal crack, and involving the cleavage of chemi- cal bonds of active functional groups in coal (Aich et al. 2019; Zhang et al. 2018; Zeng and Li 2022). This heat shows action on coal, resulting in the CSC phenomenon. At the same time, the process of coal mining will yield oil shale coal gangue and other wastes, increasing the loss caused by CSC (Chabukdhara and Singh 2016; Ju et al. 2019; Zhang Forty percent of the world’s coal mine production is the use of open pit mining. As a big country in coal demand, China also has considerable number of open pit coal mines, and the country continues to vigorously promote the improve- ment of coal mine production capacity. Compared with 1 School of Safety Science and Engineering, Xi’an University of Science and Technology, Xi’an 710054, China (0121 3456789) 3 Page 2 of 15 S. Lu et al. 75 expeditious working as adoption of reasonable correspond- ing measures. et al. 2022). To forestall accidents, it is necessary to fully explore the thermal characteristic parameters of minerals in coal mines. expeditious working as adoption of reasonable correspond- ing measures. The existing studies mainly focus on the dynamics of kerosene mother shale and coal gangue. Through Search Engine Marketing (SEM) and X-Ray Diffraction (XRD) tests, the mineral evolution process in the pyrolysis process of oil shale was analyzed and it was found that with the increase of organic matter, the number and area of the oil shale increased, which is conducive to the in-situ develop- ment of oil shale (Huang et al. 2023; Liu et al. 2023; Zhai et al. 2023). DSC experiment can study the heat transfer process of oil shale and 500 °C semi-coke. 2.1 Coal sample Raw coal, oil shale, and coal gangue were collected from the open pit mine as experimental samples, and the selected coal samples of the open pit mine were long-flame coal. The experimental subjects were divided into two groups, one group was raw coal, the other group was the mixed sample made of raw coal, oil shales, redistilled oil shale and coal gangue according to the ratio of 1:1:1:1. The collected coal samples were prepared for crushing, screenings, and seal- ing. To forestall the oxidation of the coal with oxygen on the coal surface and affect the test results, the middle part of the coal samples was selected as the test sample, and the coal samples of 80–120 mesh were screened for the experiment. The industrial analysis and elemental analysis experimen- tal results are listed in Fig. 1. Raw coal with 5% moisture, which belongs to low moisture coal, low ash, high volatile, indicated coal sample’s flammable high calorific value. 2.3 Differential scanning calorimetry test The TA-SDTQ600 thermal analyzer with sample size 80–120 mesh, mass 2 mg, heating rate 10 °C/min, 79% nitro- gen + 21% oxygen, from room temperature to, 1000 °C, was used. 2.2 Specific surface area and microscopic pore test The specific surface area test used Autosorb-iq-c automatic physical chemical adsorption analyzer, sample particle size of 80–120 mesh; the experiment was conducted at 10 °C below room temperature, and heating rate of 5 °C/min. The experimental atmosphere was nitrogen, and temperature at − 196 °C (nitrogen critical point, 77 K). The microscopic pore testing was performed with a German-Zeiss-SIGMA HD scanning electron microscope with sample particle size 80–120 mesh and dried in experimental settings 70,000, 50,000, 20,000, and 10,000 four magnifications. To sum up, numerous scholars have only selected a single product as the research object in the study of pit minerals, analyzed the change law of functional groups, and rarely studied the change of spontaneous combustion characteristic parameters after the mixing of various minerals. Therefore, in this paper, the raw coal and raw coal shale mixed oil shale and gangue samples were selected as the research object. Microscopic characteristics were investigated by specific surface area and SEM experiments, through physical and chemical adsorption by differential scanning calorimetry (DSC) and in-situ infrared technology, the functional group of the two samples was scrutinized in different oxidation stages with temperature. The apparent activation energy of functional groups in different oxidation stages was cal- culated by Achar differential method and Coats-Redfern integration method, providing theoretical basis for accurate determination of mineral spontaneous combustion state and 1 Introduction During the com- bustion of oil shale, four peaks are the heat transfer peaks (30–300 °C), coke combustion (300 °C), carbonate decom- position (700–800 °C) and the volatile organic matter and the heat transfer peak of coke and carbonate. The basic characteristics of oil shale and its pyrolysis products were assessed, and the relative strength of the methyl functional groups in raw semi-coke and residual ore decreased with the pyrolysis. The aliphatic carbon chain is more likely to cleave, and the carbon–oxygen double bond structure is not easy to decompose, and it is more likely to occur in shale oil (Alexander et al. 2023; Jiang et al. 2023; Jin et al. 2023). Using thermal mass-Fourier transform infrared spectros- copy of typical pyrolysis oil shale, the oil shale pyrolysis process can be divided into four stages. Pyrolysis reaction and volatile release mainly occur in the second stage. The more fat hydrocarbon content, the more methane released in the process of pyrolysis, and the formation of methane is by a desorption process with the results of four chemi- cal reactions (Onifade and Genc 2019; Wang et al. 2023; Zheng et al. 2023). The mixed combustion characteristics of biomass and gangue were studied, and the maximum com- bustion ratio of gangue and 200 °C baking products was obtained (Zhu et al. 2020). 3 Experimental results and analysis specific surface area of the two samples is shown in Fig. 2. The calculated specific surface area of raw coal was 39.38 m2/g, and the mixed surface area of the composite samples was 4.46 m2/g. 2.4 In‑situ infrared test Using the INVENIO type Fourier transform infrared spec- trometer of Bruker and in-situ infrared of Specac Selector TM, the sample of 80–120 was 1 mg. 1 3 3 Apparent activation energy of mineral in open pit mine based upon the evolution of active… Page 3 of 15 75 75 Mad (%) Aad (%) Vad (%) FCad (%) FCad/Vad 0 10 20 30 40 50 Raw coal sample 0 10 20 30 40 50 60 70 Composite sample N (%) C (%) H (%) O (%) S (%) -10 0 10 20 30 40 50 60 70 80 Raw coal sample 0 10 20 30 40 Composite sample (a) Results of the proximate analysis experiments (b) Elemental analysis of the experimental results Fig. 1 Results of the proximate analysis experiments and elemental analysis N (%) C (%) H (%) O (%) S (%) -10 0 10 20 30 40 50 60 70 80 Raw coal sample 0 10 20 30 40 Composite sample (b) Elemental analysis of the experimental results Mad (%) Aad (%) Vad (%) FCad (%) FCad/Vad 0 10 20 30 40 50 Raw coal sample 0 10 20 30 40 50 60 70 Composite sample (a) Results of the proximate analysis experiments Composite sample Raw coal sample Raw coal sample (b) Elemental analysis of the experimental results (a) Results of the proximate analysis experiments Fig. 1 Results of the proximate analysis experiments and elemental analysis Fig. 1 Results of the proximate analysis experiments and elemental analysis 3.1.1 Mineral‑specific surface area analysis For the tested adsorption curve, the BET (Brunauer, Emmett, and Teller) theoretical method was used to calculate the spe- cific surface area of the BET equation as shown in Eq. (1): (1) p Q ⋅(p0 −p) = 1 Qm ⋅C + C −1 Qm ⋅C ⋅p p0 (1) In Eq. (1), p is nitrogen fractional pressure, mmHg; p0 is the saturated vapor pressure of the lower nitrogen gas, mmHg; Q is the amount of nitrogen actually adsorbed on the surface of the experimental sample, cm3/g; Qm is the nitrogen saturation of the experimental sample, cm3/g; C is the constant related to the adsorption capacity. Equation (2) can be obtained from dividing the left and bottom of the equation by P0: 3.1 Analysis of mineral surface area and microscopic pore of pit Overall, the raw coal surface area was larger, to 39.38 m2/g. At room temperature and pressure, and oxygen coal composite reaction transverse comparison can be found. According to the specific surface area from small to large arrangement for four mixed raw coal, shale’s surface area decreased, after oil distillation. Because of the volatile materials in the process of distillation, particle pore struc- ture changed, leading to composite specific surface area being less than the raw coal. 3.1.2 The aperture analysis There were various classification criteria for pore struc- tures, in which IUPAC tissue is divided into three catego- ries according to the size of pore size range and solid gas, micropores (< 2 nm) mesoporous (2–50 nm), and large pores (> 50 nm). The less the content of large pores and medium pores, the tighter the structure, and the easier it is to react with oxygen at room temperature. According to Table 1, the majority of coal samples were mesoporous, the majority of rock samples were large pores, and the content of micropores in all the samples was extremely small, indi- cating that the molecular structure of these two samples was unstable, so oxidation and adsorption were more likely under normal temperature ventilation onditions (Fig. 3). (2) p/p0 Q ⋅(1 −p/p0 ) = 1 Qm ⋅C + C −1 Qm ⋅C ⋅p p0 (2) According to the linear relationship of (p/p0)[Q(1 − p/ p0)] and p/p0, the slope and intercept are obtained, the Qm is solved, and then the specific surface area is deter- mined based upon the area occupied by a single nitrogen gas molecule on the surface. The calculation curve of the 1 3 Page 4 of 15 S. Lu et al. 75 Fig. 2 Specific surface area measured by multi-point BET method for a Raw coal and b Composite Fig. 2 Specific surface area measured by multi-point BET method for a Raw coal and b Composite Fig. 2 Specific surface area measured by multi-point BET method for a Raw coal and b Composite Table 1 Pore size distribution for raw coal and composite samples Coal sample Micropore (%) Mesoporous pore (%) Macropore (%) Raw coal sample 0.62 56.15 43.23 Composite sample 0.22 40.43 59.35 0 500 1000 1500 2000 2500 0.00 0.02 0.04 0.06 0.08 0.10 Raw coal Four types of mixing Average diameter (mm) Accumulated pore volume (cm3/g) 0.00 0.02 0.04 0.06 0.08 0.10 Accumulative pore volume (cm3/g) Fig. 3.1.2 The aperture analysis 3 Change pattern of the cumulative hole volume of the raw coal and composite samples Table 1 Pore size distribution for raw coal and composite samples Coal sample Micropore (%) Mesoporous pore (%) Macropore (%) Raw coal sample 0.62 56.15 43.23 Composite sample 0.22 40.43 59.35 Table 1 Pore size distribution for raw coal and composite samples From the SEM map of the above experiments, it was found that both samples had different numbers of stomatal structures at different magnifications. From the SEM scan- ning image of the raw coal sample, we can see that the raw coal sample was completely flat, and a small amount of clay minerals and coal debris accumulation appeared. From the SEM scanning image of composite samples, it can be observed that the sample had the structural characteristics of each sample at the same time, such as the large amount of clay ore on the surface of coal silk-carbon structure oil shale. As mixed samples’ common granular chip debris attached to the sample surface, the surface was rough, uneven with a large number of pores, and silk carbon structure, stripes, silk carbon, leading to a larger surface area. The pores’ morphol- ogy, a circular ellipsoid and irregular shape, connectivity between pores was poor, and on the surface can clearly be seen part of kaolinite cover. After comparing the raw coal with the composite sample, it can be found that the raw coal has more pore structures. More pore structures increase the oxygen channels of the raw coal, and the surface is covered by some minerals, providing conditions for saving heat. 0 500 1000 1500 2000 2500 0.00 0.02 0.04 0.06 0.08 0.10 Raw coal Four types of mixing Average diameter (mm) Accumulated pore volume (cm3/g) 0.00 0.02 0.04 0.06 0.08 0.10 Accumulative pore volume (cm3/g) Fig. 3 Change pattern of the cumulative hole volume of the raw coal and composite samples 0 500 1000 1500 2000 2500 0.00 0.02 0.04 0.06 0.08 0.10 Raw coal Four types of mixing Average diameter (mm) Accumulated pore volume (cm3/g) 0.00 0.02 0.04 0.06 0.08 0.10 Accumulative pore volume (cm3/g) Fig. 3 Change pattern of the cumulative hole volume of the raw coal and composite samples 3.2 Analysis of mineral exothermic characteristics in mines Fig. 3 Change pattern of the cumulative hole volume of the raw coal and composite samples 3.2.1 Analysis of heat flow Through the observation of experimental data, it can be found that in the DSC curve (Fig. 4), the sample curve with the increase of the temperature possessed the same trend in the initial stage of heat flow being decreased. The above- mentioned was due to water evaporation, that adsorbed heat. During the evaporation process, the heat was produced by 3.1.3 Micromorphology analysis However, oxygen quickly occupied the surface of the sample and reacted with a large number of small molecules produced by cracking, releasing an enormous amount of heat, which was constantly greater than the heat adsorption. Here, the DSC curve showed a ris- ing trend until the peak of heat flow was reached.if adsorption effect was gradually enhanced, with the heat released being greater than the physical adsorption effect. Therefore, the heat flow value began to rise gradually. Mean- while, this stage was in the process of heat release. In the rapid heating phase, two samples DSC curve increased with temperature until the peak heat flow, then the heat flow curve commenced to drop. This phenomenon was ascribed to the fact that the sample contained a variety of functional groups. The high activity functional group first reacted with oxygen, then released an immense amount of gas, and finally the low activity functional group in the process of oxidation was gradually activated to participate in the reaction. In the high- temperature combustion stage, the DSC curve of the two samples decreased with the temperature, and then rose to the second peak heat flow. The peak heat flow was between 462 and 478 °C. In addition, the heat flow value started to decrease after reaching the peak. This was because with the rising temperature, there was not only a pyrolysis reaction inside the sample, but also an oxidation combustion reac- tion. A large number of unstable bridge bonds in the sample underwent bond breaking reactions, cracking at high-tem- perature, and producing a huge number of small molecule structures and pyrolysis products. The process required more energy, adsorbing part of the reaction to release heat, leading to the reduction of DSC curve. However, oxygen quickly occupied the surface of the sample and reacted with a large number of small molecules produced by cracking, releasing an enormous amount of heat, which was constantly greater than the heat adsorption. Here, the DSC curve showed a ris- ing trend until the peak of heat flow was reached.if raw coal samples was greater than the composite sample. When the temperature of the second exothermic peak was roughly 460 °C, the heat flow value of the raw coal sample was greater than the composite sample. It can be observed that the heat flow of the raw coal sample was the highest. 3.1.3 Micromorphology analysis The reaction was exothermic, the heat released was larger, and the raw coal was more prone to spontaneous combustion and produced more harmful gases (Fig. 5). 3.1.3 Micromorphology analysis The pore and fissure development of the two samples at different magnifications (raw coal on the left and four mix- ture on the right) were tested, as shown in Fig. 4. Page 5 of 15 75 Apparent activation energy of mineral in open pit mine based upon the evolution of active… Page 5 of 15 75 Fig. 4 SEM of raw coal and composite samples During the cold oxidation phase, the DSC curves of the two samples increased with temperature. The heat adsorp- tion effect caused by water evaporation under low-temper- ature oxidation conditions was weakened, and the chemical coal’s physical adsorption, where heat release was relatively small. Therefore, this stage in the heat adsorption process as the temperature rising DSC curve first rose followed by the downward trend. 1 3 Page 6 of 15 S. Lu et al. 75 75 adsorption effect was gradually enhanced, with the heat released being greater than the physical adsorption effect. Therefore, the heat flow value began to rise gradually. Mean- while, this stage was in the process of heat release. In the rapid heating phase, two samples DSC curve increased with temperature until the peak heat flow, then the heat flow curve commenced to drop. This phenomenon was ascribed to the fact that the sample contained a variety of functional groups. The high activity functional group first reacted with oxygen, then released an immense amount of gas, and finally the low activity functional group in the process of oxidation was gradually activated to participate in the reaction. In the high- temperature combustion stage, the DSC curve of the two samples decreased with the temperature, and then rose to the second peak heat flow. The peak heat flow was between 462 and 478 °C. In addition, the heat flow value started to decrease after reaching the peak. This was because with the rising temperature, there was not only a pyrolysis reaction inside the sample, but also an oxidation combustion reac- tion. A large number of unstable bridge bonds in the sample underwent bond breaking reactions, cracking at high-tem- perature, and producing a huge number of small molecule structures and pyrolysis products. The process required more energy, adsorbing part of the reaction to release heat, leading to the reduction of DSC curve. 3.2.2 Heat release analysis The structure can be determined only if these characteristic peaks can be identified.f heat release continued to increase until the peak heat release embarked on to reach plateau. In low-temperature oxidation stage, at the beginning of the reaction, two types of sample heat were less. Even the heat curve showed slightly down- ward trend, due to water evaporation; it adsorbed heat and release of heat was generated by physical adsorption. Here, exothermic heat was relatively small. Accordingly, in the heat adsorption process, the previous DSC curve tended to be stable and consistent. Then, with the change of tempera- ture, heat started to increase in rapid heating stage. As can be clearly observed, two types of sample heat commenced to present exponential growth. In addition, as the tempera- ture presented the characteristics of highly active functional group that first reacted with oxygen, and then released an immense amount of gas, low active functional group in the process of oxidation gradually activated to participate in the reaction. Therefore, with the rise of temperature, the sam- ple participated in the reaction of active functional quantity, followed by the increase of heat intensity high-temperature combustion stage. For two samples of heat, the temperature continued to increase until the heat peak began to smooth. This was due to a large number of small molecules active agent structure and combustible materials reacting with oxy- gen, constantly releasing a large amount of heat, and yield- ing an enormous amount of gases. i From the infrared spectrum in Fig. 7, there are differences in the absorption peak intensities of different samples, result- ing in the functional groups contained in the experimental samples being basically the same, but with varying quanti- ties. The functional group with greater activity during the oxidation process is the hydroxyl group. The inter-molec- ular association hydroxyl hydrogen bonds, inter-molecular hydroxyl hydrogen bonds, and free hydroxyl groups con- tained in different coal samples were the existing differences. Both methyl and methylene are present in all experimental samples, and the methyl deformation vibration absorption intensity is high. In addition, the content of methylene is slightly higher than that of methyl, indicating that methyl- ene is more active during the oxidation process. The methyl absorption intensity of the raw coal sample is higher than that of the composite sample. 3.2.2 Heat release analysis The main active group during the oxidation process is the oxygen-containing functional group, which has a significant impact on the chemical prop- erties of the sample. From Fig. 6, it can be seen that the C–O–C functional groups contained in the composite sam- ple had the highest absorbance, while the residual C–O–C oxygen-containing functional groups in the raw coal sample were relatively low. Because of the fact that the C=C double 3.2.2 Heat release analysis The total heat release of raw coal was 4714.40 J/g, and the total heat release of composite sample coal was 1795.63 J/g. The total heat release of the raw coal sample was the largest, indicating that there were relatively more active functional groups in the raw coal sample, which was easy to partici- pate in the oxidation reaction and generated more deleteri- ous gases. The change of heat release of both samples with temperature is shown in Fig. 6. The heat release of the two samples had the same change pattern with the temperature increase. The heat release of the sample was exceptionally small in the low-temperature oxidation stage. With the increase of coal temperature, the heat release gradually increased in the expeditious heating stage, and the growth rate accelerated, increasing exponen- tially. The difference between the two curves is in the rapid heating stage. When the sample is in the rapid heating stage, the growth rate of the raw coal heat release is greater than that of the composite sample, denoting that the raw coal is sensitive to the temperature in this stage, and the oxi- dation reaction is more intense than that of the composite sample. During the high-temperature combustion phase, the l Overall, when the fixed heating rate of different coal samples was 10 °C/min, the first exothermal peak tempera- ture was aprox. 326 °C. Furthermore, the heat flow value of Fig. 6 Plots of heat release of raw coal and composite sample coal with temperature 1 3 Fig. 5 Mixed heat flow curve of raw coal and composite samples Fig. 6 Plots of heat release of raw coal and composite sample coal with temperature Fig. 6 Plots of heat release of raw coal and composite sample coal with temperature Fig. 5 Mixed heat flow curve of raw coal and composite samples 1 3 Apparent activation energy of mineral in open pit mine based upon the evolution of active… Page 7 of 15 75 75 in the molecule and the influence of the adjacent groups, reflecting the characteristics of the molecular structure. Because of the various vibration modes of various groups, a large number of adsorption peaks appeared in the infrared spectrogram. Four types of adsorption bands of hydroxyali- phatic aromatics and oxygen-containing functional groups were taken as the characteristic peaks of the sample. 3.3.1 Infrared spectrogram analysis The position and intensity of the infrared adsorption spec- trum peak depended on the vibrational form of the groups Fig. 7 The IR spectrum of raw coal and composite sample Fig. 7 The IR spectrum of raw coal and composite sample 3 S. Lu et al. 75 Page 8 of 15 75 Table 2 Peak position distribution of active functional groups for raw coal and composite sample Functional group Raw coal sample Composite sample Free hydroxyl–OH 3690, 3650 3697, 3649 Intramolecular hydrogen bond–OH 3620 3620 Intermolecular association hydrogen bond–OH 3420 3449 Methylene symmetric stretching motion 2852 2852 Methylene asymmetric stretching motion 2920 2923 Methylene shear vibration 1448 1461 Methyl shear vibration 1374 C=C 1605 1645 C–O 1263 C–O–C 1032, 1098 1035, 1093 Aromatic hydrocarbons Ar–CH 794, 871 794 Table 2 Peak position distribution of active functional groups for raw coal and composite sample consumed, and other functional groups reacted with oxygen to generate hydroxyl groups. bond was the main structure of the aromatic ring and the raw coal had not been oxidized, the double bond structure was stable, the content was high, and the peak intensity was high, resulting in a strong C=C double shoulder absorption spectrum of the raw coal sample. Table 2 summarizes the assigned peaks of different functional groups for different coal samples. During the low-temperature oxidation stage, the inter- molecular hydrogen bond strength of inter-molecular hydroxyl groups in raw coal maintains an upward trend. In the composite sample, the strength of inter-molecular hydro- gen bonds tends to stabilize while the absorption strength of free hydroxyl groups increases. Meanwhile, some unsatu- rated hydrocarbons were oxidized to form hydroxyl groups, but the formation rate is greater than the consumption rate. Therefore, there was an upward trend at this stage. In the rapid heating stage, in the composite of the raw coal sam- ple, the light absorption intensity of hydrogen bonds with wave numbers 3690 and 3620 cm−1 decreases, while the light absorption density of free hydroxyl groups with a wave length of approximately 3650 cm−1 basically increases. In 3.3.2 Analysis of functional group transfer law The curves of different groups with temperature are shown in Figs. 8, 9, 10 and 11. From Fig. 8, it can be seen that hydroxyl groups existed in the oxidation process of each sample. As the temperature increased, the number of hydroxyl groups kept changing, as hydroxyl groups con- tinuously participated in oxidation reactions and were 1 Fig. 8 Curve of the hydroxyl group with temperature for raw coal and composite samples Fig. 8 Curve of the hydroxyl group with temperature for raw coal and composite samples 3 Apparent activation energy of mineral in open pit mine based upon the evolution of active… Page 9 of 15 75 h hi h b i b f h l ib i d d bili d i li h l F h d Fig. 9 Curve of fat hydrocarbon with temperature for a Raw coal sample and b Composite sample Fig. 10 Curves of oxygen-containing functional groups with temperature for a Raw coal sample and b Composite sample Fig. 9 Curve of fat hydrocarbon with temperature for a Raw coal sample and b Composite sample Fig. 9 Curve of fat hydrocarbon with temperature for a Raw coal sample and b Composite sample Fig. 10 Curves of oxygen-containing functional groups with temperature for a Raw coal sample and b Composite sample Fig. 10 Curves of oxygen-containing functional groups with temperature for a Raw coal sample and b Composite sample vibration tended to stabilize and rise slightly. Fatty hydrocar- bon in samples generally existed in the form of long bonds, general chain bond long activity performance was higher. In low-temperature oxidation stage, as the temperature increased, fatty hydrocarbon attacked by oxygen, fractured into more fatty hydrocarbon, methyl was oxidized, generat- ing CO, CO2, and hydrocarbon gas. When methyl and meth- ylene production was greater than the consumption absorb- ance rising, otherwise downward trending. In the stage of rapid heating, the absorbance of the methyl deformation the high-temperature combustion stage, because of the later reaction, free hydroxyl groups and intramolecular hydrogen bonds, as well as the continuous reaction between the final oxygen-containing functional groups and water, the adsorp- tion strengths of the two types of coal samples were 3690, 3650, and 3620 cm−1, respectively. In the low-temperature oxidation stage, the absorbance of the methyl deformation vibration of the raw coal sample gradually decreased. 3.3.2 Analysis of functional group transfer law While in the mixed sample, the light absorption intensity of the methyl asymmetric expansion 1 3 1 S. Lu et al. Page 10 of 15 75 75 Fig. 11 Curves of aromatic hydrocarbons with temperature for a Raw coal sample and b Composite sample Fig. 11 Curves of aromatic hydrocarbons with temperature for a Raw coal sample and b Composite sample In the low-temperature oxidation stage, in the raw coal sample, the expansion and vibration absorbance of fat ether with a wavenumber of about 1032 cm–1 basically showed an upward trend, while the absorbent intensity of C–O showed an upward trend, while the absorbent intensity of fat ether at about 1083 cm–1 had a downward trend. In the mixed samples, the adsorption intensity of the fat ether exhibited a decreasing trend. The light absorption intensity of the fat ether was due to the secondary group containing C–O–C in the oxidation process, which caused the absorbance to rise. In the rapid heating stage for in the raw coal sample, the light absorption intensity of the fat ether expansion vibration bond increased, while the absorbance of C–O also showed an upward trend. In the composite sample mixture, the absorption intensity of the fat ether showed a downward trend. In the high-temperature combustion stage, in the raw coal sample and composite sample mixture, the light adsorp- tion intensity of the fat ether showed a downward trend. In the raw coal sample, the absorbance of C–O also became downward. vibration of raw coal samples showed a downward trend, while the methyl deformation vibration of mixed samples showed a horizontal trend, with a slightly downward trend. With the rise of temperature, side chain and oxygen com- posite generated more CO and water vapor precipitation, leading to reducing fat hydrocarbon side chain, light adsorp- tion intensity reduced high-temperature combustion stage. In raw coal, methylene asymmetric vibration methyl symmetry vibration methyl deformation vibration absorbance dropped swiftly, then fluctuation absorbance increased, then reduced. In the composite sample, the methyl asymmetric expansion vibration commenced to maintain a relatively stable trend, and then started to decline quickly, while the absorbance of the methyl deformation vibration appeared to rise with the temperature, and decreased expeditiously after reaching the peak absorbance. When the 600 °C value was reached, the intensity of methylene asymmetry was already less than 0.1. While the intensity of methyl deformation vibration was slightly greater than 0.1. 3.3.2 Analysis of functional group transfer law This indicated that during the oxi- dation process, the adipose hydrocarbon of the expansion vibration peak was mainly consumed, and that the methylene consumption was greater than the methyl group. In the low-temperature oxidation stage, the vibration absorbance of the deformation of about 1605 cm–1 and the deformation of 794 cm–1 was decreasing, and the absorb- ance of the deformation of the substituted aromatics around 871 cm–1 was rising. The vibration absorbance of C=C dou- ble bonds and various substituted aromatics in the compos- ite sample mixture was decreasing. The C=C double bond was related to the oxidation reaction of the fat hydrocarbon side chain. Because of the participation of the fat hydrocar- bon side chain in the oxidation reaction, which led to the decrease of its content, the aromatic ring structure of various Oxygen-containing functional groups accounted for a large proportion in the total functional groups, and the more active functional groups mainly included carboxyl hydroxyl ether bond fat ether. The main spectrum of the infrared spectrum was fat ether expansion vibration C–O–C, mainly manifested by the wave number of 1093 and 1032 cm–1, in which the coal contains additional phenolic alcohol ether C–O characteristic absorption peak, mainly manifested for aprox. 1263 cm–1. 3 3 3 Page 11 of 15 75 Apparent activation energy of mineral in open pit mine based upon the evolution of active… 75 (2) Achar differentiation substituted aromatic hydrocarbons began to react and then consume. At this time, the consumption was greater than the production amount, resulting in a downward trend. In the rapid heating stage for in the raw coal sample, the vibra- tion absorbance of C=C double bond with wave number of about 1605 cm–1 and wave number of about 871 cm–1 kept rising, and the vibration absorbance of a deformation of substituted aromatics of rough 794 cm–1 was decreasing. In the composite sample mixture, the absorbance of C=C began to decrease, and the vibration absorbance of various substituted aromatics continued to decrease. (5) ln d훼 f(훼)dT = ln A 훽−Ea RT (5) In Eq. (5), when the heating rate of β is certain, map 1000/T with ln[dα/dT/f(α)], and then make a linear fitting, and obtain the apparent activation energy Ea from the slope of the resulting quasi-t line. The light absorption intensity of the functional group was used to calculate the conversion degree. (6) A = (An −A1 ) (An −AEnd ) The generation and consumption of C=C double bonds were almost equal, so the absorbance remained constant, exceeding the production amount with increasing tempera- ture, resulting in a decreasing curve. In the high-temperature combustion stage, in the composite sample of raw coal sam- ples, the vibration absorbance of various substituted aro- matics with wave number of roughly 1605 cm–1 and wave- number of approximately 794 cm–1 was decreasing. In the raw coal sample, the vibration absorbance of deformation of various substituted aromatics with wave number of ca. 794 cm–1 decreased. In the composite sample mixture, the C=C double bond wave number and the vibration absorb- ance of various substituted aromatic hydrocarbons showed a decreasing trend. Owing to the increasing temperature, the stable functional group structure fractured, producing a large number of substituted hydrocarbon group. Meanwhile, the absorbance slightly exhibited an upward trend, while the deformation vibration of a variety of substituted aromatic hydrocarbons still decreased with the continuous reaction. (6) In Eq. (6), the absorptive intensity of the functional group at the beginning of the initial stage; A1 is the absorp- tion intensity of the functional group at time T; AEnd is the absorptive intensity of the functional group at the end of the stage. 3 The original data and the commonly used gas–solid reac- tion mechanism function f(α) and g(α) put into the integral Eqs. (3) and (5), a series of apparent activation energy is properly selected (Zhao et al. 2023). If reasonable f(α) and g(α) can reflect the real reaction, the apparent activation energy is similar, and the correlation coefficient of the fit- ting curve is excellent. Therefore, similar apparent activation energy and high correlation of a set of mechanism functions were chosen as the most universal mechanism function in this stage. The hydrogen bond of the association of shear vibration methyl shear in aromatic ring C= C stretching vibration C–O, C–O–C aromatic hydrocarbon Ar–CH, respectively, solved the mechanism function of different oxidation stages, selected the highest fit as the reactions apparent activation energy, and took the free hydroxyl group in composite sam- ple as an example. The integration method and differential method of different mechanism functions of the apparent activation energy and fit degree are summarized in Figs. 12, 13, and 14. 3.4 Analysis of the pit mineral dynamics The data of the main functional groups analyzed by in–situ infrared spectroscopy were evaluated by Achar differential method and Coats-Redfern integral method. (1) The Coats-Redfern integration method. Coats-Redfern is a method for calculating kinetic param- eters based on the results of heating rate test. 13, and 14. From the data in Figs. 12, 13 and 14, it can be clearly observed that the free hydroxyl group in the hydroxyl group of the mixed sample adopted the 2D diffusion Valensi equa- tion of the integral method during the low-temperature oxi- dation stage. The fitting coefficient R2 reached the maxi- mum value, e.g., 0.999. That is, the diffusion model best reflected the apparent activation energy of the functional groups during the reaction. By the Avrami-Erofeev equa- tion (n = 4), the fitting coefficient R2 reached the maximum value, e.g., 0.984. That is, the random nucleation and growth models can best reflect the apparent activation energy of the functional groups during the reaction. The one-dimensional (3) ln g(훼) T2 = −Ea RT + ln [ AR 훽Ea ] (3) In Eq. (3), the conversion degree (α) of coal at time T; T is the temperature, K; also Ea is the apparent activation energy, kJ/mol; R is the universal gas constant, R = 8.314 J/ (mol·K); A refers to the prefactor, S–1; β is the heating rate, K/min. 1000/T using ln (g (α)/T2) to obtain the apparent activation energy Ea. (4) Ea = −KR Ea = −KR (4) 1 3 S. Lu et al. Page 12 of 15 75 Fig. 12 Mechanism functions of the four mixed free hydroxyl groups are solved during the low-temperature oxidation phase Fig. 12 Mechanism functions of the four mixed free hydroxyl groups are solved during the low-temperature oxidation phase Fig. 13 Mechanism functions of the four mixed free hydroxyl groups during the rapid heating phase Fig. 13 Mechanism functions of the four mixed free hydroxyl groups during the rapid heating phase 1 3 Apparent activation energy of mineral in open pit mine based upon the evolution of active… Page 13 of 15 75 75 Fig. 14 The mechanism functions of the four mixed free hydroxyl groups during the high-temperature combustion phase 14 The mechanism functions of the four mixed free hydroxyl groups during the high-temperature combustion phase diffusion parabolic rule of the integral method was adopted in the high-temperature combustion phase. The fitting coef- ficient R2 reached the maximum value, for instance, 0.977. As planned, the results showed that the diffusion model best reflected the apparent activation energy of the func- tional groups during the reaction for the remaining active functional groups. This study took the same approach. 13, and 14. The apparent activation energy was calculated by integral and differential methods, by selecting the most highly fitted reac- tion model as the mechanism function. Using this apparent activation energy as the basis for the subsequent analysis, the apparent activation energies of each functional group at different stages are shown in Fig. 11. oxygen atoms slowly decomposed. At the same time, the organic matter in oil shale root for shale oil, combined with four mixed with coal gangue, exacerbated the difficulty of the chemical reaction; the energy increased, the comprehen- sive influence of various factors eventually led to its apparent activation energy which was higher than raw coal. Because of the small quartz mineral content in raw coal, the less fatty hydrocarbon structure, inter-molecular aromatic ring layer stack height was small. The loose microcrystalline struc- ture, aliphatic structure content, aromatic ring condensation degree, and layer structure were relatively orderly. Here, a variety of conditions in the chemical reaction played a vital role in promoting, and mixing the sample of high quartz content and raw coal; on the other hand, the apparent activa- tion energy of aromatic coal was greater than composited. f As can be seen from Fig. 15, the Ea of the functional groups in the raw coal and the composite samples were small in the low-temperature oxidation stage, indicating that the functional activity of the two samples was high in this stage, the low-temperature oxidation stage was 0–220 °C, the rapid heating stage was 220–580 °C, and the high-temperature combustion stage was more than 580 °C. Mixed samples of hydroxyl fatty hydrocarbon oxygen functional group of three types of groups apparent activation energy were substan- tially higher than raw coal. This was because in the process of coal oxygen composite reaction, as the temperature rose, adsorption on the sample surface of oxygen molecules and oxygen functional groups, such as carboxyphenolic hydroxyl 4 Conclusions The oxidation dynamics of open-pit minerals were stud- ied from both microscopic and macroscopic perspectives. Combined with pore characteristics and exothermic charac- teristics, the oxidation stage of coal spontaneous combus- tion process was divided, and the migration and conversion rules of functional groups with temperature were analyzed. In summary, the following conclusions were obtained: 1 3 75 Page 14 of 15 S. Lu et al. Page 14 of 15 75 Fig. 15 Ea of different functional groups of raw coal mixed with composite samples ig. 15 Ea of different functional groups of raw coal mixed with composite samples Fig. 15 Ea of different functional groups of raw coal mixed with composite samples (1) The raw coal sample possessed high fixed carbon con- tent. After the composite samples were affected by coal gangue, the fixed ash content was high, the element content decreased with temperature, and the specific surface area was abated. The large aperture distribution was slightly higher than that of the middle pore, the micropore was exceedingly low, and the gas adsorption capacity was weaker than that of raw coal. The oxygen channels of raw coal were more than the composite samples, and the heat storage capacity was weaker than the composite samples. ite samples after mixing DSC curve possessed simi- lar trend. With the rising temperature, the DSC curve increased in the downward trend. This was due to the active functional group in the sample of a large number of small molecular structures and oxygen reaction. The sample released an enormous amount of heat, then the DSC curve began to rise. (3) (3) The free hydroxyl group and inter-molecule hydro- gen bonds appeared in the inter-molecular hydrogen bond. However, the methyl light absorption intensity was higher than the other samples. The highest absorb- ance of the C–O–C functional groups contained in the mixed samples was found in the oxidation process. The free hydrogen bond –OH in the mixed samples demon- strated a trend of first rising and then decreasing. The intensity of the inter-molecular association hydrogen (3) The free hydroxyl group and inter-molecule hydro- gen bonds appeared in the inter-molecular hydrogen bond. However, the methyl light absorption intensity was higher than the other samples. The highest absorb- ance of the C–O–C functional groups contained in the mixed samples was found in the oxidation process. 4 Conclusions The free hydrogen bond –OH in the mixed samples demon- strated a trend of first rising and then decreasing. The intensity of the inter-molecular association hydrogen (2) Using DSC curve, two samples of combustion process were divided into low-temperature oxidation, expedi- tious heating and high-temperature combustion with three stages of raw coal sample of DSC curve and heat. The surface of the coal active functional group was more likely to react with oxygen. However, compos- 1 3 Page 15 of 15 75 Apparent activation energy of mineral in open pit mine based upon the evolution of active… 75 bond –OH was mainly due to the influence of the alkyl side chain of the sample. bond –OH was mainly due to the influence of the alkyl side chain of the sample. Deng J, Ma XF, Zhang YT et al (2015) Effects of pyrite on the spon- taneous combustion of coal. Int J Coal Sci Technol 2:306–311 Huang XD, Kang ZQ, Zhao J et al (2023) Experimental investigation on micro-fracture evolution and fracture permeability of oil shale heated by water vapor. Energy 277:127677 (4) The apparent activation energy of the main active func- tional groups in raw coal and composite samples exhib- ited a trend from low to high with the development of oxidation stage, the composite sample increased the main functional group reaction’s apparent activation energy, improved the coal activity in low-temperature oxidation phase, and made the less need of heat for oxidation reaction. (4) The apparent activation energy of the main active func- tional groups in raw coal and composite samples exhib- ited a trend from low to high with the development of oxidation stage, the composite sample increased the main functional group reaction’s apparent activation energy, improved the coal activity in low-temperature oxidation phase, and made the less need of heat for oxidation reaction. Jiang HY, Liu S, Wang J et al (2023) Study on evolution mechanism of the pyrolysis of chang 7 oil shale from Ordos basin in China. Energy 272:127097 Jin JF, Sun JS, Lü KH et al (2023) Catalytic pyrolysis of oil shale using tailored Cu@zeolite catalyst and molecular dynamic simulation. Energy 278:127858 Ju Y, Zhu Y, Xie HP et al (2019) Fluidized mining and in-situ trans- formation of deep underground coal resources: a novel approach to ensuring safe, environmentally friendly, low-carbon, and clean utilisation. Declarations Wang XM, Wang Q, Pan S et al (2023) The non-isothermal thermal decomposition evolution of the Fushun oil shale kerogen based on ReaxFF molecular dynamics simulation. J Analy Appl Pyroly 169:105869 Competing interests The authors declare that we do not have any com- mercial or associative interest that represents a conflict of interest in connection with the work submitted. Zeng Q, Li S (2022) Experimental study on the oxidation kinetics of coal in typical coal mining areas of the Southern Junggar coal- field, Xinjiang, China. Int J Coal Sci Technol 9:78 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/. i Zhai YM, Yang TH, Zhang Y et al (2023) Co-pyrolysis characteris- tics of raw/torrefied corn stalk and oil shale. J Analy Appl Pyrol 171:105967 Zhang L, Qi SC, Takeda N et al (2018) Characteristics of gas evolution profiles during coal pyrolysis and its relation with the variation of functional groups. Int J Coal Sci Technol 5:452–463 Zhang LJ, Zhou X, Zhou Y et al (2022) Surface coal mining impacts on land use change and ecological service value: a case study in Shengli coalfield, Inner Mongolia. Int J Coal Sci Technol 9:65 i Zhao JY, Ming HQ, Guo T et al (2022) Semi-enclosed experimental system for coal spontaneous combustion for determining regional distribution of high-temperature zone of coal fire. Int J Coal Sci Technol 9:62 Zhao JY, Xiao YY, Song JJ et al (2023) Kinetic properties of non- caking coal spontaneous combustion by evolution of its functional groups. Fuel 354:129428 4 Conclusions Int J Coal Sci Technol 6:184–196 Acknowledgements Financial support for this study was kindly pro- vided by the National Natural Science Foundation Project of China (5217-4202), Young Elite Scientists Sponsorship Program of China Association for Science, and Technology (2021QNRC001). Liang YT, Yang YL, Guo SD et al (2023) Combustion mechanism and control approaches of underground coal fires: a review. Int J Coal Sci Technol 10:24 Liu YM, Xue LF, Ma JX et al (2023) Three-dimensional numerical simulation, energy efficiency and economic benefit estimation of oil shale in situ pyrolysis process. Geoenergy Sci Eng 227:211804 Author contributions LSP: Validation, Resources Writing-review and editing, ZJY: Validation, Funding acquisition. SJJ: Conceptualization, Data curation. CJM: Data curation. C-MS: Supervision and editing. All authors read and approved the final manuscript. Onifade M, Genc B (2019) Spontaneous combustion liability of coal and coal-shale: a review of prediction methods. Int J Coal Sci Technol 6:151–168 Tang YG (2020) Special issue on coal geology in China. Int J Coal Sci Technol 7:217–219 Data availability All data included in this study are available in the anuscript. Tang YG, Li RQ, Wang SQ (2020) Research progress and prospects of coal petrology and coal quality in China. Int J Coal Sci Technol 7:273–287 References Zheng Y, Lei GL, Yao CJ et al (2023) Characteristics and kinetics of Maoming oil shale pyrolysis in the presence of CoCl2 assisted steam. Fuel 338:127279 Aich S, Nandi BK, Bhattacharya S (2019) Effect of weathering on physico-chemical properties and combustion behavior of an Indian thermal coal. Int J Coal Sci Technol 6:51–62 Zhu T, Wang RN, Yi NJ et al (2020) CO2 and SO2 emission character- istics of the whole process industry chain of coal processing and utilization in China. Int J Coal Sci Technol 7:19–25 Alexander VB, Yuan CD, Mikhail AV et al (2023) In-situ combus- tion technique for developing fractured low permeable oil shale: experimental evidence for synthetic oil generation and successful propagation of combustion front. Fuel 344:127995i 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. Cao DY, Wang AM, Ning SZ et al (2020) Correction to: coalfield structure and structural controls on coal in China. Int J Coal Sci Technol 7:417 Chabukdhara M, Singh OP (2016) Coal mining in northeast India: an overview of environmental issues and treatment approaches. Int J Coal Sci Technol 3:87–96 1 3
https://openalex.org/W2558654066	http://dinamikahukum.fh.unsoed.ac.id/index.php/JDH/article/download/411/399	English	null	ROLE OF LAW IN CONSTRUCTION AND DEVELOPMENT OF SMALL SCALE INDUSTRIES THROUGH NORMATIVE PERSPECTIVE	Dinamika Hukum/Jurnal Dinamika Hukum	2,015	cc-by	4,831	1 Sri Redjeki Hartono, 2007, Orientasi Ke Arah Pengelo- laan Investasi (Sebuah Landasan Pemikiran Awal), Bandar Lampung: Lampung University, page 2. ROLE OF LAW IN CONSTRUCTION AND DEVELOPMENT OF SMALL SCALE INDUSTRIES THROUGH NORMATIVE PERSPECTIVE Endang Sutrisno Magister of Legal Studies Programme Swadaya Gunung Jati University of Cirebon E-mail: endangsutrisno94@gmail.com Abstract The presence of law has become an absolute prerequisite that must exist in the dynamics of civil society. It is to achieve justice, certainty, and expediency, so the works of it will not be separated from such a noble mission. On the other side, the law is likely inseparable from the fields of meta- juridical, including economics. The expectations of the interference of law into economy, makes the existence of justice for the business players can be realized through the enacted product legislation. Regulations concerning investments and partnerships have the intent to build self-reliance and empowerment for small industry players so as to compete in the era of economic globalization. Laws employed as the instrument of social change to strengthen the capitalization of small industry and business empowerment through the training and development of small industries, as normatively mandated by law. Key words: progressive law, normative dimention, small industry 2 Satjipto Rahardjo, 2010, Ilmu Hukum, Bandung: Alumni, page 5-6. Abstrak Kehadiran hukum menjadi prasyarat mutlak yang harus ada dalam dinamika kehidupan masyarakat. Hukum hadir untuk mencapai tujuan keadilan, kepastian dan kemanfaatan, sehingga aktivitas bekerjanya hukum tidaklah akan terlepas dari misi luhur tersebut. Pada sisi lain hukum-pun tidak mungkin dipisahkan dari bidang-bidang meta yuridis, termasuk ekonomi. Harapan masuknya hukum ke tataran ekonomi, menjadikan eksistensi keadilan untuk pelaku-pelaku bisnis dapat diwujudkan khususnya melalui produk perundang-undangan yang diberlakukan. Regulasi menyangkut investasi dan kemitraan mengandung maksud membangun kemandirian dan pemberdayaan untuk pelaku industri kecil sehingga mampu bersaing di era globalisasi ekonomi. Hukum dijadikan sarana untuk melakukan perubahan sosial, yang berakibat pada aspek penguatan permodalan industri kecil serta pemberdayaan usaha melalui pembinaan dan pengembangan industri kecil, sebagaimana yang diamanatkan oleh undang-undang secara normatif. Kata Kunci : hukum progresif, dimensi normatif, industri kecil Introduction munities as well as validating the changes that have occurred in the past.2 In the end there are 5 (five) prerequisite conditions in order for the legal support of economic development which can be described in figure 1. The law has a very important role in terms of the legal relations of the parties nor the le- gality of economic activity itself, which means that business activity should be in good-setting so that inequality and injustice does not hap- pen.1 The function of the law on the one hand can be used as a means to change the socie- ty in order to become better and on the other hand to maintain the pattern of existing com- Based on Figure 1 above, this illustrates that the legal system essentially sought to have the task to keep existing interests in the grow- th of community development that competes each other, so that it is required factors, which are stability through a secure state of attempt- 318 Jurnal Dinamika Hukum Vol. 15 No. 3, September 2015 ple sovereignty, can be made a "hypothesis"6 that the sovereignty of the law was not intend- ed solely for the benefit of the law itself, but must rather be addressed and stood for com- munity interests. Normative provisions concern- ing small businesses, making the law should really be showing face alignments on a small marginalized community interests. This became the key to the direction of the development of the law that is most essential goals of law exis- tence in the life of the community. It is so fully realized that so far law development in this country tends to move in the artificial space and without direction.7 Indonesia today is faced with a very "unique" problem of law perfor- mance regarding the formal truth treated as the most dominant consideration of legal deci- sion embracing reine Rechtslehre Kelsenian’s way of thinking. 7 Dayanto, “Rekonstruksi Paradigma Pembangunan Nega- ra Hukum Indonesia Berbasis Pancasila”, Jurnal Dinami- ka Hukum, Vol. 13 No. 3 September 2013, Purwokerto: Law Faculty Jenderal Soedirman University, page 498. g, p g 9 Ibnu Artadi, “Dekonstruksi Pemahaman Penyelesaian Sengketa Bisnis (Ekonomi dan Keuangan) Beraspek Pida- na melalui Prosedur Perdamaian: Menuju Proses Peradi- lan Pidana Rekonsiliatif”, Jurnal Hukum Responsif, Vol. 1, No. 1 Year 2011, Cirebon: Law Faculty, Swadaya Gu- nung Jati University, page 33-34. y y, p g 8 Endang Sutrisno, “Tracing the Performance of Law in Indonesia (A Perspective of Thomas Kuhn’s “Normal Science”, Journal of Law, Policy and Globalization, International Institute for Science, Technology & Edu- cation Accelerating Global Knowledge Creation and Sharing, page 126. p g 6 Bambang Widjojanto, “Negara Hukum, Korupsi dan Hak Asasi Manusia: Suatu Kajian Awal”, Jurnal Hukum Prio- ris, Vol. 3 No.1 Year 2012, Jakarta: Law Faculty, Tri- sakti University, page 30. 3 Romli Atmasasmita, ”Tiga Paradigma Hukum dalam Pembangunan Nasional”, Jurnal Hukum Prioris, Vol. 3 No. 1 Year 2012, Jakarta: Law Faculty, Trisakti Univer- sity, page 5. y p g 4 Bahria Prentha, “Filsafat Hukum dan Nilai-Nilai Panca- sila”, Jurnal Ilmiah Kebijakan Hukum, Vol. 5 No. 2 Au- gust 2011, Jakarta: Pusat Pengkajian dan Pengembang- an Kebijakan Kementerian Hukum dan HAM RI, page 177. 5 Yunus Bureni, “Moralitas Pembentukan Peraturan Dae- rah dalam Upaya Mencapai Keadilan Substantif (Morali- ty Formation of Local Regulations in An Effort to Ensure Substantive Justice)”, Jurnal Legislasi Indonesia, Vol.10 No.2 June 2013, Jakarta: Direktorat Jenderal Peraturan Perundang-undangan Kementerian Hukum dan HAM RI, page 125. Substantive Justice)”, Jurnal Legislasi Indonesia, Vol.10 No.2 June 2013, Jakarta: Direktorat Jenderal Peraturan Perundang-undangan Kementerian Hukum dan HAM RI, page 125. 3 Romli Atmasasmita, ”Tiga Paradigma Hukum dalam Pembangunan Nasional”, Jurnal Hukum Prioris, Vol. 3 No. 1 Year 2012, Jakarta: Law Faculty, Trisakti Univer- sity, page 5. 4 Bahria Prentha, “Filsafat Hukum dan Nilai-Nilai Panca- sila”, Jurnal Ilmiah Kebijakan Hukum, Vol. 5 No. 2 Au- gust 2011, Jakarta: Pusat Pengkajian dan Pengembang- an Kebijakan Kementerian Hukum dan HAM RI, page 177. 5 Yunus Bureni, “Moralitas Pembentukan Peraturan Dae- rah dalam Upaya Mencapai Keadilan Substantif (Morali- ty Formation of Local Regulations in An Effort to Ensure Substantive Justice)”, Jurnal Legislasi Indonesia, Vol.10 No.2 June 2013, Jakarta: Direktorat Jenderal Peraturan Perundang-undangan Kementerian Hukum dan HAM RI, page 125. 6 Bambang Widjojanto, “Negara Hukum, Korupsi dan Hak Asasi Manusia: Suatu Kajian Awal”, Jurnal Hukum Prio- ris, Vol. 3 No.1 Year 2012, Jakarta: Law Faculty, Tri- sakti University, page 30. 7 Dayanto, “Rekonstruksi Paradigma Pembangunan Nega- ra Hukum Indonesia Berbasis Pancasila”, Jurnal Dinami- ka Hukum, Vol. 13 No. 3 September 2013, Purwokerto: Law Faculty Jenderal Soedirman University, page 498. 8 Endang Sutrisno, “Tracing the Performance of Law in Indonesia (A Perspective of Thomas Kuhn’s “Normal Science”, Journal of Law, Policy and Globalization, International Institute for Science, Technology & Edu- cation Accelerating Global Knowledge Creation and Sharing, page 126. 9 Ibnu Artadi, “Dekonstruksi Pemahaman Penyelesaian Sengketa Bisnis (Ekonomi dan Keuangan) Beraspek Pida- na melalui Prosedur Perdamaian: Menuju Proses Peradi- lan Pidana Rekonsiliatif”, Jurnal Hukum Responsif, Vol. 1, No. 1 Year 2011, Cirebon: Law Faculty, Swadaya Gu- nung Jati University, page 33-34. Introduction An approach that is still in further discussion through a more holistic al- ternative paradigm.8 The teaching of legal po- sitivism which is monistic, where it only admits one kind of Justice, that justice is born from positive law.9 ed law can play a role in the motion dynamics of community development, the existence of condition that is able to predict the changes occured, the existence of values of honesty in development activities, education aspect and ability of the legal profession that are able to anticipate problems arising in consequence of the development changes. Development of In- donesia's legal system since the time of colonial up to Romli Atmasasmita currently, distinguish in 4 (four) of the law models; first, highly rep- ressive collonial law model; second, develop- ment law model; third, progressive law model; and fourth, integrative law model.3 Figure 1 : Five Prerequisite of Legal Conditions Supporting Economic Development  Stability  Predictability  Fairness In order to make  Education; and system able to ma-  Ability of legal pro- intain intercompe fession which titive importance increases  Stability  Predictability  Fairness  Education; and  Ability of legal pro- fession which increases In order to make system able to ma- intain intercompe titive importance The development of small industries in fulfilling their needs, especially in terms of overcoming the limitations, the partnership be- came the only option that needs to be done by small industries with medium and large indus- tries. Introduction In partnership, there is cooperation bet- ween the major industry and medium scale in- Related to efforts actualizing the effec- tive legal system need realignment of institut- ional law supported by the quality of the hu- man resources and legal awareness of society and culture that continue to rise, along with the renewal of legal materials are structured harmoniously without conflict and overlapping and law continuously is updated in accordance with the demands of development needs.4 It is necessary to realize that legal justice is required to create an active role of the vari- ous parties ranging from the establishment of a legal product to enforcement of legal pro- ducts.5 The law has a sovereignty based on peo- Role of Law in Construction and Development of Small Scale Industries through Normative Perspective 319 Role of Law in Construction and Development of Small Scale Industries through Normative Perspective 319 legislation, product policy should be under- stood in its realization in all jurisdictions-and in all steps is supposed to be an entity that is au- thentic. The definition of authentic here is meant more as an entity that genuine compli- ance with the existence, based on Gustav Rad- bruch, the nature’s existence of law can be simplified into several goals to be achieved i.e. justice, legal certainty and benefit11. It aims to achieve the target as the ideals of Justice as a manifestation of popular sovereignty. Justice is something abstract that is in sollen world grown in human’s biological skepticism, but could not be overlooked that everyone crave justice. In the science of law, justice is the idea and law purpose but literally, justice could not be defined by science of law; thus, justice should be examined with theoretic and philosophy perspective.12 dustry and small industry, this can be form of home industry, basically mutual benefit for both parties who perform such cooperation. This reason is based on a logical consideration of economic development being one way in prospering community, in the economic deve- lopment required friendly bureaucracy towards capital investment. 10 Law No. 10 Haris Budiman & Suwari Akhmaddhian, “Implementasi Reformasi Birokrasi Bidang Perizinan Penanaman Modal di Kabupaten Kuningan”, Jurnal Ilmu Hukum Unifikasi, Vol. 1, No. 1 October 2013, Kuningan: Law Faculty, Ku- ningan University, page 2. Introduction 25 of 2007 about Capital Invest- ment, in Chapter VIII: Development Capital In- vestment for Micro, Small, Medium Scale En- terprises and Cooperatives Article 13 paragraph (2) mentions that the Government does the asistance and development of micro, small, medium scale enterprises and cooperatives th- rough partnership programs, improvement of competitiveness, giving of the encouragement of innovation and market expansion, as well as the dissemina-tion of the information widely. This regulation clearly became quite good dri- ving factor as a juridical foundation in the basis of the latest arrangement of a partnership, be- sides as set in Government Regulation No. 44 in 1997 about Partnership. The partnership concept formulated in article 26 The Govement Regulation No.44 in 1997 about partnership as follows. First, me- dium and large business to have partnership re- lations with small business either have or do not have a business relationship. Second, im- plementation of the partnership referred to in subsection strived towards the attainment of business relations. Third, partnerships imple- mented with an accompanying coaching and development in one or more fields and product- ion managers, marketing, human resources, ca- pital and technology. Fourth, in the exercise of the relationship of the two sides have equal legal position. The problem can be discuss in this article are: first, how does the normative landscape set in relation to the contract of the partner- ship done towards parties as construction and development of small industries in perspective of capital investment?, and second, how does the partnership patterns conducted by the par- ties in the framework of the construction and development of human resources in relation to Law No. 25 of 2007 and Government Regulation No. 44 in 1997 about the Partnership? Various studies in developing of small bu- sinesses in Indonesia show that small businesses experiencing weakness in almost every aspects, 11 Yudi Kristiana, “Ketika Hukum Tidak Lagi Otentik”, Jur- nal Hukum Supremasi, Vol. IV, No. 1 October 2010- March 2011, Jakarta: Pusat Studi Hukum Bisnis, Law Fa- culty, Sahid University, page 741-742. Read also Endang Sutrisno, “Implementasi Pengelolaan Sumber Daya Pesi- sir Berbasis Pengelolaan Wilayah Pesisir Secara Terpadu untuk Kesejahteraan Nelayan (Studi di Perdesaan Nela- yan Cangkol Kelurahan Lemahwungkuk Kecamatan Le- mahwungkuk Kota Cirebon)”, Jurnal Dinamika Hukum, Vol. 14, No. 1 January 2014. Purwokerto: Law Faculty, Jenderal Soedirman University, page 9. 12 Bahder Johan Nasution, “Kajian Filosofis tentang Kon- sep Keadilan dari Pemikiran Klasik sampai Pemikiran Modern”, Jurnal Hukum Yustisia, 89 eds May-August 2014, Tahun XXIII, page 119. y, p g 12 Bahder Johan Nasution, “Kajian Filosofis tentang Kon- sep Keadilan dari Pemikiran Klasik sampai Pemikiran Modern”, Jurnal Hukum Yustisia, 89 eds May-August 2014, Tahun XXIII, page 119. 11 Yudi Kristiana, “Ketika Hukum Tidak Lagi Otentik”, Jur- nal Hukum Supremasi, Vol. IV, No. 1 October 2010- March 2011, Jakarta: Pusat Studi Hukum Bisnis, Law Fa- culty, Sahid University, page 741-742. Read also Endang Sutrisno, “Implementasi Pengelolaan Sumber Daya Pesi- sir Berbasis Pengelolaan Wilayah Pesisir Secara Terpadu untuk Kesejahteraan Nelayan (Studi di Perdesaan Nela- yan Cangkol Kelurahan Lemahwungkuk Kecamatan Le- mahwungkuk Kota Cirebon)”, Jurnal Dinamika Hukum, Vol. 14, No. 1 January 2014. Purwokerto: Law Faculty, Jenderal Soedirman University, page 9. Small, Medium and Large Business Partners- hipps in Normative Level Dimension. The notion of partnership according to the Act No.44 of 1997 i.e. cooperation effort between small business and medium-sized busi- ness or with great effort by observing the prin- ciple of mutual need, mtually reinforcing and mutually benefecial. The law in the form of the 320 Jurnal Dinamika Hukum Vol. 15 No. 3, September 2015 320 Jurnal Dinamika Hukum Vol. 15 No. 3, September 2015 Competitive conditions will be more stri- ngent in line with the development of the wor- ld economy, and the partnership became one of the keys to deal with it. The era of ASEAN Economic Community (AEC) is formed of a sing- le market in Southeast Asia. MEA aims to in- creasing the competition and improving the quality of ASEAN citizens to be able to have competitiveness with people outside ASEAN, it could be imagine what will happen when the current MEA is already running. Increasing com- petition demands products competition both goods and services, a threat that is counter- productive if the state in this case the govern- ment does not provide guarantees and protect- ion as well as referrals or restriction in the con- stitution on the implementation of MEAs13. such as the providing of raw materials, produc- tion techniques, capital management, market- ing and human resources. In line with expecta- tions for small businesses to obtain the positive benefits of the ongoing globalization of world trade, there are at least two major dimensions that should be done by the government. The first dimension, related to the busi- ness of preparing the company's internal condi- tion of small businesses to be ready to facing the open market opportunities. The second di- mension, which must be prepared by the go- vernment lies in the macro framework of the creation of the climate of healthy competition between small businesses and large enterprises in the form of competition policies. It will sure- ly have an impact on the reduction of the faci- lity that is much enjoyed by big business. Com- petition policy itself does not always mean that small businesses need protection from the go- vernment all the time. Thing which needed is the government's drive to large companies, who enjoyed the facility, in order to drain the subsi- dy to small busines-ses while having a share (subcontracting) whch in turn removes the sub- sidy after small businesses powerful and effi- cient. 13 Oly Viana Agustine, “Konstitusi Ekonomi menghadapi Masyarakat Ekonomi ASEAN (MEA) Tahun 2015”, Jurnal Konstitusi, Volume 11 No. 4, December 2014, page 778. Small, Medium and Large Business Partners- hipps in Normative Level Dimension. Partnership as a form to face the competition because the context of Role of Law in Construction and Development of Small Scale Industries through Normative Perspective 321 Role of Law in Construction and Development of Small Scale Industries through Normative Perspective 321 to identifying the law itself.15 The meaning of investment which starts from a premise that in- vestment is a need, so it activity is recognized by a very complex business activity which have huge influence and impact both individual, gro- up, social, economy and various effects, obliga- tions, and responsibility, including the right that arise are always presented first.16 an economic competition has three benefits, the first is the economic, technological and tra- de aspects.14 Essentially, the partnership em- phasizing into to three basic principles which must be implemented that require mutual, mu- tually reinforcing and mutually beneficial to the parties who enter into a contract of part- ner-ship as outlined in the normative order that the Act No. 25 of 2007 and Constitution No. 44 of 1997. A partnerships model as stipulated in Go- vernment Regulation number 44 of 1997, is the pattern of plasma core, sub-contracting, gene- ral trading, agency and franchising. Further de- tails about the partnership model of which are a pattern of plasma core; sub-contract; general trade; agency and franchise. First, the pattern of Plasma core is a partnership of relationship between small businesses with a medium or lar- ge business, which includes medium or large businesses to act as the core and small busines- ses as a plasma. The company is carrying out development ranging from the provision of pro- duction inputs, technical guidance, and the marketing products. Partnership Pattern in Perspective of Act No. 25 of 2007 and No. 44 of 1997 about Partner- ship. Partnership Pattern in Perspective of Act No. 25 of 2007 and No. 44 of 1997 about Partner- ship. The presence of investment, especially for foreign investments give the effect of posi- tive and negative aspects. Positive aspects are those it helps to increase the rate of economic growth through the management of economic resources, providing technology transfer, mana- gement capabilities, improve the skill or ability to manage the equipment containing the char- ge of high technology and the development of science, which in turn can open up opportuni- ties pitch greater work again. Small, Medium and Large Business Partners- hipps in Normative Level Dimension. Partnership in terms of large enterprises and medium-sized enterprises or small busines- ses that take place within the framework of sub-contracts to produce goods or services. Bu- sinesses large or medium-sized businesses do- nated. First, the opportunity to spend a por- tion of the production and or components. Se- cond, the widest possible opportunity in obtain- ing raw materials it produces an ongoing basis with the amount and reasonable price. Third, guidance and technical capabilities of product- ion or management. Fourth, acquisition, con- trol and improvement of the necessary techno- logy. Fiveth, defrayal. The emergence of the globalization of world trade can be used as an opportunity for the small businesses in Indonesia, if the gover- nment and the small business willing to jointly prepare themselves through economic policy that taken. From the Government, the determi- nation of boundaries and criteria of the Small Business that are similar and comprehensive for all agencies in Indonesia coupled with the for- mulation of competition policy is an action that is very urgent to be realized, meanwhile, small businesses themselves need to continue to im- prove and overcome the internal problem of the company for the entire aspects that beco- me an obstacle for small businesses for exam- ple raw materials, production techniques, ma- nagement, financing, marketing and human re- sources. It is the role of government that can solve all the problems of small businesses with the issuance of Government Regulation 44 of 1997 concerning the Partnership. Small and medium enterprises in this re- gard has been proving resilient to the current economic crisis, other things that can be ex- plained that the debts incurred are debts cong- lomerate that jammed does not have the abili- ty to pay, while for small and medium enter- prises this issue does not occur so which should be given more pressure is increased professio- nalism of small and medium-sized enterprises, which in turn is expected to serve as the back- bone of the country's economy as well as in Ar- ticle 33 of the Republic of Indonesia Constitu- tion Year 1945 mandates. 14 Moh. Saleh, “Persaingan Usaha yang Sehat dalam Pers- pektif Perlindungan Konsumen”, Jurnal Media Hukum, Volume 14 No. 3 November 2007. Yogyakarta: Law Fa- culty, Muhammadiyah University, page 26. 15 Sigit Irianto, “Paradigma Filsafati dalam Mengkaji Ilmu Hukum”, Jurnal Hukum, Volume XVII 2007 Special Editi- on, Semarang: Law Faculty, Sultan Agung Islamic Unive- rsity, page 47. 16 Sri Redjeki Hartono, op.cit, page 6. Small, Medium and Large Business Partners- hipps in Normative Level Dimension. Negative aspect sought by all means to achieve a profit as much as possible through unnatural practices as smuggling taxes, controlling market with mono- poly and in line with the development of globa- lization foreign investment wishes to remain grounded in order of values liberalization, car- rying when later investment will be made so that the investment recipient country must ma- ke room for regulation that accommodates the interests of economy and trade liberalization. This pattern has been implemented bet- ween large and medium-sized enterprises, for example by the farmers. Farmers in the plasma position, while a large and medium-sized enter- prises as the core to provide guidance and de- velopment of agriculture, which in terms of: provision and preparation of agricultural land or plantations; provision of production faciliti- es; the provision of technical assistance, busi- ness management and production; acquisition and other necessary assistance which is need to the efficiency and productivity of business. Second, the Sub Contract pattern of part- nerships between small businesses with a me- dium or large business, which includes small businesses producing components required by medium or large businesses as part of its pro- duction. The patterns of large or medium-sized sub-contract enterprises are expected to provi- de assistance to small business/small industry include: the opportunity to spend a portion of In accordance with the above description of the legal aspects of a dominant position to maintain and regulate the balance of interests among the parties involved in the investment. Including other interests, which are the consu- mer, the environment and society at large. One major focus that emerged in studying in order to get clear about what the law means, then the efforts cannot be separated from the steps 322 Jurnal Dinamika Hukum Vol. 15 No. 3, September 2015 322 Jurnal Dinamika Hukum Vol. 15 No. 3, September 2015 322 Jurnal Dinamika Hukum Vol. 15 No. 3, September 2015 A partnerships made between big, medi- um and small industries implies cooperation between large businesses, medium to small in- dustrial or domestic industry based on 3 (three) main principles that need each other, mutually reinforcing and mutually beneficial for parties enter into a contract of partnership in the form of coaching and development on as mandated by the Act 25 of 2007 and Government Regula- tion 44 of 1997. References Agustine, Oly Viana. “Konstitusi Ekonomi meng- hadapi Masyarakat Ekonomi ASEAN (MEA) Year 2015”. Jurnal Konstitusi, Vol. 11 Number 4, December 2014; Artadi, Ibnu. “Dekonstruksi Pemahaman Penyel- esaian Sengketa Bisnis (Ekonomi dan Ke- uangan) Beraspek Pidana melalui Prose- dur Perdamaian: Menuju Proses Peradilan Pidana Rekonsiliatif”. Jurnal Hukum Res- ponsif, Vol. 1, No. 1 of 2011. Cirebon: Fakultas Hukum Univ. Sunan Gunung Jati; Atmasasmita, Romli. ”Tiga Paradigma Hukum dalam Pembangunan Nasional”. Jurnal Hukum Prioris, Vol. 3 No. 1 of 2012. Ja- karta: Law Faculty, Trisakti University,; Budiman, Haris & Suwari Akhmaddhian. “Imple- mentasi Reformasi Birokrasi Bidang Per- izinan Penanaman Modal di Kabupaten Kuningan”, Jurnal Ilmu Hukum Unifikasi, Vol. 1, No. 1 October 2013. Kuningan: Law Faculty, Kuningan University; Bureni, Yunus. “Moralitas Pembentukan Peratu- ran Daerah dalam Upaya Mencapai Ke- adilan Substantif”. Jurnal Legislasi Indo- nesia, Vol. 10 No. 2 June 2013. Jakarta: Direktorat Jenderal Peraturan Perundang undangan Kementerian Hukum dan HAM RI. Dayanto. “Rekonstruksi Paradigma Pembangun- an Negara Hukum Indonesia Berbasis Pancasila”. Jurnal Dinamika Hukum, Vol. 13 No. 3 September 2013. Purwokerto: Jenderal Soedirman University; Small, Medium and Large Business Partners- hipps in Normative Level Dimension. The patterns of partnership that can be done for the benefit of the partner parties is able to form models as stipulated in Government Regulation 44 of 1997, namely the plasma core pattern, sub-contracting, general trading, agency and franchise. both the production or components; opportuni- ties as possible to small businesses in obtaining raw materials are produced continuously with the amount and at a reasonable price; provide technical assistance or management of produc- tion; conducting the process of acquisition, ma- stery and improvement of the technology need- ed; business financing. Third, the Patterns of General Trade are a partnerships between small businesses with a medium or large business that includes a me- dium or large businesses to promote the pro- duct of small businesses. In the end, this part- nership model is expected to be mutually bene- ficial, mutually reinforce and support each ot- her. Fourth, the pattern of relations agency in- clude the partnership in small businesses which are given the exclusive right to promote their goods and services from medium or large busi- ness partners. A small business as an agent is a spearheading of the marketing of large or me- dium-sized businesses. This means that small businesses have a direct contact with the con- sumer, therefore the aspects of marketing ma- nagement are an important things that must be mastered by the agent. A large or medium-si- zed businesses are obliged to provide these skills by increasing the professionalism of agen- ts through training. The higher of sales techni- ques, more products that will be sold and the profits is not only enjoyed by the agents but al- so by a large or medium-sized businesses. The- re could also a downside, whereas the big busi- ness as the stronger burden or specific targets to be achieved. On the other hand, it is justifi- able to successful marketing and spur the spirit of agent, but on the other hand it is the exploi- tation of small businesses and this should not happen, because of the ethical aspects of busi- ness may be a serious problem. Fifth, the Fran- chise Pattern is a partnership in which the fran- chisor gives the right to use licenses, trade- marks and the company distribution channels to franchisees with management guidance assis- tance. Conclusion Role of Law in Construction and Development of Small Scale Industries through Normative Perspective 323 Role of Law in Construction and Development of Small Scale Industries through Normative Perspective 323 Saleh, Moh. “Persaingan Usaha yang Sehat da- lam Perspektif Perlindungan Konsu-men”. Jurnal Media Hukum, Vol. 14 No. 3 No- vember 2007. Yogyakarta: Law Faculty Muhammadiyah University; Hartono, Sri Redjeki. 2007. Orientasi Ke Arah Pengelolaan Investasi (Sebuah Landas- an Pemikiran Awal). Bandar Lampung: Lampung University; Irianto, Sigit. “Paradigma Filsafati dalam Meng- kaji Ilmu Hukum”. Jurnal Hukum, Vol. XVII special edition of 2007. Semarang: Law Faculty, Sultan Agung Islamic Uni- versity Semarang; Sutrisno, Endang. “Tracing the Performance of Law in Indonesia (A Perspective of Tho- mas Kuhn’s “Normal Science”. Journal of Law, Policy and Globalization, May, 2015. International Institute for Science, Techn-ology & Education Accelerating Global Knowledge Creation and Sharing; Kristiana, Yudi. “Ketika Hukum tidak Lagi Oten- tik”. Jurnal Hukum Supremasi, Vol. IV, No. 1 October 2010-March 2011. Jakarta: Pusat Studi Hukum Bisnis Law Faculty of Sahid University; -------. “Implementasi Pengelolaan Sumber Da- ya Pesisir Berbasis Pengelolaan Wilayah Pesisir Secara Terpadu untuk Kesejahte- raan Nelayan (Studi di Perdesaan Nelayan Cangkol Kelurahan Lemahwungkuk Keca- matan Lemahwungkuk Kota Cirebon)”. Jurnal Dinamika Hukum, Vol. 14, No.1 January 2014. Purwokerto: Jenderal Soe- dirman University Nasution, Bahder Johan. “Kajian Filosofis ten- tang Konsep Keadilan dari Pemi-kiran Kl- asik sampai Pemikiran Modern”. Jurnal Hukum Justisia, 89 May-August 2014 of XXIII edition; Prentha, Bahria. “Filsafat Hukum dan Nilai-Nilai Pancasila”. Jurnal Ilmiah Kebijakan Hu- kum, Vol. 5 No. 2 August 2011. Jakarta: Pusat Pengkajian dan Pengembangan Ke- bijakan Kementerian Hukum dan HAM RI; Widjojanto, Bambang. “Negara Hukum, Korupsi dan Hak Asasi Manusia: Suatu Kajian Aw- al”. Jurnal Hukum Prisoris, Vol. 3 No.1, 2012. Jakarta: Fakultas Hukum Trisakti; Rahardjo, Satjipto. 2010. Ilmu Hukum. Ban- dung: Alumni;
https://openalex.org/W3096640519	https://www.frontiersin.org/articles/10.3389/fnut.2021.672372/pdf	English	null	Influence of nutritional status on eating habits and food choice determinants among Brazilian women during the COVID-19 pandemic	medRxiv (Cold Spring Harbor Laboratory)	2,020	cc-by	5,622	BRIEF RESEARCH REPORT published: 13 May 2021 doi: 10.3389/fnut.2021.672372 Poor Eating Habits and Selected Determinants of Food Choice Were Associated With Ultraprocessed Food Consumption in Brazilian Women During the COVID-19 Pandemic Fabiana Infante Smaira 1,2†, Bruna Caruso Mazzolani 1,2†, Gabriel Perri Esteves 1,2, Heloisa C. Santo André 3, Milla Cordeiro Amarante 1,2, Daniela Fernandes Castanho 1,2, Karen Jennifer de Campos 1,2, Fabiana Braga Benatti 1,2,3, Ana Jéssica Pinto 1,2, Hamilton Roschel 1,2, Bruno Gualano 1,2,4 and Carolina Ferreira Nicoletti 1,2* Edited by: Specialty section: This article was submitted to Eating Behavior, a section of the journal Frontiers in Nutrition Specialty section: This article was submitted to Eating Behavior, a section of the journal Frontiers in Nutrition Specialty section: This article was submitted to Eating Behavior, a section of the journal Frontiers in Nutrition Received: 25 February 2021 Accepted: 31 March 2021 Published: 13 May 2021 Received: 25 February 2021 Accepted: 31 March 2021 Published: 13 May 2021 Edited by: 1 Applied Physiology & Nutrition Research Group, Rheumatology Division, School of Physical Education and Sport, Faculdade de Medicina FMUSP, Universidade de São Paulo, São Paulo, Brazil, 2 Laboratory of Assessment and Conditioning in Rhematology, Faculdade de Medicina FMUSP, Disciplina de Reumatologia, Universidade de São Paulo, São Paulo, Brazil, 3 School of Applied Sciences, State University of Campinas, Limeira, Brazil, 4 Food Research Center, University of São Paulo, São Paulo, Brazil Reviewed by: Erica Schulte, University of Pennsylvania, United States Ana Beatriz Harb, University of the Rio dos Sinos Valley, Brazil Background: The aim of this study was to investigate possible associations between food consumption and eating habits and food choice determinants in women during COVID-19 pandemic. Correspondence: Carolina Ferreira Nicoletti carolina.nicoletti.ferreira@usp.br Methods: This is a cross-sectional survey conducted in Brazil between June and September, 2020, during which time social distancing measures were in place. †These authors have contributed equally to this work and share ﬁrst authorship Results: Participants (n = 629) were aged 34.0 years and mostly within normal weight according to BMI (60.4%). “Snacking” and “liking” associated with increased energy (β = 164.27 and β = 110.24) and carbohydrate intake (β = 1.97 and β = 1.80), and with reduced protein intake (β = −1.54 and β = −1.18). In contrast, “dieting” and “weight control” associated with reduced energy (β = −162.57 and β = −111.49) and carbohydrate intake (β = −2.78 and β = −2.07), and with increased protein intake (β = 3.78 and β = 1.65). “Dieting” (β = 7.27), “need and hunger” (β = 3.34), and “health” (β = 4.94) associated with an increased consumption of unprocessed and minimally processed foods, whereas “replacing main meals with snacks” (β = −8.98), “snacking” (β = −6.92) and binge eating symptoms (β = −0.34) associated with reduced consumption of foods within this processing level. In contrast, “use of delivery services” (β = 3.39), “replacing main meals with snacks” (β = 5.49), “visual appeal” (β = 2.17), “social norms” (β = 2.19) and “affect regulation” (β = 2.01) associated with increased ultraprocessed food consumption. Overall, associations were more frequent and pronounced when analyzing food consumption by processing level rather than by macronutrient intake. Edited by: Kelly Costello Allison, University of Pennsylvania, United States Outcomes Macronutrient intake and food consumption by processing level were assessed using a 1-day food diary. Participants were instructed to fully report the quantity and type of foods and beverages that they consumed within the previous 24 h. Participants were provided with a food diary template. Analysis was then performed using the Dietbox software (online version). Food preparations (e.g., soups, puree, pies, sandwiches) were broken down into foods and ingredients, according to standardized recipes. Total energy intake (kcal) and macronutrient intake [grams and percentage of total energy intake (%TEI)] were calculated. Energy contribution (%TEI) and frequency of food consumption (times/day) were calculated for each processing level, according to the NOVA classiﬁcation, and within the following categories: (i) unprocessed and minimally processed foods, such as edible parts of plants or animals, or natural foods altered by simple processes, such as fruits, vegetables, meat, egg, milk, but without adding substances, such as salt, sugar or fat; (ii) culinary ingredients, such as salt, sugar, butter or vegetable oils; (iii) processed foods, such as canned vegetables and fruits, salted nuts and seeds, cured or smoked meats; cheeses The current analysis aimed to investigate possible associations between eating habits and food choice determinants with macronutrient intake and food consumption by processing level. We hypothesized that changes in eating habits and food choice determinants would have both positive and negative inﬂuences on selected aspects of food consumption (i.e., macronutrient intake and food processing level). Citation: Smaira FI, Mazzolani BC, Esteves GP, André HCS, Amarante MC, Castanho DF, Campos KJ, Benatti FB, Pinto AJ, Roschel H, Gualano B and Nicoletti CF (2021) Poor Eating Habits and Selected Determinants of Food Choice Were Associated With Ultraprocessed Food Consumption in Brazilian Women During the COVID-19 Pandemic. Front. Nutr. 8:672372. doi: 10.3389/fnut.2021.672372 Conclusion: Some eating habits and food choice determinants (“snacking,” “replacing meals with snacks,” “use of delivery services”) observed during the COVID-19 pandemic May 2021 \| Volume 8 \| Article 672372 Frontiers in Nutrition \| www.frontiersin.org Smaira et al. Food Consumption During the COVID-19 Pandemic were associated with an unhealthy diet (high energy and carbohydrate consumption increased ultraprocessed food consumption and reduced unprocessed/minimal processed foods consumption) in Brazilian women. Keywords: eating behavior, food processing level, SARS-CoV-2, quarantine, macronutrient intake INTRODUCTION This study was approved by the local ethical committee and was conducted in accordance with the Helsinki declaration. An approved Informed Consent Form was signed digitally by all participants before initiating the survey. INTRODUCTION order to determine macronutrient intake and food consumption by processing level. Data from the survey were reported on a previous manuscript (14), except for the 1-day food diary. The determinants of food consumption are factors that aﬀect the choice of food through individual thoughts and feelings, and refer to why and how people eat, which foods they eat, and with whom they eat, as well as the ways people obtain, store, use, and discard food (1–3), resulting in actions or behaviors toward food consumption (i.e., eating habits) (4). As decisions related to eating habits and food choices are performed daily and in similar contexts, they likely result from a habitual response (5, 6), which, despite being relatively stable during adulthood (7), are prone to variation due to changes in daily routine and environment (8). Participants were recruited through advertisements on social media platforms (Facebook R⃝, WhatsApp R⃝, Instagram R⃝, and Twitter R⃝), press release, television, and radio. Inclusion criteria were as follows: women aged ≥18 years, currently living in Brazil, with ability to read, and with internet access. All participants completed an online survey using the Google R⃝Forms platform (Google R⃝LLC, Menlo Park, CA, USA). Data related to their demographic, socioeconomic, and anthropometric characteristics, eating habits, food choice determinants, psychological symptoms, and food consumption were obtained. g y Social distancing measures necessary to contain the spread of SARS-CoV-2, changed lifestyle behaviors across the globe, including eating habits (9–12). In fact, unhealthier eating habits and food choices have been reported during the COVID- 19 pandemic, such as overeating, snacking, replacing main meals with snacks, increased use of delivery services, and high ultraprocessed food intake (12–16). In a recent publication (17), we reported that Brazilian women increased eating habits such as cooking, use of delivery services, eating at the table, and snacking, during the COVID-19 pandemic, whereas they decreased participation in grocery shopping and dieting. Nonetheless, these ﬁndings are not unanimous, and some positive changes have also been observed, such as a rise in frequency of cooking, mirrored by an increase in consumption of home-cooked meals (13, 18). Importantly, it is currently unclear how these mixed changes in eating habits and food choices associate with food consumption during the COVID-19 pandemic. Exploring these aspects is key to understanding which changes may be potentially beneﬁcial or detrimental to overall health. Evaluation Tool The online survey included questions about age, ethnicity, marital status, educational level, smoking, and chronic medical conditions, as well as anthropometric data [i.e., self-reported weight and height, which was then used to calculate body mass index (BMI)], eating habits, food choice determinants, eating attitudes, psychological symptoms, and food consumption. Frontiers in Nutrition \| www.frontiersin.org Study Design and Participants This is an exploratory analysis comprising a subset of participants from a larger cross-sectional survey (17), conducted between June and September, 2020, a period in which a set of social distancing measures intended to contain the spread of COVID- 19 were in place in Brazil. Considering the number of researchers and working capacity of our team, a subsample (n = 629) of participants from our original study (n = 1,183) (14) was randomly selected with the aim of analyzing 1-day food diaries in May 2021 \| Volume 8 \| Article 672372 Frontiers in Nutrition \| www.frontiersin.org 2 Food Consumption During the COVID-19 Pandemic Smaira et al. TABLE 1 \| General characteristics of participants. Total (n = 1,183) Sub-sample (n = 629) p Age (years) 34.6 (33.9, 35.3) 34.0 (33.0, 35.0) 0.356 BMI (kg/m2) 24.8 (24.5, 25.1) 24.8 (24.4, 25.2) 0.963 Self-related ethnicity White 921 (77.8%) 497 (79.3%) 0.560 Yellow 41 (3.5%) 26 (4.2%) Brown 159 (13.4%) 76 (12.1%) Black 51 (4.3%) 25 (4.0%) Indigenous 6 (0.5%) 3 (0.5%) Marital status Married 436 (36.9%) 223 (35.5%) 0.629 Single 657 (55.5%) 357 (56.9%) Divorced 78 (6.6%) 43 (6.9%) Widow 12 (1.0%) 5 (0.8%) Educational level Elementary school 12 (0.1%) 6 (1.0%) 0.225 High school degree 314 (26.5%) 181 (28.8%) University degree 857 (72.4%) 438 (69.8%) Presence of chronic diseases Hypertension 50 (4.2%) 22 (3.5%) 0.454 Diabetes mellitus 17 (1.4%) 10 (1.6%) 0.795 Dyslipidemia 77 (6.5%) 40 (6.4%) 0.909 Thyroid disorders 109 (9.2%) 60 (9.6%) 0.375 Cardiovascular diseases 21 (1.8%) 15 (2.4%) 0.375 Smoking habit No 1,120 (94.8%) 600 (95.5%) 0.422 Yes 63 (5.3%) 28 (4.5%) Data presented as mean and 95% conﬁdence interval (95% CI) or absolute and relative frequency (n[%]).BMI, body mass index. TABLE 1 \| General characteristics of participants. and unpackaged freshly made bread; (iv) ultraprocessed foods, such as carbonated drinks, ice-cream, cookies, pre-prepared pasta, pie or pizza, hot dogs, burgers, or instant soup noodles (19). Eating habits (i.e., “participation in grocery shopping,” “cooking,” “use of delivery services,” “replacing main meals with snacks,” “eating at the table,” “eating in front of television/tablet/cellphone,” “snacking,” and “dieting”) were classiﬁed as binary outcomes (i.e., “yes,” if participant reported certain eating habit, or “no,” if participant reported the absence of certain eating habit). Study Design and Participants Food choice determinants (i.e., “liking,” “habits,” “need and hunger,” “health,” “convenience,” “pleasure,” “traditional eating,” “natural concerns,” “sociability,” “price,” “visual appeal,” “weight control,” “social norms,” “social image,” and “aﬀect regulation”) were assessed by The Eating Motivation Survey (TEMS) (20), which comprises 45 questions preceded by “I eat what I eat,...”. The answers are given in a seven-point scale ranging from 1 (“never”) to 5 (“always”), with higher scores representing a higher impact of a given food choice determinant. Eating attitudes were assessed by the Binge Eating Scale (BES), which evaluates symptoms of binge eating episodes (21, 22), and by the Disordered Eating Attitude Scale (DEAS), which evaluates eating attitudes (23, 24). Higher scores represent more symptoms of binge eating episodes (BES score range: 0–46), and more dysfunctional eating attitudes (DEAS score range: 17–75). Psychological symptoms (i.e., depression, anxiety and stress symptoms, and loneliness) were assessed by Depression Anxiety Stress Scale- 21 (DAS-21) (25, 26) and by the UCLA Loneliness Scale (UCLA-LS) (27, 28), with higher scores representing more symptoms (DAS-21 score range for: 0–28; UCLA-LS score range for: 1–8). Statistical Analysis Descriptive data are presented as mean ±95% conﬁdence interval (95% CI) for continuous variables and absolute and relative frequency (n [%]) for categorical variables. The association (linear regression) between eating habits/food choice determinants with macronutrient intake and with processing level were also tested, assuming eating habits/food choice determinants (e.g., “cooking,” “snacking,” “liking”) as independent variables, and macronutrient intake and processing level (e.g., energy, carbohydrate and unprocessed and minimally processed food consumption) as dependent variables. Additionally, we also tested the association between macronutrient intake and food consumption by processing level and was tested using linear regression models, assuming processing level (e.g., unprocessed and minimally processed, processed, and ultraprocessed food consumption) as independent variables, and macronutrients (e.g., carbohydrate, protein and fat intake) as dependent variables. All regression models were adjusted for age, BMI, educational level, ethnicity, marital status, and number of comorbidities. Data are presented as β (95% CI). All analyses were performed using the statistical package SAS (version 9.4). The level of signiﬁcance was set at p ≤0.05. RESULTS Out of 1,183 women who participated in the original study (17), 629 were randomly selected and were included in this analysis. Among them, 129 participants did not properly report portion sizes, and their data regarding macronutrient intake and energy contribution were not included. Participant’s age ranged from 18 to 72 years (34.0 [95% CI: 33.0, 35.0] years) and most participants were within normal weight according to BMI (60.4%), white (79.3%), single (56.9%), and had a high educational level (69.8%). Subsample characteristics were not diﬀerent from the total sample (Table 1). Linear regression models showed “snacking” and “liking” associated with increased energy (β = 164.27 [61.09, 267.46], p = 0.002 and β = 110.24 [37.67, 182.81], p = 0.003) and carbohydrate intake (β = 1.97 [0.15, 3.78], p = 0.033 and β = 1.80 [0.53, 3.07], p = 0.005), and with reduced protein intake (β = −1.54 [−2.88, −0.21], p = 0.023 and β = −1.18 [−2.12, −0.24], p = 0.013). In contrast, “dieting” and “weight control” were associated with reduced energy (β = −162.57 [−289.49, −35.66], May 2021 \| Volume 8 \| Article 672372 Frontiers in Nutrition \| www.frontiersin.org 3 Food Consumption During the COVID-19 Pandemic Smaira et al. FIGURE 1 \| Associations between food consumption (dependent variables) and eating habits and determinants (independent variables). Data presented as standardized β (95% CI); p < 0.05. UNMP, unprocessed and minimally processed foods; PR, processed foods; UPR, ultraprocessed foods. FIGURE 1 \| Associations between food consumption (dependent variables) and eating habits and determinants (independent variables). Data presented as standardized β (95% CI); *p < 0.05. UNMP, unprocessed and minimally processed foods; PR, processed foods; UPR, ultraprocessed foods. “Dieting” (β = 7.27 [3.30, 11.24], p < 0.005), “need and hunger” (β = 3.34 [1.20, 5.49], p = 0.002) and “health” (β = 4.94 [3.23, 6.65], p < 0.0001) associated with an increased consumption of unprocessed and minimally processed foods, whereas “replacing main meals with snacks” (β = −8.98 [−12.32, p = 0.012 and β = −111.49 [−170.46, −52.52], p < 0.001) and carbohydrate intake (β = −2.78 [−5.00, −0.56], p = 0.014 and β = −2.07 [−3.10, −1.04], p < 0.0001), and with increased protein intake (β = 3.78 [2.17, 5.39], p < 0.0001 and β = 1.65 [0.89, 2.40], p < 0.0001) (Figure 2). RESULTS May 2021 \| Volume 8 \| Article 672372 Frontiers in Nutrition \| www.frontiersin.org Frontiers in Nutrition \| www.frontiersin.org 4 Food Consumption During the COVID-19 Pandemic Smaira et al. FIGURE 2 \| Associations between macronutrient intake (dependent variables) and food consumption by processing level (independent variables). Data presented as standardized β (95% CI); •p < 0.05; ◦p > 0.05. UNMP, unprocessed and minimally processed foods; PR, processed foods; UPR, ultraprocessed foods. −5.63], p < 0.0001), “snacking” (β = −6.92 [−10.14, −3.70], p < 0.0001) and binge eating symptoms (β = −0.34 [−0.57, −0.12], p = 0.002) associated with reduced consumption of foods within this processing level. In contrast, “use of delivery services” (β = 3.39 [0.37, 6.42], p = 0.028), “replacing main meals with snacks” (β = 5.49 [2.23, 8.76], p = 0.001), “visual appeal” (β = 2.17 [0.25, 4.10], p = 0.027), “social norms” (β = 2.19 [0.19, 4.19], p = 0.031) and “aﬀect regulation” (β = 2.01 [0.35, 3.68], p = 0.017) associated with increased ultraprocessed food consumption (Figure 1). Overall, visual inspection of Figure 1 indicates that the number and magnitude of associations were greater when analyzing consumption by food processing levels rather than by macronutrient intake. Average carbohydrate, fat, and protein intake were 45.4 [44.5, 46.2], 36.7 [36.0, 37.4], and 17.9 [17.3, 18.6] %TEI, respectively. The most prevalent food group consumed was unprocessed/minimally processed food (43.9 [42.3, 45.4] %TEI), followed by processed food (22.8 [21.2, 24.6] %TEI), ultraprocessed food (20.8 [19.3, 22.3] %TEI), and culinary ingredients (13.1 [12.5, 13.8] %TEI). Interestingly, the consumption of unprocessed or minimally processed food was associated with decreased energy (β = −8.50 [−11.21, −5.78], p < 0.001), carbohydrate (β = −0.13 [−0.17, −0.08], p < 0.001) and fat (β = −0.04 [−0.08, −0.003], p = 0.036) intake, and with increased protein intake (β = 0.16 [0.13, 0.20], p < 0.001). In contrast, the consumption of ultraprocessed food was associated with increased energy (β = 6.71 [3.83, 9.59], p < 0.001) and carbohydrate intake (β = 0.08 [0.03, 0.13], p = 0.003) and with reduced protein intake (β = −0.08 [−0.12, −0.05], p < 0.001). Processed food consumption was only associated with increased carbohydrate intake (β = 0.05 [0.01, 0.10], p = 0.024) (Figure 2). FIGURE 2 \| Associations between macronutrient intake (dependent variables) and food consumption by processing level (independent variables). RESULTS Data presented as standardized β (95% CI); •p < 0.05; ◦p > 0.05. UNMP, unprocessed and minimally processed foods; PR, processed foods; UPR, ultraprocessed foods. Frontiers in Nutrition \| www.frontiersin.org DISCUSSION The main ﬁndings of this study were that: (i) associations were stronger and more numerous when analyzing food consumption by processing level as opposed to macronutrient intake; and, more importantly, (ii) consumption of unprocessed and minimally processed foods were associated with healthier dietary patterns (e.g., food choice determined by “need and hunger” and “health”), whereas consumption of ultraprocessed foods associated with poorer dietary patterns (e.g., “snacking,” “use of delivery services” and “replacing main meals with snacks”). Evidence indicates that eating habits have changed during the COVID-19 pandemic in several countries (9–14, 17, 42, 43). Increases in snacking have been consistently reported (12, 13, 17, 43), which could be attributed to an attempt to cope with the emotional stress of social isolation (44, 45). Herein, we showed that “snacking” and “replacing meals with snacks” were associated with nutritionally inadequate diets, such as those characterized by higher consumption of carbohydrate and ultraprocessed foods. In contrast, several other eating habits and food choice determinants associated with indicators of healthier dietary patterns. For instance, “dieting” and choosing foods based on “health” and “weight control” associated with a more nutritionally balanced diet. These ﬁndings are in line with the literature, which shows that people concerned with health- and weight-related matters are more prone to report a higher consumption of fruits and vegetables, and a lower consumption of red meat, snacks and sweets (18, 46, 47). In our sample, consumption of ultraprocessed foods was relatively low (∼21 %TEI) when compared to other populations (range: 25.8–59.7 %TEI) (29–33). Nonetheless, a higher consumption of foods in this processing level was still associated with a nutritionally inadequate diet (i.e., higher energy and carbohydrate intake and lower protein intake), which is in line with other studies that also reported an association between ultraprocessed food consumption and less desirable dietary patterns (34–36). Particular attention should be given to high consumers of ultraprocessed foods during the COVID-19 pandemic, as this type of food is associated with increased adiposity (37–40), even in a short period of time (41). May 2021 \| Volume 8 \| Article 672372 5 Food Consumption During the COVID-19 Pandemic Smaira et al. foods, which, in turn, could be detrimental to overall health (34–36). Interestingly, associations with eating habits and food choice determinants were more frequent and pronounced when analyzing food consumption by processing level rather than by macronutrient content. DISCUSSION These ﬁndings could, at least in part, be attributed to the fact that focusing on macronutrients may result in a narrower view of food consumption as it may not fully capture the heterogeneity of eating behaviors; ultimately, people usually select what they eat based on food availability and preference rather than nutrients per se (48–50). ETHICS STATEMENT p Our study is strengthened by the fact that data were collected when the most restrictive stay-at-home orders were in place, thus allowing for a more representative view of this unprecedented social context on diet. However, this study is not without limitations. Firstly, food consumption data were only analyzed for a subset of participants; however, it is important to highlight that the participants included in this study were considered representative of the total sample, as they were randomly selected, and their demographic characteristics did not signiﬁcantly diﬀer from the broader data set. Additionally, we used 1-day food diaries to assess food consumption, which may have introduced reporting bias in our study as some participants may have reported food consumption for an unusual day. Finally, our sample is predominantly composed of women with university degrees, which may limit the generalization of the present ﬁndings to other populations. The studies involving human participants were reviewed and approved by Ethics Committee for the Analysis of Research Projects of Clinical Hospital of FMUSP, Presentation Certiﬁcate for Ethical Appreciation number 33561720.2.0000.0068. The patients/participants provided their written informed consent to participate in this study. FUNDING FS, BM, GE, AP, and BG were supported by São Paulo Research Foundation—FAPESP (Grants #2019/14819-8, #2019/14820-6, #2020/07860-9, #2015/26937-4, and #2017/13552-2). CN, HR, and BG were supported by National Council for Scientiﬁc and Technological Development—CNPq (Grant #402123/2020- 4, #301571/2017-1, and #301914/2017-6). DATA AVAILABILITY STATEMENT The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. ACKNOWLEDGMENTS We are thankful to Dr. Eimear Dolan for proofreading this manuscript. AUTHOR CONTRIBUTIONS FS and BM: conceptualization, investigation, writing-original draft, visualization, project administration, formal analysis, and funding acquisition. GE: investigation, formal analysis, writing-review and editing, and funding acquisition. HA, MA, DC, and KC: investigation, formal analysis, and writing-review and editing. FB and HR: writing-review and editing. AP: formal analysis, visualization, writing-original draft, and funding acquisition. BG: conceptualization, writing-original draft, supervision, and funding acquisition. CN: conceptualization, investigation, writing-review and editing, funding acquisition, project administration, and supervision. All authors contributed to the article and approved the submitted version. In conclusion, some eating habits and food choice determinants (e.g., “snacking,” “replacing meals with snacks” or “use of delivery services”) observed during the COVID-19 pandemic (17) associated with unhealthy dietary patterns (e.g., high energy and carbohydrate consumption, increased ultraprocessed food consumption and reduced unprocessed/minimally processed foods consumption) in Brazilian women. Interestingly, these associations were more frequent and pronounced when analyzing food consumption by processing level rather than by macronutrient content. The comprehensive understanding of the complex interplay between eating habits, food choice determinants, and food consumption may guide health professionals to identify at-risk individuals for an unhealthier diet pattern during the COVID-19 pandemic. From a practical perspective, clinical and public health interventions focused on mitigating poor eating habits (e.g., “snacking,” “replacing main meals with snacks”) should be implemented since these behaviors are associated with an increased intake of ultraprocessed 6. Sobal J, Bisogni CA. Constructing food choice decisions. Ann Behav Med. (2009) 38:s37–46. doi: 10.1007/s12160-009-9124-5 REFERENCES (2016) 65:68– 75. doi: 10.1590/0047-2085000000105 29. Baraldi LG, Steele EM, Canella DS, Monteiro CA. Consumption of ultra- processed foods and associated sociodemographic factors in the USA between 2007 and 2012: evidence from a nationally representative cross-sectional study. BMJ Open. (2018) 8:e020574. doi: 10.1136/bmjopen-2017-020574 10. 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https://openalex.org/W2802514849	https://link.springer.com/content/pdf/10.1007/s12393-018-9178-x.pdf	English	null	Effect of Vacuum Frying on Quality Attributes of Fruits	Food engineering reviews	2,018	cc-by	8,999	Abstract Vacuum frying of fruits enables frying at lower temperatures compared to atmospheric frying, thereby improving quality attributes of the fried product, such as oil content, texture, retention of nutrients, and color. Producing high-quality vacuum-fried fruit is a challenge, especially because of the high initial water content of fruits that requires long frying times. Factors influencing vacuum-fried fruit quality attributes are the type of equipment, pre-treatments, processing conditions, fruit type, and fruit matrix. Pre-treatments such as hot air, osmotic drying, blanching, freezing, impregnation, anti-browning agents, and hydrocolloid application strongly influence the final quality attributes of the products. The vacuum-frying processing parameters, namely frying time, temperature, and vacuum pressure, have to be adjusted to the fruit characteristics. Tropical fruits have different matrix properties, including physical and chemical, which changed during ripening and influenced vacuum-fried tropical fruit quality. This paper reviews the state of the art of vacuum frying of fruit with a specific focus on the effect of fruit type and matrix on the quality attributes of the fried product. Keywords Vacuum frying . Fruit . Quality attributes . Matrix . Phytochemicals Effect of Vacuum Frying on Quality Attributes of Fruits Received: 6 December 2017 /Accepted: 12 April 2018 /Published online: 25 April 2018 # The Author(s) 2018 * Ruud Verkerk ruud.verkerk@wur.nl Food Engineering Reviews (2018) 10:154–164 https://doi.org/10.1007/s12393-018-9178-x Food Engineering Reviews (2018) 10:154–164 https://doi.org/10.1007/s12393-018-9178-x REVIEW ARTICLE * Ruud Verkerk ruud.verkerk@wur.nl 1 Food Quality and Design, Wageningen University & Research, Bornse Weilanden 9, 6708 WG Wageningen, Netherlands 2 Department of Nutrition Science, Faculty of Medicine, Diponegoro University, Semarang, Indonesia 3 Center of Nutrition Research, Diponegoro University, Jalan Prof. Soedarto, SH Tembalang, Semarang, Jawa Tengah 1269, Indonesia Introduction Vacuum frying is a frying process below atmospheric pressure (~ 100 kPa). At reduced pressure, the boiling point of oil and water is lower compared to atmospheric pressure [31]. Due to a lower frying temperature, vacu- um frying better preserves the nutritional value, aroma, and color of the fried product compared to atmospheric frying [2]. Fried products are appreciated by all age groups and play an important role in consumer’s diet because of their unique fla- vor and texture. However, it is difficult to combine fried foods with the contemporary consumer trends toward healthier and low-fat products. There is an increased demand for healthy snack products with good taste, texture, and appearance [38]. This demand offers the opportunity to design novel fried products that have higher health properties such as fruit-based products. Increasing fruit consumption is promoted in all parts of the world to increase public health. Fruit implicitly has a strong health awareness based on the content of (micro) nutri- ents, fibers, and numerous bioactive phytochemicals [19, 51]. Some anecdotic findings from existing studies highlighted several advantages vacuum frying might have over atmo- spheric frying: & Oil uptake in vacuum-fried apple chips is lower compared frying at atmospheric pressure [44]; & Color of vacuum-fried mango was lighter compared to atmospheric frying [17]; & Carotenoid retention was higher in vacuum-fried mango compared to atmospheric frying [50]; * Ruud Verkerk ruud.verkerk@wur.nl & Vacuum-fried mango was more uniform and crispier compared to soggy, burnt, and oily for atmospheric fried mango [50]. 1 Food Quality and Design, Wageningen University & Research, Bornse Weilanden 9, 6708 WG Wageningen, Netherlands The multiple factors influencing the quality attributes of vacuum-fried fruit can be distinguished in vacuum-frying equip- ment (type and specifications), properties of the raw fruit (fruit matrix), pre- and post-treatments, and processing conditions. 155 Food Eng Rev (2018) 10:154–164 Time, temperature, and vacuum pressure influenced color, tex- ture, nutrients, and oil content of fried fruits [2, 14, 73]. the end of vacuum frying, the vacuum breaking period pro- duces a higher outside pressure then the pore pressure. The oil content of vacuum-fried apples was lower com- pared to atmospheric fried one. Apple absorbed 1.2–2.0 times more oil by atmospheric frying compared to vacuum frying [29, 44]. This difference was explained by the lower temper- atures during vacuum frying due to the lower vapor pressure of water. Introduction This low temperature will reduce temperature- induced tissue matrix degradation that increases the oil ab- sorption. Dueik et al. [30] found that atmospheric fried apple had a larger portion of small pores and absorbed more oil by capillary suction compared to vacuum-fried apple. Larger pore formation was related to the higher specific volume of water vapor at lower pressure. These studies provided a con- vincing explanation about the mechanisms behind the reduced oil absorption of vacuum-fried products. Another relevant aspect is the fruit matrix such as the fruit type and ripening stage that are affecting the vacuum-fried product quality attributes [20, 30, 84]. Pre-treatments such as blanching, drying, freezing, antioxidant, and coating applica- tions have been used to preserve color, improve texture, and reduce oil absorption [9, 22, 24]. The use of post-frying steps such as centrifugation has a major effect on the oil content of fried product [47]. Some recent papers dealt with different aspects of vacuum- frying technology. The strategies to reduce oil absorption of vacuum-fried products have been studied intensively by Moreira [46], including optimizing temperature, pressure, pre-treatment, pressurization speed, and de-oiling time. The recent review by Diamante et al. [26] discussed the product and process optimization, oil uptake, oil quality, as well as packaging and storage of vacuum-fried fruits without men- tioning matrix factors. Dueik and Bouchon [28] and Ayustaningwarno and Ananingsih [11] compared the quality changes comparing atmospheric and vacuum frying as well as the oil quality and packaging of fried products. Dueik and Bouchon [28] put emphasis on the microstructure, methods to reduce oil uptake, oil quality, bioactive compound degrada- tion, and toxic compound generation. Andres-Bello et al. [8] reviewed the vacuum-frying processing for producing high- quality fried products, focusing on equipment types, pre-treat- ments, and vacuum-frying conditions. Shyu and Hwang [61] showed that oil absorption was high- ly correlated with moisture loss in vacuum-fried apple slices. At the beginning of the frying procedure, the outer surface of the product is dried, the moisture inside the product is con- verted into steam, and a pressure gradient is created. By prolonging the frying, the dried surface becomes more hydro- phobic which facilitates the absorption of oil. This can explain the observed oil content that was increased from 33.64% in first 5 min of vacuum frying to 39.38% after 30 min of vacu- um frying. Introduction This oil absorption mechanism is different com- pared with atmospheric frying in which most of the oil is absorbed after frying during the cooling period [12]. Based on this existing background information, this review will consider the effects of vacuum frying on changes in quality attributes of tropical fruits with a focus on the role of the fruit matrix, since this is a very relevant but underexposed factor. The situation found in apple is different as found in plantain and mango: as in plantain [2] and mango [17], vacuum frying resulted in a higher oil content compared to atmospheric fry- ing. This difference could be attributed by matrix differences of apple with plantain and mango. Wexler et al. [80] explained that at the end of vacuum frying of papaya, capillary absorp- tion of surface oil was favored to be absorbed inside the prod- uct when the vacuum was broken to restore the system into atmospheric pressure. Additionally, vacuum-fried plantain had less gelatinized starch due to the lower temperature, there- by having more pores and absorbed more oil compared to atmospheric frying [2]. Vacuum Frying Versus Atmospheric Frying The main difference between vacuum frying and atmospheric frying is the lower boiling point of water at lower pressures that enables to fry at lower temperatures. For that reason, vacuum frying has many advantages over atmospheric frying in relation to product quality attributes. Several comparative studies between vacuum and atmospheric frying were done on apple, plantain, banana, and mango. In general, a higher nutrient retention is expected with a lower temperature of vacuum frying. Ascorbic acid content of apple was found 1.7–1.9 times higher after vacuum frying compared to atmospheric frying [29]. Additionally, carotenoid retention in vacuum-fried mango was two times higher com- pared to atmospheric frying. High retention of carotenoid was attributed by the absence of oxygen, which induce oxidation in atmospheric frying [50]. In addition, a lower temperature of vacuum frying compared to atmospheric frying will have an effect on the nutrition degradation. A less pronounced effect was observed by Da Silva and Moreira [17] who found that vacuum-fried mango had 20–50% higher carotenoids com- pared to atmospheric fried mango. Vacuum-Frying Process The vacuum-frying process consists of several steps as sum- marized in Fig. 1. These steps include fruit preparation, peel- ing and slicing, pre-treatment, vacuum-frying process, and removal of excess oil. Vacuum frying usually uses raw mate- rials as fresh fruits. However, fruit paste also can be used by preparing a dough made up with fruit pulp and starch or flour [73]. Utilization of fresh fruit has some advantages as well as disadvantages. The product could be recognized by the con- sumer as the original fruit, but fresh fruits usually have a variety of shapes and irregularities resulting in uneven heat distribution during frying and a subsequent inhomogeneity in color and texture [36]. On the other side, using fruit paste a homogenous product in size and shape can be obtained, but the characteristic of the original fruit is lost [73]. Fig. 1 Flow chart of vacuum-frying process In this section, common pre-treatments used for vacuum- frying processing will be mentioned briefly and discussed further in separated sections. The pre-treatments that are re- ported in literature are blanching, pre-drying, impregnation, and freezing [20, 22, 23, 35, 43, 44, 50, 61, 64]. Blanching is used to minimize enzymatic browning [44, 61] and also to pre-gelatinize starch. Pre-drying is used to reduce the initial water content before frying and thus reduce frying time [44]. Osmotic dehydration is used to introduce salt or sugar to re- duce initial water content [17, 22, 23, 50, 61, 64]. Application of anti-browning agents prevents browning reactions [44]. Freezing can be used to create a porous and spongy matrix in vacuum-fried fruit [61]. In this section, common pre-treatments used for vacuum- frying processing will be mentioned briefly and discussed further in separated sections. The pre-treatments that are re- ported in literature are blanching, pre-drying, impregnation, and freezing [20, 22, 23, 35, 43, 44, 50, 61, 64]. Blanching is used to minimize enzymatic browning [44, 61] and also to pre-gelatinize starch. Pre-drying is used to reduce the initial water content before frying and thus reduce frying time [44]. Osmotic dehydration is used to introduce salt or sugar to re- duce initial water content [17, 22, 23, 50, 61, 64]. Application of anti-browning agents prevents browning reactions [44]. Freezing can be used to create a porous and spongy matrix in vacuum-fried fruit [61]. Slicing of the fruit has a large influence on the final product characteristics. Color, Texture, and Sensory Attributes Natural fruit color preservation is an important product quality attribute for vacuum-fried fruit [46]. This color preservation can be attributed to the low pressure and temperature of the vacuum-frying process. A low pressure means a low oxygen level, thereby reducing oxidation processes, which could lead to darkening of the color. In addition, low temperature slows down non-oxidative browning reaction. Vacuum frying better preserved the lightness and redness of apple chips compared to atmospheric frying [29, 44]. Similar results for lightness and redness were found in plantain [2] and mango [17, 50]. Vacuum frying of plantain produced a crispier product compared with plantain fried in atmospheric pressure, in- dicated by a lower maximum breaking force value [2]. Less effect was observed in mango which had no maximum breaking force value difference between vacuum and atmo- spheric fried mango [17]. Based on sensory analysis, vacuum-fried plantain chips have significantly higher scores on sensory attributes as taste, aroma, overall appearance (color), and texture (crispiness/ crunchiness) [2]. Similar observations were done by [17], showing that vacuum-fried mango has significantly higher sensory score in color, odor, texture, flavor, and a higher over- all quality than perceived for atmospheric fried mango. Oil and Nutrient Content The mechanism of oil uptake in atmospheric frying and vac- uum frying is different. Oil uptake occurs mainly after frying: by the lower pressure in the pores, the oil present on the sur- face of the products is sucked into the pores. During atmo- spheric frying, this lower pressure in the pores is created by the evaporative cooling after frying [12]. On the other hand, at Food Eng Rev (2018) 10:154–164 156 Vacuum Frying Peeling and slicing Fresh Fruits Blanching Immersing Freezing Pre-drying Vacuum Fried Fruits Pulping and dough making Centrifugation Fig. 1 Flow chart of vacuum-frying process Blanching Blanching was used to minimize enzymatic browning in vacuum-fried apple chips [44, 61]. Enzymatic browning in fruits is the result of oxidation reactions of polyphenols with catalytic action of polyphenol oxidase (PPO) enzyme [58]. During blanching, PPO in mango can be inactivated by a 5- min treatment at 94 °C. However, blanching for more than 5 min resulted in color loss [49], even before frying. Blanching of jackfruit produced a negative effect on oil con- tent and texture; a higher porosity matrix was formed during the vacuum frying causing a higher oil absorption compared to non-blanched jackfruit [20]; however, the mechanism be- hind the porosity formation is not clear. Nevertheless, Hasimah et al. [35] describe that blanched vacuum-fried pine- apple at 100 °C for 3 min has shrunken cell due to air lost by blanching, and consequently produce a hard product. Blanching was used to minimize enzymatic browning in vacuum-fried apple chips [44, 61]. Enzymatic browning in fruits is the result of oxidation reactions of polyphenols with catalytic action of polyphenol oxidase (PPO) enzyme [58]. During blanching, PPO in mango can be inactivated by a 5- min treatment at 94 °C. However, blanching for more than 5 min resulted in color loss [49], even before frying. Blanching of jackfruit produced a negative effect on oil con- tent and texture; a higher porosity matrix was formed during the vacuum frying causing a higher oil absorption compared to non-blanched jackfruit [20]; however, the mechanism be- hind the porosity formation is not clear. Nevertheless, Hasimah et al. [35] describe that blanched vacuum-fried pine- apple at 100 °C for 3 min has shrunken cell due to air lost by blanching, and consequently produce a hard product. Vacuum-Frying Equipment Vacuum frying is carried out in a closed system below atmo- spheric pressures. Schematic of a batch vacuum fryer can be observed in Fig. 2. Conceptually, different devices in batch and semi-continuous mode were used in the experimental studies. The batch vacuum frying is suitable for small produc- tion sizes [63], as well as for a larger capacity. Vacuum fryers with a low capacity (2–10 L) are also often used for research [14, 30, 50], while Diamante et al. [21] used a large capacity fryer (460 L) for their research. Producing a high-quality vacuum-fried fruit which has desirable product quality attributes is a challenge in vacuum-fried fruit production, especially because of the high initial water content of fruits that requires long frying times. High oil absorptions, burnt product, and low crisp- ness are the possible product quality attributes that are consequences of this high water content. Pre-treatments such as blanching, hot air pre-drying, immersion drying, freezing, anti-browning agent, and hydrocolloid applica- tion can limit these problems (Table 1). On the other hand, vacuum frying is also possible using a semi-continuous method, which is a batchwise process with aspects of continuous processing [63]. This process was adopted by Perez-Tinoco et al. [54], who used a conveyor belt frying system inside a vacuum chamber. A small vacuum fryer usually not includes a centrifuge inside the vacuum chamber like larger vacuum fryer do. A centrifugation before breaking the vacuum is desired to remove the surface oil that will oth- erwise get sucked into the pores. A centrifugation after break- ing the vacuum could lead to higher oil content then when the centrifugation is done before. A high-capacity industrial fryer also usually includes several heat exchangers to maintain a constant and equally distributed oil temperature and an oil filter to maintain oil quality. Vacuum-Frying Process Fruit could be sliced into thin pieces from 1.5- to 7.5-mm thickness that need a relative short frying time. Fruit with thicker slices needs longer frying times to lower the water content, to get the desired crispiness and shelf life, leading to an elevated degradation of nutrients and bioactive compounds [17, 23]. After the pre-treatment, fruits are ready to be fried. In a small-scale fryer, the process will start by placing the fruits inside a basket and placed in the vacuum chamber after which the vacuum pump is started. After the oil has reached the desired temperature and the chamber has the desired pressure, the basket is submerged in the oil to start the frying process. At the end of the frying time, the basket is lifted from the oil and shaken or spun to drain the surface oil. The pressure is Pre-treatments can be used to further improve quality attri- butes of the fried product, such as oil content, appearance, texture, taste, and retention of nutrients and phytochemicals. 157 Food Eng Rev (2018) 10:154–164 of vacuum-frying parameters includes studies which applied pre-treatment in their method. The main parameters for the frying process are temperature, time, and pressure. However, the pre-treatments play a crucial role in the improvement of quality attributes as well. Therefore, the discussion of effects of pre-treatment and vacuum-frying parameters was separated into two sections. gradually increased, and the product is centrifuged to elimi- nate part of the surface oil. Different setups could be found in larger scale and industrial scale vacuum fryer. Pre-drying Several strategies have been applied to reduce the initial water content of fruit such as pre-drying with hot air and osmotic dehydration. Hot air-drying as a pre-treatment at 80 °C, which produced final moisture content of 64% (wb), preserved apple slice color, which remains similar to that of raw apple [44]. This color preservation corresponds to lower water activity after hot air drying, which further inhibits non-enzymatic browning. Additionally, at 80 °C, hot air drying could de- crease enzymatic activity which might reduce enzymatic browning. Hot air drying reduces moisture and form a crust which produce a high resistance to oil absorption during vac- uum frying. Osmotic dehydration by 30–40% fructose resulted in crispy texture of apple chips measured as low maximum breaking force [61]. Additionally, Diamante et al. [22] observed immersion with dextrose 55% increases crunchy texture of gold kiwifruit. The osmotic dehydration in fructose solution also produced chips with uniform porosity and reduced surface shrinkage of apple chips resulting in a smoother surface [61]. The negative effect of the osmotic dehydration with fruc- tose on vacuum-fried fruits is the impact on color. Fructose application decreased the lightness of products because of the Maillard reaction during vacuum frying of apple [61]. A sim- ilar result was also found by Diamante et al. [22] whose ap- plication of 55% maltodextrin increased the browning index of gold kiwifruit. Surprisingly, at higher maltodextrin concen- tration, the browning index decreased; the mechanism behind this is still unclear. Vacuum-Frying Pre-treatments Vacuum frying is an integral process which consists of pre- treatment, frying, and post-treatment. There are a few studies that described vacuum frying without a pre-treatment, but it cannot exclude the post-treatment. The discussion on effects a b c d e f g h i Fig. 2 Schematic representation of a vacuum fryer. a Vacuum chamber. b Frying basket. c Electric motor. d Oil filter. e Oil heater. f Oil cooler. g Condenser. h Vacuum pump. i Centrifuge Food Eng Rev (2018) 10:154–164 158 Table 1 Pre-treatment effect on vacuum-fried product quality attributes Pre-treatments Quality attributes References Oil content Texture Nutrient Color Blanching Negative – N.A. N.A. [20] – Negative – – [35] – Negative – – [61] Hot air drying Positive N.A N.A. Positive [44] Osmotic dehydration Positive Positive – Negative [61] Positive – – – [50] Positive – Neutral – [23] – Positive – Negative [22] Freezing N.A. Positive N.A. N.A. [61] Anti-browning agent N.A. N.A N.A. Positive [44] Hydrocolloids Positive Negative N.A. Positive [43, 64] N.A. data not available Table 1 Pre-treatment effect on vacuum-fried product quality attributes Osmotic dehydration reduced the initial water content by 10–70% depending on the process condition and fruit proper- ties [45]. After osmotic dehydration with 40–65% maltodex- trin, mango chip will have lower initial moisture content, and thus time needed to reach same final frying time will be shorter [50]. On the other hand, in vacuum-fried apple, oil content was decreased as the concentration of fructose was increased from 30 to 40% [61]. Additionally, Nunes and Moreira [50] explained that the oil content reduction was af- fected by the water loss during the osmotic dehydration of mango by 40–65% maltodextrin in 5 h. On the other hand, blanching was found to limit oil uptake since the gelatinization leads to starch swelling and prevent oil to enter the product, as found in atmospheric fried tortilla chip [37], vacuum-fried sweet potato chips [57], and atmospheric- fried potato slices [3]. Osmotic Dehydration Osmotic dehydration can be applied for reducing the initial water content by applying sugars like fructose, maltodextrin, and salts like NaCl [17, 22, 23, 50, 61]. Osmotic dehydration is a mass transfer process, which removes partially water and simultaneously increases the soluble solid content of fruit by immersion in an osmotic solution (OS). An activity gradient between the fruit and OS causes a flow of water across fruit cell membranes which act as semi-permeable films [68]. The process results in modification of the fruit tissue which can be tailored toward compositional, textural, and sensorial quality of vacuum-fried fruit. Vacuum-Frying Parameters Vacuum-frying process is mainly characterized by time- temperature and vacuum pressure as the main parameters, which should be adjusted to the fruits characteristics to pro- duce high-quality vacuum-fried fruit. Vacuum-frying temper- ature for fruits ranged in a wide interval from 72 to 136 °C, as well as frying time (from 0.5 to 90 min), and the vacuum pressure (from 1.3 to 98.7 kPa). Freezing is also used to preserve the raw material prior the frying process. During slow freezing processes, large ice crys- tal forms that damages the cell membranes and is causing water to leach upon thawing [18]. However, fruits have a different susceptibility to freezing injury. This difference is caused by the ability of cell membrane to adapt or resist the phase change during freezing which is different for each fruit [60]. Apricots, banana, and peaches are very susceptible, while apple, grapes, and pears are moderately susceptible and dates are least susceptible for freezing damage [77]. Clearly, increasing temperature from 70 to 90 °C and time from 35 to 65 min results in an increased oil content for gold kiwi fruit [23]. On the other hand, increasing temperature from 112 to 136 °C and time from 3 to 9 min results insignificant increase of oil content in plantain [2]. Mariscal and Bouchon [44] found that increasing temperature from 95 to 115 °C induces structural changes such as tissue degradation that en- hanced the oil absorption in apple chips. Additionally, Shyu and Hwang [61] explained that the increase of oil content when temperature increase from 90 to 110 °C was caused by a higher speed of water escaping from the matrix of apple. When the water is removed from the matrix, the process will damage the cells and make the surface hydrophobic, and thus oil can absorb into the damaged sites. Anti-browning Agent The application of an anti-browning agent could prevent fur- ther browning reaction in susceptible fruits. Pre-treatment by tartaric acid, cysteine, and calcium chloride have been used to prevent non-enzymatic browning in banana. Synergistic effect was observed by combining tartaric acid-ascorbic acid, calci- um chloride-ascorbic acid, and cysteine-citric acid. However, using 1% cysteine-citric acid resulted in the highest overall preference evaluation in vacuum-fried banana [9]. Citric acid at 5.8% can also be applied to prevent non-enzymatic brow- ning in vacuum-fried apple [44], and it was also able to reduce the rate of quinone formation and color development [5]. The maximum breaking force of the vacuum-fried apricot [25] increased as the temperature and time were increased from 70 to 90 °C and 35 to 65 min; similar effect was observed in plantain [2]. Accordingly, Shyu and Hwang [61] found that increasing of frying time (from 5 to 30 min) leads to a higher crispness of apple chips. However, Yamsaengsung et al. [84] found that increasing temperature from 100 to 120 °C did not affect the crispness of banana chips. At the beginning of the frying, fruit tissue becomes soft due to cell rupture and solubi- lization of the middle lamellae and leads to rubbery and soggy products. Continuing the frying, the rapid loss of moisture from the surface leads to crust formation and an increase of the max- imum breaking force. In the final stages of the process, the crust thickened until the end of the process [2, 25, 84]. Freezing Freezing is an alternative pre-treatment strategy to achieve a crispy fruit chips matrix in vacuum-frying processing [23, 25, 61]. Shyu and Hwang [61] found that freezing at −30 °C overnight formed a porous sponge-like matrix in vacuum- fried apples. In fact, due to fast heat transfer to frozen tissue, 159 Food Eng Rev (2018) 10:154–164 ice crystal inside the frozen cells sublimed under vacuum con- dition leaving pores in the food matrix accelerated the mois- ture loss and sequentially decrease the final moisture content. Albertos et al. [4] found that moisture content in vacuum-fried carrot was lower in sample with – 20 °C blast freezing follow- ed by overnight freezing pre-treatment compared to not frozen sample. To obtain the desired benefit of freezing, water in the fruit matrix should be in frozen condition, without thawed, to enable it for sublimed and left the matrix. created a rigid, resistant film, protecting the inner matrix. Similar observation was made by Maity et al. [43] in jackfruit, showing that arabic gum was effective to reduce oil absorption up to 35.3%; on the other hand, it increased chip toughness, thus decreased crispness was observed. Different hydrocolloids produce different effects when ap- plied to the vacuum-fried fruits. CMC and other cellulose coat- ings produce a protective layer which induced gelatinization at 60 °C and subsequently prevent moisture loss and oil absorption. Meanwhile, guar gum reduces the formation of pores and cracks in the fried food, thereby reduce oil penetration [39]. Freezing rate could affect vacuum-fried fruit. Slow freezing produces big size crystal, which damages the cell [7, 16]. Then it could increase oil penetration, since oil could penetrate into damaged cell during the frying [72]. Thus, fast freezing is preferred to minimize oil uptake. Hydrocolloids Dipping the fruits in a solution of hydrocolloids such as guar gum and xanthan gum, pectin, carboxymethyl cellulose (CMC), gum arabic, and sodium alginate is a common fruit pre-treatment before vacuum frying to improve product qual- ity attributes. Sothornvit [64] described that 1.5% of guar gum is able to reduce oil absorption by 25% and 1.5% of xanthan gum by 17% in banana chips. The application of hydrocol- loids was not significantly improving the color of vacuum- fried banana chips. In the same paper, it was reported that hydrocolloid application increases the maximum breaking force. However, the differences were not observed during sen- sory study. This is explained because the hydrocolloids Vitamin C content of the vacuum-fried gold kiwifruit [23] and apple [29] was decreased as the temperature increased from 70 to 90 °C (gold kiwifruit) and 160 to 180 °C (apple) because of heat sensitivity of vitamin C. However, an Food Eng Rev (2018) 10:154–164 160 the oil content by 24% compared to without centrifugation. In general, data show that increasing centrifugation speed de- creased the oil uptake. However, the centrifugation speed has to be limited according to the product hardness to prevent product breakage. increasing frying time from 35 to 55 min of vacuum-fried gold kiwifruit was found to have only a slight effect on vitamin C [23]. Diamante et al. [24] found that in apricot, the β-carotene content increased upon frying temperature increase from 70 to 90 °C; they attributed this to the higher accessibility of the β- carotene by the oil which penetrates the fruit. The color of the fruit chips was affected as the temperature- time of the frying process is increased. Lightness and yellowness values decreased, and redness increased as found in plantain, gold kiwifruit (from 70 to 90 °C and from 35 to 65 min), apple, and mango (from 100 to 120 °C, and from 30 to 90 s) [2, 22, 61, 73]. No significant color change was found by Dueik and Bouchon [29], Mariscal and Bouchon [44], and Diamante et al. [25], who found that there was no difference in color when the frying temperature was increased for apple (from 160 to 180 °C), mango, and apricot. Moreover, Mariscal and Bouchon [44] and Diamante et al. [25] found that frying time does not influence the color of the vacuum-fried apple (between 2 and 15 min) and apricot. Hydrocolloids The a* and L* values as indicators of the browning reaction were similar to the value of raw product. As the frying time increased for plantain and apple (from 5 to 30 min), the Maillard reaction was more pronounced; and as the moisture removed, the lightness was decreased while redness and yellowness were increased [2, 61]. Effect of Matrix to Vacuum-Fried Fruit Quality The matrix of food products is defined as Bthe whole of the chemical components of food and their molecular relation- ships, the chemical composition of food, and the way those components are structurally organized at micro-, meso-, and macroscopic scales^ [15]. Tropical fruits have diverse matrix characteristics that could have different effects on vacuum- fried fruit quality. Those characteristics include cell size, cell wall, flesh thickness, firmness, intracellular spaces, sugar con- tent, fiber content, and fiber type. Some matrix characteristics of the fruits that are usually quantified and processed by vac- uum frying are described in Table 2. The effect of different matrix characteristic will be discussed in this chapter. Fruits can have two possible types of ripening. The first are called climacteric fruits, whose respiration and ethylene bio- synthesis rates increase during ripening. The second are non- climacteric fruits, whose respiration and ethylene biosynthesis rates do not increase during ripening [32]. This characteristic is important to select which fruit is suitable for frying. A char- acteristic of climacteric fruits will change substantially over time during storage. The characteristics of non-climacteric fruits will stay more constant after harvest. Another vital processing parameter is the pressure: decreas- ing the frying pressure which decreases the oil content. A lower pressure (from 13.14 to 26.54 kPa) produces a faster moisture removal, reducing the rate of oil diffusion into the pores of vacuum-fried plantain [2]. On the other hand, a lower pressure (from 40 to 60 Pa) leads to decrease of the texture quality and darker color in vacuum-fried plantain and mango [2, 73]. Ripening stage has an important role on the vacuum-fried fruit quality attributes: as a general rule, the riper the fruit, the higher the oil content in the vacuum-fried chips [20]. Yashoda et al. [86] explained that during the early ripening stage of mango, the cell wall is compact and rigid, and as the ripening continues, the cell become more loose and expanded. This expansion is due to the movement of water into the voids that form after pectin solubilization. Pectin is important because of its role in gluing the adjacent cell which results in tissue rigid- ity and firmness. Moreover, pectin is essential to maintain the matrix cohesiveness during frying [1]. Vacuum-Frying Post-treatment Centrifugation for removing the surface oil is an important part of the post-frying process and can be part of the frying equipment. Centrifugation done while the pressure is still low will significantly decrease the amount of surface oil that can penetrate the porous products when breaking the vacuum. Tarmizi and Niranjan [66] found that centrifugation under high vacuum following moderate vacuum frying has potency to reduce oil uptake in potato slices. Furthermore, Tarmizi and Niranjan [67] also found that potato chip, centrifuged under vacuum, has a significantly lower oil content than atmospheric centrifuged chip (56.85-g oil/100 g and 35.01-g oil/100 g defatted dry matter, respectively). The effect of differences in ripening stages on the texture of vacuum-fried banana has been described by Yamsaengsung et al. [84]. They found that at the first stage of ripening, sugar to starch ratio was 2.95 and the vacuum-fried banana chips have the highest maximum breaking value as an indicator of compactness and hardness of the chips. This high maximum breaking value was caused by the high content of starch which helps forming a crust [87]. At the second stage of ripening, the maximum breaking value is lower than early ripening stage as an indicator of crispy and porous matrix. At this stage, sugar to starch ratio was 8.75, which is the most optimum value to produce crispy vacuum-fried banana. However, at the third On the other hand, atmospheric centrifugation is also prom- ising. Sothornvit [64] compared two atmospheric centrifuga- tion speeds 140 and 280 rpm to remove oil after vacuum frying of banana. They found centrifugation at 280 rpm re- duced oil content, 17.3% higher than at 140 rpm. Similar findings were reported by Dueik et al. [30] who found centri- fugation of vacuum-fried apple at 400 rpm for 3 min reduced 161 Food Eng Rev (2018) 10:154–164 Table 2 Fresh tropical fruit matrix characteristic Fruits Fruit ripeninga Firmness Water contentb Porosity References Apple Non-climacteric 4.0 N 85.5 0.15 [70, 78, 62] Avocado Climacteric 5.5 N/mm 73.2 0.16 [41, 27, 69] Banana Climacteric 12.0 N/mm 71.8 0.06 [13, 56, 9, 85] Dragon fruits Non-climacteric 7.0 N/mm 83.6 N.A. [79, 74, 42] Jackfruit Climacteric 14.0 N 73.5 N.A. [82, 59] Longan Non-climacteric 18.2 N/g 81.9 N.A. [88, 75] Mango Climacteric 22.2–35.6 N 83.0 0.05 [48, 83, 85] Pineapple Non-climacteric 11.2 N 85.7 0.11 [52, 53, 85] Rambutan Climacteric 1.5 N 80.0 N.A. Vacuum-Frying Post-treatment [34, 76, 10] Snake fruit Non-climacteric 32.7 N 81.0 N.A. [65] Watermelon Non-climacteric 24.1 N 91.5 N.A. [6, 55] N.A. data not available a Wongs-Aree et al.[81] b US Department of Agriculture. Agricultural Research Service. Nutrient Data Laboratory [71] ble 2 Fresh tropical fruit matrix characteristic fruit may be affected by the sugar content that increased during ripening. Yashoda et al. [86] described that the alcohol-soluble sugar in unripe mango is mostly oligosaccharides; on the other hand, in ripe mango, it is mainly glucose and fructose. The increasing content of glucose and fructose will increase the Maillard reaction that produces brown color. A similar finding was found by Li et al. [40] in banana in which the sugar content was increasing as the starch content was decreasing. stage of ripening, the maximum breaking value is increased again, and the product is becoming hard and compact. At this ripening stage, the sugar to starch ratio was decreasing again to 4.05. The sugar to starch ratio should be increasing during the ripening process; this reverse effect could be because of the high biological variance in the banana. The high sugar content slows down the gelatinization process, thus produces a shrunk banana chip [84]. Similar results were found in mango. Starch and pectin concentrations in mango are decreasing during ripening. On the other hand, sugar is increasing during ripening. Unripe mango has 18% starch, 1.9% pectin, and 1% total soluble sugar. However, after ripening, mango has 0.1% starch, 0.5% pectin, and 15% of total soluble sugar [86]. This com- position changes during ripening could have effect on texture of vacuum-fried mango. References 16. Charoenrein S, Owcharoen K (2016) Effect of freezing rates and freeze-thaw cycles on the texture, microstructure and pectic sub- stances of mango. Int Food Res J 23:613–620 1. Aguilar CN, AnzalduaMorales A, Talamas R, Gastelum G (1997) Low-temperature blanch improves textural quality of French-fries. J Food Sci 62:568–571. https://doi.org/10.1111/j.1365-2621.1997. tb04432.x 17. Da Silva PF, Moreira RG (2008) Vacuum frying of high-quality fruit and vegetable-based snacks. LWT-Food Sci Technol 41: 1758–1767. https://doi.org/10.1016/j.lwt.2008.01.016 18. David SR, Diane MB (2004) Fruit Freezing. In: Barrett DM, Somogyi L, Ramaswamy H (eds) Processing fruits. CRC Press. https://doi.org/10.1201/9781420040074.ch8 2. Akinpelu OR, Idowu MA, Sobukola OP, Henshaw F, Sanni SA, Bodunde G, Agbonlahor M, Munoz L (2014) Optimization of process- ing conditions for vacuum frying of high quality fried plantain chips using response surface methodology (RSM). Food Sci Biotechnol 23: 1121–1128. https://doi.org/10.1007/s10068-014-0153-x 19. Dembitsky VM, Poovarodom S, Leontowicz H, Leontowicz M, Vearasilp S, Trakhtenberg S, Gorinstein S (2011) The multiple nu- trition properties of some exotic fruits: biological activity and active metabolites. Food Res Int 44:1671–1701. https://doi.org/10.1016/j. foodres.2011.03.003 3. Al-Khusaibi MK, Niranjan K (2012) The impact of blanching and high-pressure pretreatments on oil uptake of fried potato slices. Food Bioprocess Technol 5:2392–2400. https://doi.org/10.1007/ s11947-011-0562-2 20. Diamante LM (2008) Vacuum fried jackfruit: effect of maturity, pre-treatment and processing on the physiochemical and sensory. In: Annual Scientific Meeting of the Nutrition Society of Australia. Nutrition Society of New Zealand (Inc), New Zealand, pp 138–142 4. Albertos I, Martin-Diana AB, Sanz MA, Barat JM, Diez AM, Jaime I, Rico D (2016) Effect of high pressure processing or freezing technologies as pretreatment in vacuum fried carrot snacks. Innovative Food Sci Emerg Technol 33:115–122. https://doi.org/ 10.1016/j.ifset.2015.11.004 21. Diamante LM, Presswood HA, Savage GP, Vanhanen L (2011) Vacuum fried gold kiwifruit: effects of frying process and pretreatment on the physico-chemical and nutritional qualities. Int Food Res J 18:7 5. Ali HM, El-Gizawy AM, El-Bassiouny REI, Saleh MA (2015) Browning inhibition mechanisms by cysteine, ascorbic acid and citric acid, and identifying PPO-catechol-cysteine reaction prod- ucts. J Food Sci Technol 52:3651–3659. https://doi.org/10.1007/ s13197-014-1437-0 22. Diamante LM, Savage GP, Vanhanen L (2012a) Optimisation of vacuum frying of gold kiwifruit slices: application of response sur- face methodology. Int J Food Sci Technol 47:518–524. https://doi. org/10.1111/j.1365-2621.2011.02872.x 23. Diamante LM, Savage GP, Vanhanen L (2013) Response surface methodology optimization of vacuum-fried gold kiwifruit slices based on its moisture, oil and ascorbic acid contents. J Food Process Preserv 37:432–440. Conclusions Vacuum frying is a processing method that is suitable to pro- duce high-quality fried fruit products. Several factors have been reviewed for their influence on product quality attributes like oil content, texture, color, and nutrient content. Although some contradictory results have been reported for the different fruits, there are several indications for a higher quality of vacuum-frying products compared to atmospheric frying of fruit. Different equipment used in vacuum-frying processing have different characteristics, which leads to different process- ing conditions and different product quality attributes. Pre- treatments could improve most of the product quality attri- butes; however, the treatment should be tailored on the char- acteristics of the raw material and on the desired final proper- ties. We can conclude that information about the role of the fruit matrix is a very important factor in vacuum processing, but is described very limited, fragmentary, and anecdotal in the literature. During the ripening process, the fruit matrix and chemical composition will change, which will have an effect on the texture, oil content, and color of vacuum-fried fruits. Especially, tropical fruits have quite different ripening proper- ties, firmness, texture, and porosity that will influence the quality attributes of vacuum-fried tropical fruits. More Starch content could play a major role during vacuum fry- ing of fruit and determine the final quality of fried products. Banana and plantain are examples of high-content starchy fruit, which are commonly used for vacuum frying. Starch in the fruit will be gelatinized, swollen, and prevents moisture and oil transport. Giraldo Toro et al. [33] found that 35–25- mm vacuum-packed plantain slices were gelatinized for 80% at 85 °C and the degree of gelatinization increased even more at higher temperatures. Also, the fiber content could play a significant role in the final quality of vacuum-fried fruits. Fruits with high fiber con- tent could influence the fat and water transfer to and from the product; fiber could get gelatinized, swollen, and inhibit fat entering the product [39]. After vacuum frying, fruit at an early ripening stage pro- duced a low-color-intensity product; at later ripening stage, the color of the product will be more intense, which also con- tributed by Maillard reaction. The color of the vacuum-fried 162 Food Eng Rev (2018) 10:154–164 9. Apintanapong M, Cheachuminang K, Sulansawan P, Thongprasert N (2007) Effect of antibrowning agents on banana slices and vacuum-fried slices. Conclusions J Food Agric Environ 5:151–157 systematic research into the effects of the fruit matrix on the vacuum-frying process and the quality attributes of the fried fruits is needed. By such research, the mechanistic under- standing can be used to optimize the frying process to produce high-quality vacuum-fried fruits. 10. Arenas MGH, Angel DN, Damian MTM, Ortiz DT, Diaz CN, Martinez NB (2010) Characterization of Rambutan (Nephelium lappaceum) Fruits from Outstanding Mexican Selections. Revista Brasileira De Fruticultura 32:1098–1104. https://doi.org/10.1590/ S0100-29452011005000004 Funding Information Financial support for this study was provided by the Indonesia Endowment Fund for Education (LPDP) within the Ministry of Finance, Indonesia (grant number PRJ-201/LPDP/2015). 11. Ayustaningwarno F, Ananingsih VK (2007) Vacuum frying usage on increasing food diversity. Paper presented at the International Agricultural Engineering Conference: Cutting Edge Technologies and Innovations on Sustainable Resources for World Food Sufficiency, Bangkok, Thailand Compliance with Ethical Standards 12. Bouchon PB, Aguilera JM, Pyle DL (2003) Structure oil-absorption relationships during deep-fat frying. J Food Sci 68:2711–2716. https://doi.org/10.1111/j.1365-2621.2003.tb05793.x Conflict of Interest The authors declare that they have no conflict of interest. 13. Boudhrioua N, Michon C, Cuvelier G, Bonazzi C (2002) Influence of ripeness and air temperature on changes in banana texture during drying. 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https://openalex.org/W4391357405	https://wjso.biomedcentral.com/counter/pdf/10.1186/s12957-024-03310-y	English	null	Association between preoperative sarcopenia and prognosis of pancreatic cancer after curative-intent surgery: a updated systematic review and meta-analysis	World journal of surgical oncology	2,024	cc-by	7,298	Liu et al. World Journal of Surgical Oncology (2024) 22:38 https://doi.org/10.1186/s12957-024-03310-y Liu et al. World Journal of Surgical Oncology (2024) 22:38 https://doi.org/10.1186/s12957-024-03310-y World Journal of Surgical Oncology (2024) 22:38 Liu et al. World Journal of Surgical Oncology https://doi.org/10.1186/s12957-024-03310-y Open Access Association between preoperative sarcopenia and prognosis of pancreatic cancer after curative‑intent surgery: a updated systematic review and meta‑analysis Chenming Liu1,2, Liang An3, Siyuan Zhang4, Shiqing Deng5, Neng Wang6 and Haijun Tang1* Abstract Background Sarcopenia is associated with poor outcomes in many malignancies. However, the relationship between sarcopenia and the prognosis of pancreatic cancer has not been well understood. The aim of this meta-anal- ysis was to identify the prognostic value of preoperative sarcopenia in patients with pancreatic cancer after curative- intent surgery. Methods Database from PubMed, Embase, and Web of Science were searched from its inception to July 2023. The primary outcomes were overall survival (OS), progression-free survival (PFS), and the incidence of major complica- tions. The hazard ratio (HR), odds ratio (OR), and 95% confidence intervals (CIs) were used to assess the relationship between preoperative sarcopenia and the prognosis of patients with pancreatic cancer. All statistical analyses were conducted by Review Manager 5.3 and STATA 17.0 software. Results A total of 23 retrospective studies involving 5888 patients were included in this meta-analysis. The pooled results demonstrated that sarcopenia was significantly associated with worse OS (HR = 1.53, P < 0.00001) and PFS (HR = 1.55, P < 0.00001). However, this association was not obvious in regard to the incidence of major complications (OR = 1.33, P = 0.11). Conclusion Preoperative sarcopenia was preliminarily proved to be associated with the terrible prognosis of pancre- atic cancer after surgery. However, this relationship needs to be further validated in more prospective studies. Keywords Sarcopenia, Pancreatic neoplasm, Prognosis, Meta-analysis *Correspondence: Haijun Tang tanghaijun.1988@163.com 1 Department of Hepatopancreatobiliary Surgery, Shaoxing People’s Hospital, Shaoxing, Zhejiang, China 2 Department of General Surgery, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, China 3 Department of Gastrointestinal Surgery, Shaoxing People’s Hospital, Shaoxing, Zhejiang, China 4 Department of Hepatopancreatobiliary Surgery, The First Affiliated Hospital of Jinzhou Medical University, Jinzhou, Liaoning, China 5 Department of Breast and Thyroid Surgery, General Hospital of Huainan Eastern Hospital Group, Huainan, Anhui, China 6 Department of Hepatopancreatobiliary Surgery, The Quzhou Affiliated Hospital of Wenzhou Medical University, Quzhou People’s Hospital, Quzhou, Zhejiang, China 6 Department of Hepatopancreatobiliary Surgery, The Quzhou Affiliated Hospital of Wenzhou Medical University, Quzhou People’s Hospital, Quzhou, Zhejiang, China 6 Department of Hepatopancreatobiliary Surgery, The Quzhou Affiliated Hospital of Wenzhou Medical University, Quzhou People’s Hospital, Quzhou, Zhejiang, China Introduction Inclusion criteria were as follows: patients were patholog- ically diagnosed with pancreatic cancer; sarcopenia was evaluated by cross-sectional computed tomography (CT) scan of the third lumbar (L3) vertebra with respective cut-off values defined by sex before surgery; the meas- urement method of sarcopenia included skeletal muscle index (SMI) and psoas muscle index (PMI), as described in previous studies [19, 20], which represented two most common measurement methods; the definition of cut- off values included various standards, such as receiver operating characteristic (ROC) curves, Martin’s defini- tion [21], Prado’s definition [22], and lowest quantile; outcomes were evaluated by prognostic indicators such as overall survival (OS) and/or progression-free survival (PFS) and the incidence of postoperative complications. Pancreatic cancer is a highly malignant solid tumor with 5-year survival rate less than 10% [1, 2]. In recent years, its incidence and mortality are still gradually increas- ing, and it is predicted to be the second leading cause of cancer-related death in the USA by 2030 [3]. Although the application of systemic chemotherapy and targeted therapy has greatly benefited patients with pancreatic cancer in recent years, surgery remains the only cura- tive-intent treatment strategy. However, postoperative survival rate is still unsatisfactory due to its large proba- bility of recurrence and metastasis [4, 5]. Previous stud- ies on prognosis following pancreatectomy have mainly focused on tumor-specific factors such as tumor’s dif- ferentiation, perivascular invasion, and lymph node invasion [6–8]. However, their predictive abilities were skeptical due to the instability of these indicators. Exclusion criteria were as follows: patients were patho- logically diagnosed as benign or borderline pancreatic tumors; sarcopenia was assessed by methods other than CT, such as bioelectrical analysis (BIA) and dual-energy X-ray absorptiometry (DXA); the cut-off values for sarco- penia were not clearly defined; the types of studies were conference abstracts, case reports, letters, and reviews; the time to evaluate sarcopenia took place postopera- tively or the treatment strategy was palliative. In recent years, there has been increasing interest in the association between body composition and prognosis due to its simplicity and practicality. Sarcopenia, referring to age-dependent reduction in skeletal muscle volume, was first described in 1989 [9]. Sarcopenia was a kind of pro- gressive and widespread skeletal muscle disease associated with an increased likelihood of adverse outcomes, including falls, fractures, physical disability, and death [10]. Introduction It has been found to be a potential risk factor for morbidity and mortal- ity in patients with gastrointestinal malignancies [11]. © The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecom- mons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Page 2 of 12 Liu et al. World Journal of Surgical Oncology (2024) 22:38 Liu et al. World Journal of Surgical Oncology (2024) 22:38 Inclusion and exclusion criteria Inclusion and exclusion criteria Data extraction Two investigators independently extracted the following information from each study: publishing year, the name of first author, country, sample size, perioperative treat- ment (including neoadjuvant and adjuvant therapy), the measurement approach of sarcopenia, the cut-off values for sarcopenia, and clinical outcomes. Materials and methodsh The systematic review and meta-analysis followed Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) guidelines [18]. The registra- tion number was INPLASY202390060. The protocol could be found in Inplasy Protocol 5298 – INPLASY. Outcomesh Most patients with pancreatic cancer were prone to skeletal muscle depletion, leading to reduced tolerance for postoperative adjuvant therapy [12, 13]. Several recent studies have attempted to investigate the effect of sarcopenia on the prognosis of pancreatic cancer, but the outcomes of these studies have been more or less controversial [14–17]. Evidence needs to be updated, so the aim of this systematic review and meta-analysis is to clarify the relationship between preoperative sarco- penia and the prognosis of pancreatic cancer. The primary outcomes were OS, PFS, and the incidence of major complications (grade III–IV) according to the Clavien-Dindo classification [23]. Secondary outcomes were the incidence of overall complications (grade I–IV) according to the Clavien-Dindo classification, as well as surgery-specific complications including clinically rel- evant postoperative pancreatic fistula (CR-POPF), post- pancreatectomy hemorrhage (PPH), delayed gastric empty (DGE), and surgical site infection (SSI) [24–26]. Literature search strategy Two independent investigators assessed the quality of the included studies on the Newcastle–Ottawa Scale (NOS) [27]. The contents of the scale included case selection, cohort comparison, and exposure risk assessment. Only studies with NOS score of six or higher were included in the final meta-analysis. Two independent reviewers searched PubMed, Embase, and Web of science from its inception to July 2023. The language of search results was limited to English. Subsequently, the two persons checked each other and tried to reach a consensus. The detailed search strate- gies are presented in the Additional file 1. Page 3 of 12 Liu et al. World Journal of Surgical Oncology (2024) 22:38 Fig. 1 Flow diagram of included studies Fig. 1 Flow diagram of included studies Primary outcomes The relationship between preoperative sarcopenia and OSh The relationship between preoperative sarcopenia and OS The impact of preoperative sarcopenia on OS was explored in fifteen studies. The pooled HR demonstrated that preop- erative sarcopenia was significantly associated with worse OS (HR = 1.53, 95% CI 1.41–1.67, P < 0.00001; I2 = 15%, P = 0.28) (Fig. 2). Subgroup analyses based on the measure- ment approach, region of studies, and different definitions Statistical analysis assessed for eligibility. Eventually, 23 studies were eligible for qualitative synthesis after careful examination [14–17, 28–46]. The detailed flow diagram is shown in Fig. 1. Survival data were evaluated by hazard ratio (HR) and their 95% corresponding intervals (CIs) in multivariate regres- sion analysis, and categorical variables by odds ratio (OR). The Cochrane’s Q-test and I2 statistics were used to assess statistical heterogeneity. The cut-off value of low, moderate, and high heterogeneity was 25%, 50% and 75%, respectively. When the value of total heterogeneity exceeded 50%, we used the random-effect model. Otherwise, the fixed-effect model was applied. Subgroup analyses stratified by meas- urement approach of sarcopenia (SMI or PMI), region of studies (Asia or non-Asia), and definition of cut-off values (ROC curve, Martin’s definition, Prado’s definition, and low- est quantile) were performed further to find out the source of heterogeneity. P < 0.05 was regarded as statistically signifi- cant. In order to explore the possibility of publication bias, we applied funnel plots and Egger’s test. All analyses were conducted by Review Manager 5.3 software (Copenhagen: The Nordic Cochrane Centre, The Collaboration, 2011) and STATA 17.0 software (College Station, TX). Basic characteristics of included studies A total of 5888 patients with pancreatic cancer were incorporated into our meta-analysis. Publication year of studies ranged from 2012 to 2023. Seventeen (73.9%) studies were from Asian countries and only 6 (26.1%) from non-Asian countries. The majority of studies applied SMI to measure sarcopenia. And the definition of sex-related cut-off values for sarcopenia included 5 approaches, ROC curves (30.4%), Martin’s definition (13.0%), Prado’s definition (21.7%), Contal- O’Quigley method (4.3%), and lowest quantile (30.6%). The detailed information is listed in Table 1. Study selection We searched 1538 articles from the electronic databases (PubMed, Embase, and Web of Science). After removing duplicates and unrelated studies, 119 full-text studies were Liu et al. World Journal of Surgical Oncology (2024) 22:38 Page 4 of 12 Liu et al. Study selection World Journal of Surgical Oncology Table 1 Basic characteristics of included studies Year Author Country Sample size Neoadjuvant treatment Adjuvant treatment Measurement Cut-off value Primary outcome Secondary outcome 2023 Shen [46] China 614 NA 379 (61.7%) SMI Male < 52.4cm2/m2; female < 38.5 cm2/m2 OS (HR = 1.23, 95% CI 0.93–1.62) MC NA 2022 Özkul [45] Turkey 115 NA NA SMI Male < 56.44cm2/m2; female < 43.56cm2/m2 OS (HR = 1.23, 95% CI 1.33–1.85) NA 2022 Cai [44] China 115 NA 84 (73.0%) SMI Male < 45.16cm2/m2; female < 34.65cm2/m2 OS (HR = 2.31, 95% CI 1.21–4.40) PFS (HR = 1.91, 95% CI 1.15–3.17) NA 2022 Kim [14] Korea 347 0(0.0%) 226 (65.1%) SMI Following legends behinda OS (HR = 1.40, 95% CI 0.93–2.10) PFS (HR = 1.43, 95% CI 0.96–2.13) NA 2021 Rom [15] Israel 111 3(2.7%) 73 (65.8%) SMI Male < 44.35cm2/m2; female < 34.82cm2/m2 OS (HR = 1.73, 95% CI 1.07–2.80) MC OC 2021 d’Engremont [43] France 76 NA NA SMI Male < 52.4cm2/m2; female < 38.5cm2/m2 PFS (HR = 1.78, 95% CI 1.01–3.14) NA 2020 Ryu [41] Korea 548 22(4.0%) NA SMI Male < 50.18cm2/m2; female < 38.63cm2/m2 OS (HR = 1.16, 95% CI 0.92–1.46) MC CR-POPF PPH DGE SSI 2020 Xu [42] China 152 NA NA PMI Male < 4.78cm2/m2; female < 3.46cm2/m2 MC NA 2020 Peng [40] China 116 3 (2.6%) 58 (34.9%) SMI Male < 42.2cm2/m2; female < 33.9cm2/m2 OS (HR = 2.51, 95% CI 1.03–6.12) PFS (HR = 1.00, 95% CI 0.55–1.81) MC NA 2019 Ratnayake [39] New Zealand 89 NA NA SMI Male < 43cm2/m2 (BMI < 25 kg/m2), 53cm2/ m2 (BMI > 25 kg/m2); female < 41cm2/m2 MC OC CR-POPF PPH DGE SSI 2019 Gruber [38] Austria 133 20 (15.0%) NA SMI Male < 52.4cm2/m2; female < 38.5cm2/m2 OS (HR = 1.51, 95% CI 1.04–2.19) MC CR-POPF 2018 Yamane [37] Japan 99 NA NA SMI Male < 43cm2/m2 (BMI < 25 kg/m2), 53cm2/ m2 (BMI > 25 kg/m2); female < 41cm2/m2 NA CR-POPF 2018 Wagner [36] Austria 424 NA NA PMI Male < 20.74cm2/m2; female < 14.65cm2/m2 MC OC 2018 Tankel [35] Israel 61 3 (4.9%) NA PMI Male < 4.92cm2/m2; female < 3.62cm2/m2 MC CR-POPF DGE 2018 EI Amrani [34] France 107 NA NA SMI Male < 52.4cm2/m2; female < 38.5cm2/m2 OS (HR = 2.04, 95% CI 0.93–4.47) MC CR-POPF DGE SSI 2018 Choi [16] Korea 180 NA NA SMI Male < 45.3cm2/m2; female < 39.3cm2/m2 OS (HR = 1.78, 95% CI 1.20–2.65) MC OC 2017 Takagi [33] Japan 219 NA NA SMI Male < 68.5cm2/m2; female < 52.5cm2/m2 MC CR-POPF DGE SSI Page 5 of 12 Liu et al. Study selection World Journal of Surgical Oncology (2024) 22:38 SMI Skeletal muscle index, PMI Psoas muscle index, BMI Body mass index, HR Hazard ratio, CI Confidential interval, OS Overall survival, PFS Progression-free survival, MC Major complications, OC Overall complications, CR-POPF Clinically related postoperative pancreatic fistula, PPH Post-pancreatectomy hemorrhage, DGE Delayed gastric empty, SSI Surgical site infection, NA No available BMI < 23 kg/m2, age < 65 years: male < 45.25 cm2/m2, female < 37.39 cm2/m2; BMI < 23 kg/m2, age ≥ 65 years: male < 48.86 cm2/m2, female < 38.85 cm2/m2 BMI ≥ 23 kg/m2, age < 65 years: male < 54.89 cm2/m2, female < 44.90 cm2/m2; BMI ≥ 23 kg/m2, age ≥ 65 years: male < 49.66 cm2/m2, female < 49.84 cm2/m2 a Kim et al. Table 1 (continued) Year Author Country Sample size Neoadjuvant treatment Adjuvant treatment Measurement Cut-off value Primary outcome Secondary outcome 2017 Okumura [32] Japan 301 33 (11.0%) 216 (71.2%) SMI Male < 47.1cm2/m2; female < 36.6cm2/m2 OS (HR = 1.79, 95% CI 1.24–2.58) PFS (HR = 1.60, 95% CI 1.18–2.16) MC CR-POPF 2017 Ninomiya [17] Japan 265 0 (0.0%) 174 (65.6%) SMI Male < 43.75cm2/m2; female < 38.5cm2/m2 OS (HR = 2.11, 95% CI 1.20–3.70) MC NA 2016 Nishida [31] Japan 266 22 (8.3%) NA SMI Male < 43cm2/m2 (BMI < 25 kg/m2), 53cm2/ m2 (BMI > 25 kg/m2); female < 41cm2/m2 MC CR-POPF DGE SSI 2015 Okumura [30] Japan 230 24 (10.4%) NA PMI Male < 5.90cm2/m2; female < 4.07cm2/m2 OS (HR = 1.99, 95% CI 1.37–2.90) PFS (HR = 1.60, 95% CI 1.14–2.24) MC NA 2015 Amini [29] USA 763 NA NA PMI Male < 5.64cm2/m2; female < 4.15cm2/m2 OS (HR = 1.46, 95% CI 1.11–1.92) MC OC 2012 Peng [28] China 557 NA NA PMI Male < 4.92cm2/m2; female < 3.62cm2/m2 OS (HR = 1.63, 95% CI 1.28–2.08) MC OC Liu et al. World Journal of Surgical Oncology (2024) 22:38 Page 6 of 12 Fig. 2 Forest plot of comparison in overall survival between sarcopenia and non-sarcopenia of cut-off values confirmed the similar results (all P < 0.05). And all heterogeneity was moderate or low (Table 2). of major complications (OR = 1.33, 95% CI 0.93–1.89, P = 0.11; I2 = 76%, P < 0.00001) (Fig. 4). However, interest- ingly, subgroup analysis stratified by the different defini- tions of cut-off values showed the inconsistent results. Study selection The pooled OR of those studies whose cut-off values were defined by ROC curves demonstrated preoperative sarcopenia’s strong relevance to the increased incidence of major complications (OR = 2.73, 95% CI 1.35–5.53, P = 0.005; I2 = 0%, P = 0.01), but this relevance was not shown in studies defined by the other three definitions (Fig. 5, Table 3). SMI Skeletal mass index, PMI Psoas mass index, HR Hazard ratio, CI Corresponding intervals MI Psoas mass index, HR Hazard ratio, CI Corresponding intervals The relationship between preoperative sarcopenia and PFS Six studies evaluated the association between preop- erative sarcopenia and PFS. The pooled HR showed that preoperative sarcopenia was strongly related to worse PFS (HR = 1.55, 95% CI 1.31–1.84, P < 0.00001; I2 = 0%, P = 0.67) (Fig. 3). However, we were not able to further perform subgroup analysis due to the limited available information. and the incidence of major complications Impact of preoperative sarcopenia on overall complica- tions was reported in six studies. Preoperative sarcope- nia was not obviously related to the increased incidence of postoperative overall complications (OR = 1.33, Eighteen studies including 4877 participants explored the predictive role of preoperative sarcopenia for major com- plications. Contrary to OS and PFS, preoperative sarco- penia was not obviously associated with high incidence Table 2 Subgroup analysis for overall survival SMI Skeletal mass index, PMI Psoas mass index, HR Hazard ratio, CI Corresponding intervals No. studies Samples HR 95% CI P value I2 Measurement method SMI 12 2952 1.49 1.36–1.63 < 0.00001 21% PMI 3 1550 1.62 1.38–1.91 < 0.00001 0% Region of studies Asia 12 3499 1.54 1.41–1.68 < 0.00001 31% Non-Asia 3 1003 1.51 1.22–1.86 0.0001 0% Definition of cut-off values ROC curves 5 877 1.69 1.48–1.94 < 0.00001 0% Lowest quantile 5 2159 1.45 1.27–1.65 < 0.00001 34% Prado’s definition 4 1119 1.44 1.18–1.76 0.0003 22% Table 2 Subgroup analysis for overall survival Liu et al. World Journal of Surgical Oncology (2024) 22:38 Page 7 of 12 Fig. 3 Forest plot of comparison in progression-free survival between sarcopenia and non-sarcopenia Fig. 4 Forest plot of comparison in the incidence of major complications between sarcopenia and non-sarcopenia Fig. 3 Forest plot of comparison in progression-free survival between sarcopenia and non-sarcopenia Fig. 4 Forest plot of comparison in the incidence of major complications between sarcopenia and non-sarcopenia Fig. 3 Forest plot of comparison in progression-free survival between sarcopenia and non-sarcopenia Fig. 3 Forest plot of comparison in progression-free survival between sarcopenia and non-sarcopenia Fig. 4 Forest plot of comparison in the incidence of major complications between sarcopenia and non-sarcopenia ig. 4 Forest plot of comparison in the incidence of major complications between sarcopenia and non-sarcopenia 95% CI 0.84–2.12, P = 0.23; I2 = 75%, P = 0.001). And the increased probability of surgery-related complica- tions, including CR-POPF, PPH, DGE, and SSI, was not observed to have a strong association with preopera- tive sarcopenia, either (all P > 0.05) (Additional file 2). And the results of subgroup analyses were consistent (Tables 4 and 5). after radical surgery, including OS, PFS, and the inci- dence of complications (overall complications and major complications, as well as four surgical-related compli- cations including CR-POPF, PPH, DGE, and SSI). Our results were encouraging, suggesting that preoperative sarcopenia significantly reduced survival time (OS and PFS). Publication biash Basically consistent with our results, the first meta- analysis conducted by Mintziras et al. in patients with pancreatic ductal adenocarcinoma confirmed that sarco- penia was strongly associated with worse OS (HR = 1.49, 95% CI 1.27–1.74, P < 0.001) [47]. However, they did not exclude those patients with palliative treatment. Moreo- ver, analyses of the incidence of major complications and CR-POPF in sarcopenia were not performed due to limited data. Bundred et al. showed that sarcopenia was not significantly associated with the incidence of post- operative complications or CR-POPF [48]. However, of The symmetrical distribution of funnel plots showed no significant risk of publication bias (Additional file 3). Moreover, Egger’s regression test suggested that pub- lication bias was insignificant for OS (P = 0.757), PFS (P = 0.684), and the incidence of major complications (P = 0.448). and the incidence of major complications However, our analysis did not confirm that sarcope- nia was strongly associated with high incidence of post- operative complications. SMI Skeletal mass index, PMI Psoas mass index, OR Odds ratio, CI Corresponding intervals oas mass index, OR Odds ratio, CI Corresponding intervals Discussion studies Samples OR 95% CI P value I2 The incidence of CR-POPF 8 1522 0.97 0.65–1.44 0.87 42% Region of studies Asia 5 1193 0.98 0.54–1.77 0.93 63% Non-Asia 3 329 0.87 0.50–1.50 0.61 0% The incidence of DGE 6 1290 1.18 0.67–2.08 0.56 54% Region of studies Asia 4 1094 1.26 0.53–2.99 0.60 69% Non-Asia 2 196 1.13 0.57–2.25 0.73 13% The incidence of SSI 5 1229 1.31 0.75–2.29 0.34 60% Region of studies Asia 3 1033 1.53 0.63–3.72 0.35 77% Non-Asia 2 196 1.04 0.57–1.90 0.91 0% Table 5 Subgroup analysis for the incidence of surgical related complications immune system is weakened, and the postoperative wound healing is poor, thus affecting the risk of postop- erative complications. musculature [51, 52]. Thormann et al. concluded that sar- copenia was strongly relevant to dismal prognosis in both radical and palliative settings. Unfortunately, they did not conduct further subgroup analyses to explore the sources of heterogeneity [53].h Since sarcopenia is associated with unsatisfactory postoperative survival rate and high incidence of com- plications, perioperative intervention is important to reduce these risks. Nutritional counseling and oral nutri- tional supplements may also be available as intervention options for the treatment of cachexia [59, 60]. Studies have shown that in patients with gastric cancer, preop- erative exercise and nutritional support programs can reduce the incidence of sarcopenia and improve postop- erative outcomes [61]. The mechanism of the association between sarcopenia and poor prognosis has not been well understood. Sar- copenia is not merely a loss of muscle mass or quantity, but a disorder that reflects a disorder of immune nutri- tional status, and its relationship with the tumor micro- environment is still being studied [54]. Several nutritional and immune factors were found to have an important role in people with sarcopenia. Previous studies have reported that high neutrophil–lymphocyte ratio (NLR) was an independent indicator of muscle mass loss [45]. A recent meta-analysis showed that in patients with pancreatic cancer, lower NLR had better OS and PFS in patients with pancreatic cancer [55]. In addition, several studies have demonstrated that sarcopenia was associ- ated with insulin resistance, vitamin D deficiency, ele- vated levels of inflammatory cytokines (such as tumor necrosis factor-alpha and interleukin-6), and decreased concentrations of muscle factors (such as interleukin-15) [56–58]. Discussion studies Samples OR 95% CI P value I2 Measurement method SMI 3 380 1.29 0.82–2.03 0.27 0% PMI 3 1483 1.34 0.62–2.87 0.45 75% Region of studies Asia 3 587 1.10 0.66–1.86 0.71 43% Non-Asia 3 1276 1.49 0.69–3.23 0.31 86% Table 5 Subgroup analysis for the incidence of surgical related complications OR Odds ratio, CI Corresponding intervals, CR-POPF Clinically related postoperative pancreatic fistula, DGE Delayed gastric empty, SSI Surgical site infection No. studies Samples OR 95% CI P value I2 The incidence of CR-POPF 8 1522 0.97 0.65–1.44 0.87 42% Region of studies Asia 5 1193 0.98 0.54–1.77 0.93 63% Non-Asia 3 329 0.87 0.50–1.50 0.61 0% The incidence of DGE 6 1290 1.18 0.67–2.08 0.56 54% Region of studies Asia 4 1094 1.26 0.53–2.99 0.60 69% Non-Asia 2 196 1.13 0.57–2.25 0.73 13% The incidence of SSI 5 1229 1.31 0.75–2.29 0.34 60% Region of studies Asia 3 1033 1.53 0.63–3.72 0.35 77% Non-Asia 2 196 1.04 0.57–1.90 0.91 0% Table 4 Subgroup analysis for the incidence of overall complications SMI Skeletal mass index, PMI Psoas mass index, OR Odds ratio, CI Corresponding intervals No. studies Samples OR 95% CI P value I2 Measurement method SMI 3 380 1.29 0.82–2.03 0.27 0% PMI 3 1483 1.34 0.62–2.87 0.45 75% Region of studies Asia 3 587 1.10 0.66–1.86 0.71 43% Non-Asia 3 1276 1.49 0.69–3.23 0.31 86% Table 4 Subgroup analysis for the incidence of overall complications SMI Skeletal mass index, PMI Psoas mass index, OR Odds ratio, CI Corresponding intervals Table 5 Subgroup analysis for the incidence of surgical related complications OR Odds ratio, CI Corresponding intervals, CR-POPF Clinically related postoperative pancreatic fistula, DGE Delayed gastric empty, SSI Surgical site infection No. Discussion We conducted a systematic review and meta-analysis of 23 studies to investigate the relationship between preop- erative sarcopenia and the prognosis of pancreatic cancer Liu et al. World Journal of Surgical Oncology (2024) 22:38 Page 8 of 12 Fig. 5 Forest plot of comparison in the incidence of major complications between sarcopenia and non-sarcopenia according to different definitions of cut-off values Fig. 5 Forest plot of comparison in the incidence of major complications between sarcopenia and non-sarcopenia according to different definitions of cut off values Fig. 5 Forest plot of comparison in the incidence of major complications between sarcopenia and non-sarcopenia according to different definitions of cut-off values radiation, and studies have confirmed that CT scan has been shown to be more sensitive to small changes in muscle area than DXA [49, 50]. So, based on the recent consensus from the European Working Group on Sar- copenia in Older People and the Asian Working Group for Sarcopenia, CT imaging at the level of the L3 vertebra represents a standardized method to quantify the skeletal the studies they included, only five and two, respectively, reported the incidence of major complications and CR- POPF. In addition, the generalization of their results was limited by the high heterogeneity caused by non-stand- ardized measurement methods, such as BIA and DXA. CT could make up for the unavoidable disadvantage of BIA and DXA to patients caused by repeated doses of Table 3 Subgroup analysis for the incidence of major complications SMI Skeletal mass index, PMI Psoas mass index, OR Odds ratio, CI Corresponding intervals No. studies Samples OR 95% CI P value I2 Measurement method SMI 12 2951 1.13 0.84–1.53 0.40 47% PMI 6 1926 1.86 0.77–4.51 0.17 89% Region of studies Asia 13 3359 1.30 0.86–1.97 0.21 76% Non-Asia 5 1518 1.39 0.68–2.82 0.37 79% Definition of cut-off values ROC curves 5 1223 2.73 1.35–5.53 0.005 69% Lowest quantile 7 2178 0.92 0.59–1.45 0.73 66% Martin’s definition 2 355 1.70 0.58–5.02 0.33 74% Prado’s definition 4 1121 1.00 0.69–1.45 1.00 19% Table 3 Subgroup analysis for the incidence of major complications Liu et al. World Journal of Surgical Oncology (2024) 22:38 Page 9 of 12 Table 4 Subgroup analysis for the incidence of overall complications SMI Skeletal mass index, PMI Psoas mass index, OR Odds ratio, CI Corresponding intervals No. Availability of data and materials Availability of data and materials The current study was based on the results of relevant published studies. Acknowledgements None. Acknowledgements None. Acknowledgements None. Consent for publication Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests. Received: 11 August 2023 Accepted: 13 January 2024 Received: 11 August 2023 Accepted: 13 January 2024 Authors’ contributions Conceptualization, Chenming Liu and Liang An; Literature research, Liang An; Software, Siyuan Zhang and Neng Wang; Writing–Original Draft Preparation, Chenming Liu and Liang An; Writing–Review & Editing, Haijun Tang; Project Administration, Haijun Tang; Funding Acquisition, Liang An and Haijun Tang. Declarations Ethics approval and consent to participate Not applicable. Ethics approval and consent to participate Not applicable. Competing interests Competing interests The authors declare no competing interests. The authors declare no competing interests. Preoperative sarcopenia was preliminarily proved to be significantly associated with the poor prognosis of pan- creatic cancer patients after radical surgery. However, this relationship needs to be further validated in more prospective studies. Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s12957-024-03310-y. The online version contains supplementary material available at https://doi. org/10.1186/s12957-024-03310-y. Additional file 1. Additional file 2: Supplementary Figure 1. Forest plots of comparison between sarcopenia and non-sarcopenia. (A) overall complications, (B) CR-POPF, (C) PPH, (D) DGE, (E) SSI. Additional file 3: Supplementary Figure 2. Funnel plots for examina- tion of publication bias. (A) overall survival, (B) major complications, (C) profession-free survival. Additional file 2: Supplementary Figure 1. Forest plots of comparison between sarcopenia and non-sarcopenia. (A) overall complications, (B) CR-POPF, (C) PPH, (D) DGE, (E) SSI. Additional file 3: Supplementary Figure 2. Funnel plots for examina- tion of publication bias. (A) overall survival, (B) major complications, (C) profession-free survival. We have to admit that our study has several limita- tions. First, all the studies we included were retrospective cohort studies. In the future, large-scale randomized con- trolled trials are needed to further clarify the relationship between sarcopenia and the prognosis of pancreatic can- cer. Second, due to the limited information available from the included studies, we did not conduct more subgroup analyses of other important indicators that may influence prognosis, such as tumor’s stage, gender, perioperative treatment (including neoadjuvant and adjuvant ther- apy), and surgical procedure. Finally, we did not analyze biomarkers that might affect muscle quality, such as fat infiltration and accumulation, because relevant studies were still insufficient. Sarcopenia reflects a combination of muscle quantity and mass. However, to the best of our knowledge, this study is the first meta-analysis to analyze the relationship between sarcopenia and the prognosis after radical resection of pancreatic cancer according to different definition criteria of sarcopenia cut-off values, which may provide novel direction for accurate explora- tion in the future. References Abbreviations PRISMA Preferred Reporting Items for Systematic Review and Meta-Analysis CT Computed tomography OS Overall survival PFS Progression-free survival BIA Bioelectrical analysis DXA Dual-energy X-ray absorptiometry CR-POPF Clinical related-postoperative pancreatic fistula PPH Post-pancreatectomy hemorrhage DGE Delayed gastric empty SSI Surgical site infection Discussion Under the action of the above factors, the body’s To analyze the sources of heterogeneity, we performed subgroup analyses by regions of studies (Asian or non- Asian), measurement methods of sarcopenia (SMI or PMI), and definition criteria for sex-specific cut-off values, respectively. Our subgroup analyses of differ- ent study regions and measurement methods did not change the overall results. But interestingly, our research showed that under the criteria of cut-off values defined by the ROC curve, preoperative sarcopenia was strongly associated with worse OS (HR = 1.69, 95% CI 1.48–1.94, P < 0.00001) and higher incidence of complications Liu et al. World Journal of Surgical Oncology (2024) 22:38 Page 10 of 12 SMI Skeletal muscle index PMI Psoas muscle index NOS Newcastle-Ottawa Scale HR Hazard ratio CIs Corresponding intervals OR Odds ratio ROC Receiver operating characteristic NLR Neutrophil-lymphocyte ratio SMI Skeletal muscle index PMI Psoas muscle index NOS Newcastle-Ottawa Scale HR Hazard ratio CIs Corresponding intervals OR Odds ratio ROC Receiver operating characteristic NLR Neutrophil-lymphocyte ratio (OR = 2.73, 95% CI 1.35–5.53, P = 0.005). In contrast, the relationship was less significant or non-significant based on the criteria of other definitions, such as the lowest quantile, Prado’s, and Martin’s definition. We speculate that this phenomenon may be related to the objectivity and accuracy of ROC curve based on the data itself, free from external interference. Therefore, this finding may provide a novel direction for more accurate definition of cut-off values for sarcopenia in the future. However, at present, no unanimously accepted cut-off values have been established for CT-based sarcopenia in Asian pop- ulations. Therefore, more large-scale studies are needed in the future to establish standardized cut-off values for sarcopenia in different populations and confirm these observations. Funding g The study was supported by Shaoxing Basic Public Welfare Project (No. 2022A14012), and Shaoxing Health Science and Technology Project (Labora- tory opening plan) (No. 2022SY013). Availability of data and materials Received: 11 August 2023 Accepted: 13 January 2024 Abbreviations Prognostic value of sarcopenia and myosteatosis in patients with resectable pancreatic ductal adenocarci- noma. 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https://openalex.org/W4324092099	https://www.mdpi.com/1424-2818/15/3/407/pdf?version=1678672896	English	null	The Diversity of Alien Plant Species in South Africa’s National Botanical and Zoological Gardens	Diversity	2,023	cc-by	14,293	Article The Diversity of Alien Plant Species in South Africa’s National Botanical and Zoological Gardens Thabiso M. Mokotjomela 1,2,, Sebataolo J. Rahlao 1 , Loyd R. Vukeya 3, Christophe Baltzing Lindokuhle V. Mangane 1, Christopher K. Willis 5 and Thompson M. Mutshinyalo 5 Thabiso M. Mokotjomela 1,2,, Sebataolo J. Rahlao 1 , Loyd R. Vukeya 3, Christophe Baltzinger 4 , Lindokuhle V. Mangane 1, Christopher K. Willis 5 and Thompson M. Mutshinyalo 5 1 South Africa National Biodiversity Institute (SANBI) Directorate on Biodiversity Evidence, Free State National Botanical Garden, Raytton, Dan Pienaar, P.O. Box 29036, Danhof 9310, South Africa 2 School of Life Sciences, University of KwaZulu-Natal, Pietermaritzburg 3200, South Africa 3 Free State National Botanical Garden, Raytton, Dan Pienaar, P.O. Box 29036, Danhof 9310, South Africa 4 INRAE, UR EFNO, 45290 Nogent-sur-Vernisson, France g 5 South African National Biodiversity Institute National Botanical Gardens, Pretoria National Botanical 5 South African National Biodiversity Institute National Botanical Gardens, Pretoria National Botanical Garden, Pretoria 0184, South Africa * Correspondence: mokotjomelat@yahoo.co.uk; Tel.: +27-733246118 Abstract: The management of biological invasions, which pose a growing threat to natural re- sources and human well-being, is critical for reducing associated negative impacts. As part of the process of developing a strategy for the management of biological invasions in the South African National Biodiversity Institute’s (SANBI) gardens, we collated a list of alien plant species from 13 gardens as part of a situational analysis. We requested lists of alien plant species recorded in each of the SANBI’s gardens. A total of 380 records included 225 alien plant species belonging to 73 families. A significant number of species were intentionally introduced through horticultural trade as ornamentals (49%; n = 225), while 20.9% were consumed as either food or medicine by humans. Plant life forms included woody and herbaceous plants, graminoids, succulents and ferns. Herbaceous (42.7%; n = 225) and woody plants (3.8%) were the dominant life forms. The Walter Sisulu National Botanical Garden had the highest number of alien species (88 species), followed by Kirstenbosch (61 species) and Pretoria (46 species) National Botanical Gardens, with herbaceous species constituting the largest number in all gardens (i.e., 47, 19, and 27 species, respectively). The number of species that we recorded that were listed in the National Environ- mental Management: Biodiversity Act (NEM: BA) (Act No. 10 of 2004): Alien and Invasive Species Regulations’ categories were not notably different from the number of unlisted species (58.2% vs. 42.8%). diversity diversity Article The Diversity of Alien Plant Species in South Africa’s National Botanical and Zoological Gardens The number of species listed in the different categories varied significantly across the different gardens, with a significantly higher number of unlisted species and of Category 1b species in the Walter Sisulu, Kirstenbosch and Pretoria National Botanical Gardens than in other gardens. That a significantly larger number of alien species originated from South America points to the need to improve biosecurity controls on existing relations. The results of this study provided a baseline database to help comparison between successive surveys in future. diversity diversity Citation: Mokotjomela, T.M.; Rahlao, S.J.; Vukeya, L.R.; Baltzinger, C.; Mangane, L.V.; Willis, C.K.; Mutshinyalo, T.M. The Diversity of Alien Plant Species in South Africa’s National Botanical and Zoological Gardens. Diversity 2023, 15, 407. https://doi.org/10.3390/d15030407 Citation: Mokotjomela, T.M.; Rahlao, S.J.; Vukeya, L.R.; Baltzinger, C.; Mangane, L.V.; Willis, C.K.; Mutshinyalo, T.M. The Diversity of Alien Plant Species in South Africa’s National Botanical and Zoological Gardens. Diversity 2023, 15, 407. https://doi.org/10.3390/d15030407 Academic Editor: Anatoliy A. Khapugin Academic Editor: Anatoliy A. Khapugin Received: 30 January 2023 Revised: 21 February 2023 Accepted: 21 February 2023 Published: 10 March 2023 Keywords: biodiversity conservation gardens; global change; introduction pathways 1. Introduction Botanical gardens represent the largest plant conservation network in the world [1], with diverse interconnected functions ranging from environmental education and scien- tiﬁc research to recreation [2,3]. Krishnan and Novy [4] reviewed different deﬁnitions of botanical gardens, and these deﬁnitions mainly emphasise the functions that gardens perform. For example, they were considered primarily as outdoor collections of labelled living plants in aesthetic landscapes, playing passive roles in their communities, as well as historical heritage sites. Presently, botanical gardens have evolved to include expanded programmes, such as the conservation of plant biodiversity, by serving as repositories of Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/diversity Diversity 2023, 15, 407. https://doi.org/10.3390/d15030407 Diversity 2023, 15, 407 2 of 20 2 of 20 plant germplasm for the long-term preservation of species, scientiﬁc research, and creation of urban refuges for wildlife and humans [5]. In addition, Botanic Gardens Conservation International (BGCI) [6] deﬁnes botanical gardens as permanent institutions holding doc- umented collections of living plants for the purposes of display and for education of the public. Although botanical gardens have focused strongly on plant conservation, a recent transition was noted whereby botanical gardens are valued as sentinel sites to identify pests and pathogen risks in biosecurity research for the early detection and eradication of alien pests, pathogens and plants’ screening prior to authorisation of their release to horticultural markets [7–10]. According to BGCI, one of the typical characteristics of botanical gardens should be the ability for an “Exchange of seed or other materials with other botanic gardens, arboreta or research institutions”. Consequently, botanical gardens have also been key in supporting economic botany during the 17th to 19th centuries, and this entailed the movement and introduction of new economically important plant species across the world [4]. Consistently, during the European expansion and exploration of Asia, South America and Africa, some European botanical gardens were engaged in economic botany and the cultivation of attractive plants [4,11]. The Europeans were actively involved in the collection and study, introduction and acclimatisation, cultivation, propagation and dis- semination of newly discovered and tropical crops to colonial countries [4,12]. 1. Introduction Subsequently, one of the Diversity 2023, 15, 407 3 of 20 3 of 20 strategic responses to the effective management of biological invasions in South Africa has entailed the development and enactment of legislative control via the National Environmental Management: Biodiversity Act (NEM: BA) (Act No. 10 of 2004) and the Alien and Invasive Species Regulations, hereafter called NEM:BA-A&IS Regulations [28], which are complemented by the updated 2021 NEM:BA-A&IS Regulations. It is required that SANBI must submit a report on the status of listed invasive species to the Minister of the Department of Forestry, Fisheries and the Environment (DFFE) every three years. Indeed, Zengeya and Wilson [24] pointed to the patchiness of species information as one of the main factors slowing the effective management of biological invasions in South Africa. The knowledge of alien species’ records in the SANBI gardens constitutes critical indicators for monitoring biological invasions at the national level across South Africa [29,30], which may enhance the effective management of the impacts of such species in SANBI’s gardens. p g Despite the known and increasing negative impacts of biological invasions reported in the two national status reports produced thus far [24,26,30], the effective management of biological invasions in South Africa has been difficult because of several challenges. For instance, there is no overarching national management strategy for guiding different role players in safeguarding the national biodiversity and SANBI’s national gardens from the negative impacts of biological invasions in South Africa. Above all, there is a paucity in the knowledge of the numbers of alien and invasive species, and their associated negative impacts are a nationwide problem that include SANBI’s national gardens. Zengeya et al. [31] raised the issue of low reliability of current existing estimates of the numbers of species in different contexts. In addition, the lack of human capacity and limited resources hampers the success of containing the problem of biological invasions in different contexts, such as within local municipalities [32,33] and SANBI gardens. Although the NEM:BA-A&IS Regulations [28] emphasized the development of management plans for alien species occurring on state and private land, there has generally been limited compliance, partly due to the scarcity of scientific skills in invasion biology [29]. 1. Introduction Similarly, botanical gardens play a major role in supporting the cultivation and distribution of alien plants that are used as ornamentals [12], with most of these alien species listed as the worst invasive species in the world, such as Lantana camara and Acacia mearn- sii [8,13–15]. The World Resources Institute estimated that 150 million persons visited some 1500 botanical gardens around the world in 1989 [16]. Thus, it is also possible that with increased international tourist visitation to botanical gardens in the 17th and 18th centuries, alien species from different parts of the world were accidentally spread to different areas [1,17]. In South Africa, botanical gardens perform a range of diverse functions intertwined with the conservation of flora and fauna, in alignment with international conservation treaties (e.g., the Convention for Biological Diversity), research, environmental educa- tion, horticulture and nature-based tourism [2,18–20]. The 11 botanical gardens of the South African National Biodiversity Institute (SANBI) (i.e., Free State, Harold Porter, Ka- roo Desert, Hantam, Kirstenbosch, KwaZulu-Natal, Kwelera, Lowveld, Pretoria, Walter Sisulu, and Thohoyandou; see [21]) are classified as conservation gardens that, according to the BGCI’s definition (see [2,3]), contain natural vegetation that is a national conser- vation priority (i.e., Critical Biodiversity Areas—CBAs) in addition to their cultivated collections [20–23]. A globally distinguishing attribute of the SANBI gardens is that they conserve representative biodiversity of seven of South Africa’s nine biomes, except the Desert Biome and Indian Ocean Coastal Belt [18,21,22]. In addition, the Pretoria National Zoological Garden preserves a variety of animal species, both native and alien, for public display, and the Mokopane Biodiversity Conservation Centre has overlapping functions with both botanical gardens and zoological gardens [21]. South African National Botanical Gardens are associated with conservation priority biodiversity ar- eas [21,23], and therefore warrant heightened protection from the escalating impacts of biological invasions. g To date, South Africa has recorded approximately 1880 alien species that have established within the country, some of which have become invasive (215 species, as indicated in Zengeya & Wilson [24]), and resulted in severe negative impacts on the recipient environment [23,24]. Biological invasions reduce biodiversity [25,26], which can threaten human well-being, especially for communities that rely on ecosystem goods and services in South Africa [26]. The impacts of biological invasions include the altering of habitat structures, hampering the proper functioning of ecosystems and limiting the availability of essential natural resources [26,27]. 1. Introduction In view of the above arguments, the aims of this study were to: (1) document the diversity of alien and invasive plant species occurring in SANBI’s gardens with the purpose of informing the development of a management strategy; and (2) classify different alien plant species recorded in different gardens based on continental origin, life form and their status as per the NEM:BA-A&IS regulations, to guide prioritisation in resources’ allocation for management interventions. This is the ﬁrst study to start screening and assessing the invasion risk for biodiversity conservation in South Africa’s gardens (see a case study in China: Ni & Hulme [15]). Table 1. SANBI bota / l i d bi 2.2. Data Collection use/proclaimed, biomes and preserved vegetation types. First Date of Current Land SA Province Biome Represented (Biore- i ) ( i h f d Vegetation Types Represented The species data were obtained by requesting lists of alien species for each of the 13 SANBI gardens from the curators and garden estate managers. use/proclaimed, biomes and preserved vegetation types. First Date of Current Land SA Province Biome Represented (Biore- ) ( h f d Vegetation Types Represented The species data were obtained by requesting lists of alien species for each of the 13 SANBI gardens from the curators and garden estate managers. First Date of Current Land use/Proclaimed SA Province (Town) gion) (Mucina & Rutherford, 2006) Vegetation Types Represented (Mucina & Rutherford, 2006) Free State Prov- l f Grassland (Dry Highveld Grassland) Gh 7 Winburg Grassy Shrubland Gh 8 Bloemfontein Karroid Shrub- land Several key sources were used for plant species identiﬁcation, including ﬁeld guides by Bromilow [34,35] and Henderson [36], the Invasive Species South African Database [37] and herbarium specimen collections. Due to limited evidence, some species were identiﬁed only up to the genus level. 1967 ince (Bloemfon- tein) Grassland) Azonal Vegetation (Alluvial Vegetation) land Gh 5 Bloemfontein Dry Grassland AZa 5 Highveld Alluvial Vegeta- y p g Species names were veriﬁed using the national resource: “The Status of Biological Invasions and their Management in South Africa” [24,26]. tion 2008 Northern Cape (Nieuwoudtville) Succulent Karoo (Trans-Es- carpment Succulent Karoo) Fynbos (Shale Renosterveld, and Granite and Dolerite Renosterveld) FRd 1 Nieuwoudtville-Roggeveld Dolerite Renosterveld FRs 2 Nieuwoudtville Shale Renosterveld SKt 2 Hantam Karoo 1959 Western Cape (Betty’s Bay) Fynbos (Sand Fynbos, West- ern Strandveld and Sand- stone Fynbos) Forest (Zonal & Intrazonal) FFd 6 Hangklip Sand Fynbos FFs 11 Kogelberg Sandstone Fyn- bos FOz 1 Southern Afrotemperate For- est FS 7 Overberg Dune Strandveld Freshwater (rivers) Marine biodiversity 1921 Western Cape (Worcester) Succulent Karoo (Rainshadow Valley Karoo) Fynbos (Shale Fynbos and Shale Renosterveld) FFh 4 Breede Shale Fynbos FRs 8 Breede Shale Renosterveld SKv 7 Robertson Karoo Following the criterion described in Mokotjomela et al. [33], plant species were clas- siﬁed by life form as follows: herbs, graminoids, succulents, woody for the shrubs and trees and ferns using the Botanical Database of Southern Africa (BODATSA) [38] and the Integrated Taxonomic Information System (Table A1, [39]). Pyšek et al. 2. Methods and Materials 2.1. Study Area The study was conducted in South Africa in 11 SANBI botanical gardens, 1 zoo- logical garden and the Mokopane Biodiversity Conservation Centre, which are situated in 8 provinces and 7 South African biomes (Figure 1). The SANBI national botan- ical gardens are located in eight of South Africa’s nine provinces, where there are unique vegetation types targeted for conservation (Table 1 [21]). These study sites were selected because they are all managed by SANBI and have a common function of biodiversity conservation. Diversity 2023, 15, 407 Diversity 2023, 15, x FO 4 of 20 4 of 20 Figure 1. The locations of the SANBI botanical and zoological gardens overlayed on the national vegetation map of South Africa (i.e., vegetation layers from Mucina and Rutherford [22]). Figure 1. The locations of the SANBI botanical and zoological gardens overlayed on the national vegetation map of South Africa (i.e., vegetation layers from Mucina and Rutherford [22]). Figure 1. The locations of the SANBI botanical and zoological gardens overlayed on the national vegetation map of South Africa (i.e., vegetation layers from Mucina and Rutherford [22]). Figure 1. The locations of the SANBI botanical and zoological gardens overlayed on the national vegetation map of South Africa (i.e., vegetation layers from Mucina and Rutherford [22]). Table 1. SANBI bota / l i d bi 2.2. Data Collection [40] showed that some species’ traits, especially life form, stature and pollination syndrome, may provide a method of predicting impact, regardless of the habitat and geographical region invaded. In addition, the region of origin is critical for understanding and managing the pathway of introduction for alien species (see also [41]). The data collected for each species included the gardens and their provincial locations where the species was recorded in South Africa, the reason for introduction as a proxy for the pathway pattern, and the continental origin of each species. Since the native range of the species is important for climate matching with the recipient environment during invasion risk assessment [42], we made an assumption that the species were introduced directly to South Africa from their native continent. Inasmuch as the exact country of origin of each species was identiﬁed (after [12,33]), for this study we used the continental and/or broad region to classify the recorded species as specified in the Centre for Agriculture and Bioscience International (CABI) and Global Invasive Species Database. Species were also categorised following the NEM:BA-A&IS Regulations, 2021. Diversity 2023, 15, 407 5 of 20 There are four categories—1a, 1b, 2, and 3—depending on what is permitted and the overall management goal [43,44], and the “Not listed species” (denoted by NL; 33; Tables 2 and A1). Table 1. SANBI botanical and zoological gardens’ features: area, the ﬁrst date of current land use/proclaimed, biomes and preserved vegetation types. Table 1. SANBI bota / l i d bi 2.2. Data Collection National Botanical/Zoological Garden Area (ha) First Date of Current Land Use/Proclaimed SA Province (Town) Biome Represented (Bioregion) (Mucina & Rutherford, 2006) Vegetation Types Represented (Mucina & Rutherford, 2006) Free State NBG 67 1967 Free State Province (Bloemfontein) Grassland (Dry Highveld Grassland) Azonal Vegetation (Alluvial Vegetation) Gh 7 Winburg Grassy Shrubland Gh 8 Bloemfontein Karroid Shrubland Gh 5 Bloemfontein Dry Grassland AZa 5 Highveld Alluvial Vegetation Hantam NBG 6230 2008 Northern Cape (Nieuwoudtville) Succulent Karoo (Trans-Escarpment Succulent Karoo) Fynbos (Shale Renosterveld, and Granite and Dolerite Renosterveld) FRd 1 Nieuwoudtville-Roggeveld Dolerite Renosterveld FRs 2 Nieuwoudtville Shale Renosterveld SKt 2 Hantam Karoo Harold Porter NBG 201 1959 Western Cape (Betty’s Bay) Fynbos (Sand Fynbos, Western Strandveld and Sandstone Fynbos) Forest (Zonal & Intrazonal) FFd 6 Hangklip Sand Fynbos FFs 11 Kogelberg Sandstone Fynbos FOz 1 Southern Afrotemperate Forest FS 7 Overberg Dune Strandveld Freshwater (rivers) Marine biodiversity Karoo Desert NBG 154 1921 Western Cape (Worcester) Succulent Karoo (Rainshadow Valley Karoo) Fynbos (Shale Fynbos and Shale Renosterveld) FFh 4 Breede Shale Fynbos FRs 8 Breede Shale Renosterveld SKv 7 Robertson Karoo Kirstenbosch NBG 199 1913 Western Cape (Cape Town) Fynbos (Granite Fynbos, Sandstone Fynbos, and Shale Fynbos) Forest (Zonal and Intrazonal) FFg 3 Peninsula Granite Fynbos FFh 5 Cape Winelands Shale Fynbos FFs 9 Peninsula Sandstone Fynbos FOz 1 Southern Afrotemperate Forest Freshwater (rivers) KwaZulu-Natal NBG 48 1874/1969 KwaZulu-Natal (Pietermaritzburg) Savanna (Sub-Escarpment Savanna) SVs 4 Ngongoni Veld Freshwater (river) Kwelera NBG 170 2014 Eastern Cape (East London) Forest (Zonal and Intrazonal) Azonal Vegetation (Eastern Strandveld) Albany Thicket AT 9 Albany Coastal Belt FOz 6 Southern Coastal Forest AZs 2 Albany Dune Strandveld AT 12Buffels Thicket Marine biodiversity Lowveld NBG 164 1969 Mpumalanga (Nelspruit) Savanna (Lowveld) SVl 9 Legogote Sour Bushveld SVl 10 Pretoriuskop Sour Bushveld Freshwater (river) Pretoria NBG 70 1958 Gauteng (Pretoria) Savanna (Central Bushveld) SVcb 6 Marikana Thornveld Thohoyandou NBG 89 1986 Limpopo (Thohoyandou) Savanna (Central Bushveld) SVcb21 Soutpansberg Mountain Bushveld Pretoria NZG 80 1899 Gauteng (Pretoria) Savanna (Central Bushveld) SVcb 6 Marikana Thornveld Mokopane Biodiversity Conservation Centre 1398 1979 Limpopo (Mokopane) Savanna (Central Bushveld) SVcd 20 Makhado Sweet Bushveld SVcb 23 Polokwane Plateau Bushveld Walter Sisulu NBG 276 1982 Gauteng (Roode- poort/Mogale City) Savanna (Central Bushveld) Grassland (Mesic Highveld Grassland) SVcb 9 Gold Reef Mountain Bushveld Gm 10 Egoli Granite Grassland Freshwater (river) There are four categories—1a, 1b, 2, and 3—depending on what is permitted and the overall management goal [43,44], and the “Not listed species” (denoted by NL; 33; Tables 2 and A1). 2.3.1. Species and Families 2.3.1. Species and Families From the total records of alien species obtained from different gardens (Table 1), we identiﬁed the gardens that have the highest number of alien species, the dominant plant families and the most common species across the gardens. We also determined if there was an overlap among the gardens in the alien plant species that occur in each. 2.3.2. Species Classiﬁcation: Continental Origin, Life Forms, and NEM:BA-A&IS Categories 2.3.2. Species Classiﬁcation: Continental Origin, Life Forms, and NEM:BA-A&IS Categories To compare the numbers of alien species in different classiﬁcation categories (i.e., life form, NEM:BA-A&IS Regulations’ categories, species’ continental origin and reason for plant species introduction (pathway)), species count data were analysed using a Generalised Linear Models (GLM), with a Poisson error distribution and log link in SPSS software, version 20. The counts of alien plant species were generated from total records obtained from different gardens, and they were speciﬁed as the dependent variable. Different species classiﬁcation categories were treated as predictor variables. Table 1. SANBI bota / l i d bi 2.2. Data Collection Diversity 2023, 15, 407 6 of 20 6 of 20 Table 2. Different categories of alien and invasive species following the NEM:BA-A&IS Regulations, 2021. Category NEM:BA-A&IS Description Category 1(a) Species that must be combatted and are targets for eradication Category 1(b) Species that are control targets and need a national management plan Category 2 Species requiring a permit for restricted activities Category 3 Species that are subject to exemptions “Not Listed” Unlisted species: Alien species that are not listed in the NEM:BA-A&IS Regulations but have been reported as present in natural or semi-natural ecosystems in South Africa or on offshore islands NEM:BA-A&IS Description Depending on the risk assessment outcome of each alien species in South Africa and their differential occurrence and impacts in different provinces (Kumschick et al. [42]), some species tend to have more than one listing category based on the context in which a risk assessment was carried out. To avoid allocating one species into more than one category, the most common category and/or where a species is a priority for management was considered to be the correct category in this study [33]. Additionally, the newly adopted risk assessment framework for South Africa classiﬁed species based on risk assessment for the whole country [42] instead of the contexts in each province as shown for different categories presented in the national status report and the national regulations [26]. 2.3. Data Analyses 3.1. Identiﬁcation of Alien Plant Species and Families In total, there were 380 alien species records from in 13 SANBI gardens, representing 225 unique alien plant species belonging to 73 families. Different life forms included herbs (42.7%; n = 225), woody plants (37.8%), graminoids (10.7%), succulents (6.7%) and ferns (2.2%). The most common species, occurring in more than six gardens, were: Solanum mauritianum (11 gardens), Lantana camara (10), Melia azedarach (10) and Acacia mearnsii, Jacaranda mimosifolia, Opuntia ﬁcus-indica and Ricinus communis (6 each). Among the reported plant species, a signiﬁcant number (49%; n = 225) were introduced through horticultural trade as ornamental plant species (Wald χ2 = 27.3; df = 6; p < 0.001), followed by plant species used for human consumption—medicine and food (Wald χ2 = 5.2; df = 1; p = 0.023) being more dominant. Diversity 2023, 15, 407 7 of 20 nt 3) The most represented families in the records were Fabaceae (29 species) (mainly Acacia), Asteraceae (24 species), Poaceae (23 species), Solanaceae (14 species) (mainly Solanum), Myrtaceae (11 species) (mainly Eucalyptus) and Cactaceae (11 species) (mainly Opuntia). Walter Sisulu NBG had the highest number of alien species records (88 species), followed by Kirstenbosch NBG (61 species) and Pretoria NBG (46 species) (Figure 2). The most represented families in the records were Fabaceae (29 species) (mainly Aca- , Asteraceae (24 species), Poaceae (23 species), Solanaceae (14 species) (mainly Sola- m), Myrtaceae (11 species) (mainly Eucalyptus) and Cactaceae (11 species) (mainly untia). Walter Sisulu NBG had the highest number of alien species records (88 species), fol- wed by Kirstenbosch NBG (61 species) and Pretoria NBG (46 species) (Figure 2). The most represented families in the records were Fabaceae (29 species) (mainly Acacia), Asteraceae (24 species), Poaceae (23 species), Solanaceae (14 species) (mainly Solanum), Myrtaceae (11 species) (mainly Eucalyptus) and Cactaceae (11 species) (mainly Opuntia). Walter Sisulu NBG had the highest number of alien species records (88 species), followed by Kirstenbosch NBG (61 species) and Pretoria NBG (46 species) (Figure 2). The most represented families in the records were Fabaceae (29 species) (mainly Aca- Asteraceae (24 species), Poaceae (23 species), Solanaceae (14 species) (mainly Sola- m), Myrtaceae (11 species) (mainly Eucalyptus) and Cactaceae (11 species) (mainly ntia). Walter Sisulu NBG had the highest number of alien species records (88 species), fol- ed by Kirstenbosch NBG (61 species) and Pretoria NBG (46 species) (Figure 2). 3.1. Identiﬁcation of Alien Plant Species and Families y p y yp p y p Walter Sisulu NBG had the highest number of alien species records (88 species), followed by Kirstenbosch NBG (61 species) and Pretoria NBG (46 species) (Figure 2). Opuntia). Walter Sisulu NBG had the highest number of alien species records (88 species), fol- lowed by Kirstenbosch NBG (61 species) and Pretoria NBG (46 species) (Figure 2). Figure 2. The number of alien plant species recorded in SANBI gardens. 3.2. Comparing Continental Origin of Different Alien Species, Life Forms and NEM:BA-A&IS Regulations’ Categories There was a statistically significant relationship between the number of alien species recorded across SANBI gardens and different continental regions of the world (Pearson χ2 = 199.2; df = 48; p < 0.001; Figure 3). Overall, most plant species were from the North Figure 2. The number of alien plant species recorded in SANBI gardens. 3.2. Comparing Continental Origin of Different Alien Species, Life Forms and NEM:BA-A&IS Regulations’ Categories There was a statistically signiﬁcant relationship between the number of alien species recorded across SANBI gardens and different continental regions of the world (Pearson χ2 = 199.2; df = 48; p < 0.001; Figure 3). Overall, most plant species were from the North and re 2. The number of alien plant species recorded in SANBI gardens. Figure 2. The number of alien plant species recorded in SANBI gardens. re 2. The number of alien plant species recorded in SANBI gardens. Figure 2. The number of alien plant species recorded in SANBI gardens. Comparing Continental Origin of Different Alien Species, Life Forms and NEM:BA-A&IS lations’ Categories 3.2. Comparing Continental Origin of Different Alien Species, Life Forms and NEM:BA-A&IS Regulations’ Categories g There was a statistically significant relationship between the number of alien species orded across SANBI gardens and different continental regions of the world (Pearson 199.2; df = 48; p < 0.001; Figure 3). Overall, most plant species were from the North South Americas together (50 + 66 = 106 species). A significantly greater number of cies were introduced from South America than from Asia (Wald χ2 = 7.5; df = 1; p = 6) and Australia (Wald χ2 = 49.9; df = 1; p < 0.001). 3.1. Identiﬁcation of Alien Plant Species and Families There was significantly greater number of woody species than succulents (Wald χ2 = 7.9; df = 1; p = .005) and graminoids (Wald χ2 = 3.8; df = 1; p = 0.052). However, the number of woody pecies was not significantly different than the number of herbs (Wald χ2 = 2.1; df = 1; p = .258), and ferns (Wald χ2 = 1.3; df = 1; p = 0.258). It was noteworthy that the herbaceous pecies accounted for the largest number of species (i.e., 40 out of 88 species) in Walter isulu NBG (Figure 4; Table A1). There was a statistically signiﬁcant relationship between the number of alien species recorded in SANBI gardens and life forms (Pearson χ2 = 353.5; df = 60; p < 0.001; Figure 4; Table A1). Herbaceous and woody alien species were dominant in the records. There was a signiﬁcantly greater number of woody species than succulents (Wald χ2 = 7.9; df = 1; p = 0.005) and graminoids (Wald χ2 = 3.8; df = 1; p = 0.052). However, the number of woody species was not signiﬁcantly different than the number of herbs (Wald χ2 = 2.1; df = 1; p = 0.258), and ferns (Wald χ2 = 1.3; df = 1; p = 0.258). It was noteworthy that the herbaceous species accounted for the largest number of species (i.e., 40 out of 88 species) in Walter Sisulu NBG (Figure 4; Table A1). There was a statistically significant relationship between the number of alien species recorded in SANBI gardens and life forms (Pearson χ2 = 353.5; df = 60; p < 0.001; Figure 4; Table A1). Herbaceous and woody alien species were dominant in the records. There was a significantly greater number of woody species than succulents (Wald χ2 = 7.9; df = 1; p = 0.005) and graminoids (Wald χ2 = 3.8; df = 1; p = 0.052). However, the number of woody species was not significantly different than the number of herbs (Wald χ2 = 2.1; df = 1; p = 0.258), and ferns (Wald χ2 = 1.3; df = 1; p = 0.258). It was noteworthy that the herbaceous species accounted for the largest number of species (i.e., 40 out of 88 species) in Walter Sisulu NBG (Figure 4; Table A1). Figure 4. 3.1. Identiﬁcation of Alien Plant Species and Families However, the number of species m South America was not significantly different from the number of species from Eu- e (Wald χ2 = 0.1; df = 1; p = 0.776) and North America (Wald χ2 = 1.0; df = 1; p = 0.326). There was a statistically signiﬁcant relationship between the number of alien species recorded across SANBI gardens and different continental regions of the world (Pearson χ2 = 199.2; df = 48; p < 0.001; Figure 3). Overall, most plant species were from the North and South Americas together (50 + 66 = 106 species). A signiﬁcantly greater number of species were introduced from South America than from Asia (Wald χ2 = 7.5; df = 1; p = 0.006) and Australia (Wald χ2 = 49.9; df = 1; p < 0.001). However, the number of species from South America was not signiﬁcantly different from the number of species from Europe (Wald χ2 = 0.1; df = 1; p = 0.776) and North America (Wald χ2 = 1.0; df = 1; p = 0.326). 8 of 20 8 of 20 Diversity 2023, 15, 407 Figure 3. Number of records of different alien species from different continental space (%; n = 380; (A)) and their occurrence in different SANBI gardens (B). Figure 3. Number of records of different alien species from different continental space (%; n = 380; (A)) and their occurrence in different SANBI gardens (B). Figure 3. Number of records of different alien species from different continental space (%; n = 380; (A)) and their occurrence in different SANBI gardens (B). h ll f l h b h b f l igure 3. Number of records of different alien species from different continental space (%; n = 380; A)) and their occurrence in different SANBI gardens (B). Figure 3. Number of records of different alien species from different continental space (%; n = 380; (A)) and their occurrence in different SANBI gardens (B). Figure 3. Number of records of different alien species from different continental space (%; n = 380; (A)) and their occurrence in different SANBI gardens (B). There was a statistically significant relationship between the number of alien species ecorded in SANBI gardens and life forms (Pearson χ2 = 353.5; df = 60; p < 0.001; Figure 4; able A1). Herbaceous and woody alien species were dominant in the records. 3.1. Identiﬁcation of Alien Plant Species and Families Number of records of different alien plant species in each life form (%; n = 380; (A)) and their occurrence in different SANBI gardens (B). Figure 4. Number of records of different alien plant species in each life form (%; n = 380; (A)) and their occurrence in different SANBI gardens (B). Diversity 2023, 15, 407 9 of 20 and 9 of 20 and Finally, there were no statistically signiﬁcant differences between the number of alien species listed in different NEM:BA-A&IS Regulations’ categories and the number of unlisted species (χ2 = 8.9; df = 4; p = 0.317: 58.2% vs 42.8%). We noted signiﬁcant differences in the number of alien species representing each NEM:BA-A&IS Regulations’ category in three gardens; namely, Walter Sisulu NBG, Kirstenbosch NBG and Pretoria NBG (Pearson χ2 = 151.0; df = 48; p < 0.001; Figure 5). Walter Sisulu NBG had the highest number of unlisted (38 species) species and category 1b species (39), followed by Kirstenbosch NBG (32, 24) and Pretoria NBG (25, 18), respectively. Finally, there were no statistically significant differences between the number of alien pecies listed in different NEM:BA-A&IS Regulations’ categories and the number of un- sted species (χ2 = 8.9; df = 4; p = 0.317: 58.2% vs 42.8%). We noted significant differences n the number of alien species representing each NEM:BA-A&IS Regulations’ category in hree gardens; namely, Walter Sisulu NBG, Kirstenbosch NBG and Pretoria NBG (Pearson 2 = 151.0; df = 48; p < 0.001; Figure 5). Walter Sisulu NBG had the highest number of nlisted (38 species) species and category 1b species (39), followed by Kirstenbosch NBG 32, 24) and Pretoria NBG (25, 18), respectively. Figure 5. Numbers of alien species’ records (%; n = 380) in different NEM:BA-AIS Regulations’ cat- egories for species (A) and their occurrence in different SANBI Gardens (B). Cat. = Category. Cate- gory 1a: Species that are targets for eradication. Category 1b: Species that are control targets and need a national management plan. Category 2: Species requiring a permit for restricted activities. Category 3: Species that are subject to exemptions. “Not Listed” (NL): Unlisted species: alien species that are not listed in the NEM:BA-A&IS Regulations but have been reported as present in natural or semi-natural ecosystems in South Africa or on offshore islands. Figure 5. . Discussions 4. Discussions We recorded a total of 225 alien plant species occurring in SANBI botanical gardens, nd this can be partly attributed to deliberate introductions of species to these gardens for conomic botany and ornamental and conservation research purposes [12,41,45]. Deliber- te introduction of alien species for biosecurity screening in different parts of the world was previously reported [10,15,16,41], while the accidental introduction of some species s contaminants is common in many areas, including South Africa [12,46]. In the SANBI ardens, we found a large number of plant species (49%; n = 225) that were introduced hrough horticultural trade as ornamentals [intentional introductions, 41] in South Africa nd that evidently escaped [34]. Unintentional introductions of alien species could also be ccelerated by the fact that botanical gardens attract large numbers of visitors from differ- nt parts of the world performing different activities, including grilling meat with fire- wood from unknown sources [16], and this may account for the proportion of our records hat had no specific known use (10.7 %, n = 225) in South Africa. We also suggest that, in We recorded a total of 225 alien plant species occurring in SANBI botanical gardens, and this can be partly attributed to deliberate introductions of species to these gardens for economic botany and ornamental and conservation research purposes [12,41,45]. Deliberate introduction of alien species for biosecurity screening in different parts of the world was previously reported [10,15,16,41], while the accidental introduction of some species as contaminants is common in many areas, including South Africa [12,46]. In the SANBI gardens, we found a large number of plant species (49%; n = 225) that were introduced through horticultural trade as ornamentals [intentional introductions, 41] in South Africa and that evidently escaped [34]. Unintentional introductions of alien species could also be accelerated by the fact that botanical gardens attract large numbers of visitors from different parts of the world performing different activities, including grilling meat with ﬁrewood from unknown sources [16], and this may account for the proportion of our records that had no speciﬁc known use (10.7 %, n = 225) in South Africa. We also suggest that, in part, the large diversity of alien plant species reported in this study is a likely result of their diverse uses [10,33,46] in the multi-racial South African society [46–48]. 3.1. Identiﬁcation of Alien Plant Species and Families Numbers of alien species’ records (%; n = 380) in different NEM:BA-AIS Regulations’ categories for species (A) and their occurrence in different SANBI Gardens (B). Cat. = Category. Category 1a: Species that are targets for eradication. Category 1b: Species that are control targets and need a national management plan. Category 2: Species requiring a permit for restricted activities. Category 3: Species that are subject to exemptions. “Not Listed” (NL): Unlisted species: alien species that are not listed in the NEM:BA-A&IS Regulations but have been reported as present in natural or semi-natural ecosystems in South Africa or on offshore islands. igure 5. Numbers of alien species’ records (%; n = 380) in different NEM:BA-AIS Regulations’ cat- gories for species (A) and their occurrence in different SANBI Gardens (B). Cat. = Category. Cate- ory 1a: Species that are targets for eradication. Category 1b: Species that are control targets and need a national management plan. Category 2: Species requiring a permit for restricted activities. Category 3: Species that are subject to exemptions. “Not Listed” (NL): Unlisted species: alien species hat are not listed in the NEM:BA-A&IS Regulations but have been reported as present in natural or emi-natural ecosystems in South Africa or on offshore islands. Figure 5. Numbers of alien species’ records (%; n = 380) in different NEM:BA-AIS Regulations’ categories for species (A) and their occurrence in different SANBI Gardens (B). Cat. = Category. Category 1a: Species that are targets for eradication. Category 1b: Species that are control targets and need a national management plan. Category 2: Species requiring a permit for restricted activities. Category 3: Species that are subject to exemptions. “Not Listed” (NL): Unlisted species: alien species that are not listed in the NEM:BA-A&IS Regulations but have been reported as present in natural or semi-natural ecosystems in South Africa or on offshore islands. . Discussions 4. Discussions y Other alien species are likely to have invaded the gardens by natural spread, such as through the dispersal of ﬂeshy-fruited species by birds [48–51], roadways connecting Diversity 2023, 15, 407 10 of 20 10 of 20 the gardens with different potential alien propagule sources, such as urban home gar- dens [33,52,53] and river systems that traverse most of the gardens [20,54]. Indeed, van Kleunen et al. [12] have shown that horticultural alien species tend to have spread more than many other alien species, and that they naturalise much better, which is essential for invasion [55]. Apart from this, many SANBI gardens are situated near urbanised areas, thus making them vulnerable to high alien plant propagule pressure that promotes invasion [56]. The fragmentation of habitats, especially in urban areas, shifts the ecological balance away from native species and towards favouring the human-associated alien species [45,57]. However, since SANBI gardens are protected and experience low biophysical disturbance (e.g., [20,21]), they may have some natural resistance to invasion by alien species [58]. ( g ) y y y p It has been shown that over 100 alien species have attained invasive status, with considerable direct and indirect negative impacts on the rich biotas of South Africa [59]. The fact that we recorded some of the most abundant and damaging alien woody plant species (i.e., Lantana camara and Solanum mauritianum; [26]) and Melia azedarach in the majority of the SANBI gardens (10–11) points to the possibility of undocumented impacts of these species. Above all, L. camara and Acacia mearnsii are among the worst alien invasive species in the world [13], and were recorded in some of the SANBI gardens. Additionally, the negative impacts of woody alien species (i.e., including trees and shrubs) have been documented [24,26], as well as their increasing numbers in South Africa [25,60]. In South Africa, the local biodiversity is threatened by, among others, the 141 Australian Acacia species (wattles), of which 13 are highly invasive and growing proliﬁcally both in cultivation and outside [44,61]. While M. azedarach does not have major or severe known negative impacts in South Africa [24], we argue that its abundance suggests a possible effective long-distance dispersal mediated by local vertebrates which could compromise the dispersal of native species [62]. A plausible explanation of the ﬁnding is an absence of dedicated management plans (limited compliance) and limited capacity to implement control measures [20,33]. . Discussions 4. Discussions Since the impacts of alien species have not been investigated in SANBI gardens, this study provides bases for the urgent planning and prioritisation of efforts to manage the affected gardens. g g Knowledge of the life forms and life cycles of different alien plant species and their native range can guide the prediction of invasion risk and support invasion scenario planning for management [40,63]. Our ﬁnding of a large number of herbaceous and woody species in the SANBI gardens is possibly due to the predominance of agricultural introductions for fodder in the neighbourhood farms, horticultural elements [46] and the use of graminoids for biofuel production in neighbouring farms [64]. Alien herbs and grasses are notorious in the farming sector, where they outcompete and reduce the numbers of palatable species in the pastures [65]. Nevertheless, there has been limited research on the negative impacts of herbs and grasses in South Africa [64], and speciﬁcally on the preserved vegetation in SANBI gardens as a unique land-use type (see [20]). We suggest that Walter Sisulu NBG may have the highest number of herbaceous species and other life forms because of high propagule pressure created by a water stream emerging from a catchment embedded in urbanised human settlements [66,67]. Although we could not distinguish between the intentional and unintentional species introductions, we also speculate that, in part, a high inﬂux of international tourists in Gauteng (to Walter Sisulu & Pretoria NBGs) and the Western Cape (Kirstenbosch NBG) provinces, particularly from the Americas and Europe, are some of the unintentional sources of most species in this study. Above all, these gardens are located in the economic hubs of South Africa where there are numerous activities that facilitate alien species’ spread [33], as well as a large local human population, as asserted in Pyšek et al. [68]. Biological invasions are reported to be one of the major drivers of ecological degra- dation in the grassland and fynbos biomes of South Africa [45,69,70], and, consistently, the gardens located in the fynbos and grassland biomes had a high number of alien species [20]. Most SANBI gardens have either wetlands or river systems, and indeed we recorded some of the species that have been speciﬁed as common in such environments by Diversity 2023, 15, 407 11 of 20 11 of 20 Richardson et al. [45]. . Discussions 4. Discussions Although Prosopis glandulosa is common in arid areas [45], it was not reported as present in the arid Hantum and Karoo National Botanical Garden, possibly due to effective garden management in place. In general, we recorded many similar species to those identiﬁed in different main habitats by Richardson et al. [45], and even some alien plant species that were not known in the national regulations. Consequently, we assert that systematic sampling of alien species in SANBI gardens will be critical to improving the list we presented in this study. p y While the abundance of the recorded species was not measured, the management of the existing populations is important to mitigate negative impacts. Listing the alien species in the national regulations facilitates understanding the species’ impacts and their management needs [33,42,63], and thus prioritisation of limited resources. Alien species categorised as “1b” in the national regulations are targets for containment and, consequently, their occurrence in large numbers in SANBI gardens is possibly due to the absence of management actions. It is also possible that a large number (41.8%; n = 225) of the species were not listed in the national regulations due to limited capacity in performing the alien taxa risk assessment and proﬁling [see the framework, 42]. A large number of unlisted alien plant species corroborates the recent reports that there are many alien species that have not been documented [12,24,26,33,63], with actual numbers of invasive species increasing in South Africa [25]. In addition, this ﬁnding highlights the absence or limited scientiﬁc research needed to support the science-based management of biological invasions which could partly thwart management success in South Africa [33]. The results of this study are key to improving the development and implementation of integrated management plans to protect the integrity and sustainable conservation of the gardens’ ecosystems from future impacts on botanical gardens as an important conservation strategy for the world’s ﬂora [2]. On the other hand, the horticultural plant propagations in botanical gardens can be pivotal for vegetation rehabilitation/restoration through reintroducing native species in degraded landscapes [2,4,70–72]). 5. Concluding Remarks Mokotjomelaand), L.R.V. and C.B. veriﬁed the alien species’ records, statistically analysed ﬁeld data and wrote the paper. C.K.W., T.M.M. (Thompson M. Mutshinyalo) and C.B. commented on the MS. C.B. provided reviews of the study. All authors have read and agreed to the published version of the manuscript. Funding: Funding support for this study was provided by the South African National Biodiversity Institute (SANBI). The South African Department of Forestry, Fisheries and the Environment (DFFE) are also thanked for partial funding, noting that this publication does not necessarily represent the views or opinions of DFFE or its employees. Institution Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Data are available on request. Acknowledgments: Different SANBI gardens’ managers provided valuable information on the state of management of biological invasions. Anish Dayaram provided information on the biomes of South Africa. Mbali Mkhize and Hannelie Snyman provided important species’ information from the South African Plant Invader Atlas database and Herbarium records. Conﬂicts of Interest: The authors declare that there are no conﬂict of interest. Appendix A Table A1. List of alien plant taxa recorded in different SANBI botanical and zoological gardens (follow- ing SANBI 2016: BODATSA 2016, and it is: https://www.itis.gov/servlet/SingleRpt/SingleRpt#null, accessed on 15 February 2023). Each plant species was classiﬁed according to family, life form, NEM:BA-AIS Regulations’ categories and invasion status deﬁned by Blackburn et al. [73]. Family Genus, Species and Lower Taxa Life Form NEM:BA-AIS Regs. Cat Alien Status [73] Adoxaceae Viburnum tinus L. Woody NL - Alismataceae Sagittaria latifolia Willd. Herb NL Invasive Amaranthaceae Amaranthus hybridus L. Herb NL Invasive Atriplex lindleyi F.Muell. Herb 1b Invasive Chenopodium album L. Herb NL Invasive Dysphania sect. botryoides (L.) Mosyakin & Clemants Herb NL - Salsola kali L. Herb 1b Invasive Anacardiaceae Schinus molle L. Woody NL Invasive Apiaceae Foeniculum vulgare A.W.Hill Herb NL Invasive Cyclospermum leptophyllum (Pers.) Herb NL - Apocynaceae Araujia sericifera Brot. Herb 1b Invasive Thevetia peruviana (Pers.) K. Schum. Woody 1b Invasive Vinca major L. Herb 1b Invasive Aristolochiaceae Aristolochia elegans Mast. Woody 1b Invasive Asparagaceae Agave americana L. var. americana Succulent NL Invasive Agave sisalana Perrine Succulent 2 Invasive Furcraea foetida L. Succulent 1a Invasive Yucca sp. Succulent NL - improving the management of negative impacts of the biological invasions in the preserved biodiversity as a natural asset in SANBI gardens. Data Availability Statement: Data are available on request. Acknowledgments: Different SANBI gardens’ managers provided valuable information on the state of management of biological invasions. Anish Dayaram provided information on the biomes of South Africa. Mbali Mkhize and Hannelie Snyman provided important species’ information from the South African Plant Invader Atlas database and Herbarium records. Conﬂicts of Interest: The authors declare that there are no conﬂict of interest. 5. Concluding Remarks improving the management of negative impacts of the biological invasions in the preserved biodiversity as a natural asset in SANBI gardens. Author Contributions: T.M.M. (Thabiso M. Mokotjomelaand), S.J.R. and L.V.M. conceptualized the study during the development of the strategy for management of biological invasions in SANBI Botanical and Zoological Gardens and collected ﬁeld data for situational analysis. T.M.M. (Thabiso M. Mokotjomelaand), L.R.V. and C.B. veriﬁed the alien species’ records, statistically analysed ﬁeld data and wrote the paper. C.K.W., T.M.M. (Thompson M. Mutshinyalo) and C.B. commented on the MS. C.B. provided reviews of the study. All authors have read and agreed to the published version of the manuscript. Funding: Funding support for this study was provided by the South African National Biodiversity Institute (SANBI). The South African Department of Forestry, Fisheries and the Environment (DFFE) are also thanked for partial funding, noting that this publication does not necessarily represent the views or opinions of DFFE or its employees. Institution Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Data are available on request. Data Availability Statement: Data are available on request. 5. Concluding Remarks In this study, we have presented a list of alien and invasive plant species occurring in SANBI gardens located in different parts of South Africa, which may guide the safeguarding of biodiversity conservation gardens from the threats posed by biological invasions [2]. Indeed, compiling species lists and regular monitoring of high-risk sites, including the botanical gardens, can strengthen their management if public awareness campaigns are conducted timeously and the escape of various alien species from different points of introduction is reduced. We also recognise the complexity of the relationship between human socio-economic needs and alien species, and thus advocate for increased awareness of negative impacts as a potential strategy for mitigation. The ﬁndings in this study constitute progress toward reducing the reported uncertainty of the existing alien species data sets that restrict planning management of biological invasions in South Africa [63]. While this is the ﬁrst study to collate this information, it is apparent that even the invasion status of unlisted species and potential negative impacts have not been investi- gated in SANBI gardens; a gap that Foxcroft et al. [32] identiﬁed as a major obstacle to effective science-based management of biological invasions in South Africa. The poten- tially impactful species populations will need to undergo clearing that is coupled with restoration using native plant species [72]. The ﬁnding that many plant species were not listed in the national regulations highlights the important knowledge gap in the negative impacts of the alien species in question and/or capacity constraints for compilation of the risk assessment. Further systematic surveys for alien and invasive species are required to improve the knowledge of invasion risk in biodiversity conservation gardens around the world. Considering the prominence of the role of botanical gardens in the dissemination of alien plants, the eight key research questions listed by van Kleunen et al. [12] should be explored in SANBI botanical gardens as a way of improving the current data sets and Diversity 2023, 15, 407 12 of 20 12 of 20 Diversity 2023, 15, 407 12 of 20 improving the management of negative impacts of the biological invasions in the preserved biodiversity as a natural asset in SANBI gardens. Author Contributions: T.M.M. (Thabiso M. Mokotjomelaand), S.J.R. and L.V.M. conceptualized the study during the development of the strategy for management of biological invasions in SANBI Botanical and Zoological Gardens and collected ﬁeld data for situational analysis. T.M.M. (Thabiso M. Appendix A Table A1. List of alien plant taxa recorded in different SANBI botanical and zoological gardens (follow- ing SANBI 2016: BODATSA 2016, and it is: https://www.itis.gov/servlet/SingleRpt/SingleRpt#null, accessed on 15 February 2023). Each plant species was classiﬁed according to family, life form, NEM:BA-AIS Regulations’ categories and invasion status deﬁned by Blackburn et al. [73]. Family Genus, Species and Lower Taxa Life Form NEM:BA-AIS Regs. Cat Alien Status [73] Adoxaceae Viburnum tinus L. Woody NL - Alismataceae Sagittaria latifolia Willd. Herb NL Invasive Amaranthaceae Amaranthus hybridus L. Herb NL Invasive Atriplex lindleyi F.Muell. Herb 1b Invasive Chenopodium album L. Herb NL Invasive Dysphania sect. botryoides (L.) Mosyakin & Clemants Herb NL - Salsola kali L. Herb 1b Invasive Anacardiaceae Schinus molle L. Woody NL Invasive Apiaceae Foeniculum vulgare A.W.Hill Herb NL Invasive Cyclospermum leptophyllum (Pers.) Herb NL - Apocynaceae Araujia sericifera Brot. Herb 1b Invasive Thevetia peruviana (Pers.) K. Schum. Woody 1b Invasive Vinca major L. Herb 1b Invasive Aristolochiaceae Aristolochia elegans Mast. Woody 1b Invasive Asparagaceae Agave americana L. var. americana Succulent NL Invasive Agave sisalana Perrine Succulent 2 Invasive Furcraea foetida L. Succulent 1a Invasive Yucca sp. Succulent NL - Diversity 2023, 15, 407 13 of 20 Table A1. Cont. Table A1. Cont. Family Genus, Species and Lower Taxa Life Form NEM:BA-AIS Regs. Cat Alien Status [73] Asteraceae Ageratum conyzoides (Mill.) M.Sharma Herb 1b Invasive Ageratina adenophora (Spreng.) R.M.King & H.Rob. Herb 1b Invasive Bidens bipinnata L. Herb NL Invasive Bidens pilosa L. Herb NL Invasive Campuloclinium macrocephalum (Less.) DC. Herb 1b Invasive Chromolaena odorata (L.) R.M.King & H.Rob. Herb 1b Invasive Cirsium vulgare (Savi) Ten. Herb 1b Invasive Conyza sumatrensis (Retz.) E.Walker Herb NL Invasive Cosmos bipinnatus Cav. Herb NL Invasive Flaveria bidentis (L) Kuntze Herb 1b Invasive Galinsoga ciliata (Raf.) Blake Herb NL - Hypochaeris microcephala (Sch.Bip.) Cabrera Herb NL - Hypochaeris radicata L. Herb NL Invasive Lactuca indica L. Herb NL - Schkuhria pinnata (Lam.) Kuntze ex Thell. Herb NL Invasive Sonchus oleraceus L. Herb NL Invasive Sphagneticola trilobata (L.) Pruski Herb 1b Invasive Tagetes minuta L. Herb NL Invasive Tithonia diversifolia (Hemsl.) A.Gray Herb 1b Invasive Tithonia rotundifolia S.F.Blake (Mill.) Herb 1b Invasive Zinnia peruviana L. Herb NL Invasive Basellaceae Anredera cordifolia (Ten.) Steenis Herb 1b Invasive Bignoniaceae Dolichandra unguis-cati L. (A.Gentry) Woody 1b Invasive Jacaranda mimosifolia D.Don Woody 1b Invasive Tecoma stans (L.) Juss. ex Kunth Woody 1b Invasive Boraginaceae Amsinckia menziesii var. Appendix A retrorsa (Lehm.) A.Nelson & J.F.Macbr. Herb NL Invasive Echium plantagineum L. Herb 1b Invasive Heliotropium amplexicaule Vahl Herb NL Invasive Heliotropium europaeum L. Herb NL Invasive Brassicaceae Brassica juncea (L.) Czern. Herb NL - Capsella bursa-pastoris (L.) Medik. Herb NL Introduced but not naturalized Nasturtium ofﬁcinale R.Br. Herb 2 Invasive Raphanus raphanistrum L. Herb NL Invasive Cactaceae Cereus jamacaru DC. Succulent 1b Invasive Cylindropuntia imbricata (Haw.) F.M.Knuth Succulent 1b Invasive Trichocereus spachianus(Lem.) Riccob Succulent 1b Invasive Opuntia aurantiaca Lindl. Succulent 1b Invasive Opuntia engelmannii Salm-Dyck ex Engelm. Succulent 1b Invasive Opuntia ﬁcus-indica (L.) Mill. Succulent 1b Invasive Opuntia leucotricha DC. Succulent 1b Invasive Opuntia microdasys (Lehm.) Pfeiff. Succulent 1b Invasive Opuntia pubescens J.C.Wendl. ex Pfeiff. Succulent 1a Introduced but not naturalized Diversity 2023, 15, 407 14 of 20 Table A1. Cont. Table A1. Cont. Family Genus, Species and Lower Taxa Life Form NEM:BA-AIS Regs. Cat Alien Status [73] Cannabaceae Celtis australis L. Woody 3 Introduced but not naturalized Cannaceae Canna indica L. Herb 1b Invasive Caprifoliaceae Centranthus ruber (L.) DC. Herb 1b Invasive Lonicera japonica Thunb.’Halliana’ Woody 3 Invasive Caryophyllaceae Silene gallica L. Herb NL - Cistaceae Cistus ladanifer L. Woody NL Invasive Commelinaceae Tradescantia ﬂuminensis Vell. Herb 1b Invasive Convolvulaceae Convolvulus arvensis L. Herb 1b Introduced but not naturalized Cuscuta campestris Yunck. Herb 1b Invasive Ipomoea purpurea (L.) Roth Herb 1b Invasive Crassulaceae Bryophyllum delagoense (Eckl. & Zeyh.) Schinz Succulent 1b Invasive Cucurbitaceae Diplocyclos palmatus L. Woody 1a Invasive Cyatheaceae Sphaeropteris excelsa (Endl.) R.M.Tryon Fern NL Invasive Cyperaceae Cyperus eragrostis Lam. Graminoids NL - Euphorbiaceae Euphorbia heterophylla L. Herb NL - Euphorbia peplus L. Herb NL - Homalanthus populifolius Graham. Woody 1b Invasive Mercurialis annua L. Herb NL - Ricinus communis L. Woody 2 Invasive Fabaceae Acacia cyclops A.Cunn. ex G.Don Woody 1b Invasive Acacia dealbata Link Woody 2 Invasive Acacia elata A.Cunn. ex Benth. Woody 1b Invasive Acacia longifolia (Andrews) Willd. Woody 1b Invasive Acacia mearnsii De Wild. Woody 2 Invasive Acacia melanoxylon R.Br. Woody 2 Invasive Acacia podalyriifolia A.Cunn. ex G.Don Woody 1b Invasive Acacia saligna (Labill.) H.L.Wendl. Woody 1b Invasive Caesalpinia decapetala (Roth) Alston Woody 1b Invasive Caesalpinia gilliesii Wall. ex. Hook. Woody 1b Invasive Crotalaria agatiﬂora Schweinf. Woody 1b Invasive Cytisus palmensis (Christ) Hutch. Woody NL - Desmodium sp. Woody NL - Gleditsia triacanthos L. Woody 1b Invasive Medicago lupulina L. Herb NL - Medicago polymorpha L. var. Appendix A brevispina (Benth.) Heyn Herb NL - Paraserianthes lophantha (Willd.) I.C.Nielsen Woody 1b Invasive Prosopis glandulosa var. torreyana (L.D.Benson) M.C.Johnst. Woody 1b Invasive Robinia pseudoacacia L. Woody 1b Invasive Senna bicapsularis (L.) Roxb. Woody 1b Invasive Senna septemtrionalis (Viv.) H.S.Irwin & Barneby Woody 1b Invasive Senna sp. Woody 1b - Sesbania bispinosa (Jacq.) W.Wight Woody NL - Diversity 2023, 15, 407 15 of 20 Table A1. Cont. Table A1. Cont. Family Genus, Species and Lower Taxa Life Form NEM:BA-AIS Regs. Cat Alien Status [73] Fabaceae Sesbania punicea (Cav.) Benth. Woody 1b Invasive Spartium junceum L. Woody 1b Invasive Tipuana tipu (Benth.) Kuntze Woody 3 Invasive Vicia atropurpurea L. Herb NL - Vicia sativa L. Herb NL - Fagaceae Quercus robur L. Woody NL Invasive Hypericaceae Hypericum canariense L. Woody NL - Iridaceae Iris pseudacorus L. Herb 1a Invasive Sisyrynchium sp. Herb NL - Juncaceae Juncus bufonius L. aggregate Herb NL - Lamiaceae Salvia tiliifolia Vahl. Herb 1b Invasive Lauraceae Cinnamomum camphora (L.) J.Presl Woody 1b Invasive Liliaceae Lilium formosanum Wallace Herb 1a Invasive Malvaceae Hibiscus trionum L. Herb NL Invasive Meliaceae Melia azedarach L. Woody 1b Invasive Moraceae Morus alba L. Woody 3 Invasive Morus nigra L. Woody NL - Myrtaceae Eucalyptus camaldulensis Dehnh. Woody 1b Invasive Eucalyptus grandis W.Hill ex Maiden Woody 1b Invasive Eucalyptus paniculata Sm. Woody NL Introduced but not naturalized Eucalyptus saligna Sm. Woody NL - Leptospermum laevigatum (Gaertn.) F.Muell. Woody 1b Invasive Callistemon rigidus R.Br.. Woody 1b Invasive Metrosideros excelsa Sol. ex Gaertn. Woody 1a Invasive Myrtus communis L. Woody NL - Psidium guajava L. Woody 2 Invasive Syzygium paniculatum Gaertn. Woody NL Invasive Nyctaginaceae Mirabilis jalapa L. Herb 1b Invasive Nymphaeaceae Nymphaea mexicana Zucc Herb 1b Invasive Oleaceae Ligustrum japonicum Thun. Woody 1b Invasive Ligustrum lucidum W.T. Aiton Woody 1b Invasive Ligustrum vulgare L. Woody 1b Invasive Syringa vulgaris L. Woody NL - Onagraceae Oenothera rosea L’Herit. ex Aiton Herb NL Invasive Oenothera stricta Ledeb. ex Link Herb NL Invasive Oenothera tetraptera Cav. Herb NL Introduced but not naturalized Oxalidaceae Oxalis corniculata L. Herb NL Invasive Oxalis latifolia Kunth Herb NL Invasive Papaveraceae Argemone ochroleuca Sweet Herb 1b Invasive Fumaria muralis Sond. ex Koch Herb NL Introduced but not naturalized Papaver rhoeas L. Herb NL Invasive Diversity 2023, 15, 407 16 of 20 16 of 20 Table A1. Cont. Table A1. Cont. Family Genus, Species and Lower Taxa Life Form NEM:BA-AIS Regs. Appendix A Cat Alien Status [73] Passiﬂoraceae Passiﬂora caerulea L. Herb 1b Invasive Passiﬂora edulis Sims. Herb 2 Invasive Passiﬂora ligularis Juss. Herb NL - Passiﬂora subpeltata Ortega. Herb 1b Invasive Phytolaccaceae Phytolacca americana L. Herb 1b Invasive Phytolacca dioica L. Woody 3 Invasive Phytolacca octandra L. Herb 1b Invasive Pinaceae Pinus patula Schiede ex Schltdl. & Cham. Woody 2 Invasive Pinus pinaster Aiton Woody 1b Invasive Pittosporaceae Pittosporum undulatum Vent. Woody 1b Invasive Plantaginaceae Plantago lanceolata L. Herb NL Invasive Plantago major L. Herb NL Invasive Poaceae Arundo donax L. 1753 Graminoids 1b Invasive Avena barbata Pott ex Link Graminoids NL Introduced but not naturalized Avena fatua L. Graminoids NL Invasive Brachypodium distachyon (L.) P.Beauv. Graminoids NL - Briza maxima L. Graminoids NL Introduced but not naturalized Bromus diandrus Roth Graminoids NL Introduced but not naturalized Bromus pectinatus Thunb. Graminoids NL Introduced but not naturalized Bromus rigidus Roth. Graminoids NL - Calamagrostis acutiﬂora (Schrad.) Rchb. Graminoids NL NA Cortaderia jubata (Lemoine) Stapf. Graminoids 1b - Digitaria debilis (Desf.) Willd. Graminoids NL - Eragrostis mexicana (Hornem.) Link Graminoids NL - Hordeum murinum L. Graminoids NL Invasive Imperata cylindrica (L.) Raeusch Graminoids NL - Lolium rigidum Gaudin Graminoids NL Introduced Nassella trichotoma (Nees) Hack. ex Arechav. Graminoids 1b invasive Paspalum dilatatum Poir. Graminoids NL Invasive Paspalum urvillei Steud. Graminoids NL Invasive Pennisetum clandestinum Hochst. ex Chiov. Graminoids 1b Invasive Pennisetum setaceum (Forssk.) Chiov. Graminoids 1b Invasive Pennisetum villosum R.Br. ex Fresen. Graminoids 1b - Phalaris minor Retz. (1783) Graminoids NL - Stipa capensis Thunb. Graminoids NL Introduced Vulpia myuros (L.) C.C. Gmel. Graminoids NL - Pontederiaceae Pontederia crassipes (Mart.) Solms Herb 1b Invasive Polypodiaceae Nephrolepis cordifolia L. Fern 1b Invasive Nephrolepis exaltata (L.) Schott Fern 1b Invasive 17 of 20 17 of 20 Diversity 2023, 15, 407 Table A1. Cont. Family Genus, Species and Lower Taxa Life Form NEM:BA-AIS Regs. Cat Alien Status [73] Primulaceae Ardisia crenata Sims Woody 1b Invasive Lysimachia arvensis L. Herb NL Introduced but not naturalized Pteridaceae Adiantum raddianum Presl Fern NL Introduced but not naturalized Rosaceae Cotoneaster franchetii Bois Woody 1b Invasive Cotoneaster pannosus Franch. Woody 1b Invasive Potentilla indica (Jacks.) Focke Herb NL Invasive Prunus persica (L.) Batsch Woody NL Invasive Pyracantha angustifolia (Franch.) C.K.Schneid. Woody 1b Invasive Pyracantha coccinea M.Roem. Woody 1b Invasive Rosa rubiginosa L. Woody 1b Invasive Rubus cuneifolius Pursh. Woody 1b Invasive Rubus fruticosus Lour. Woody 2 Invasive Rubus odoratus L. References Wondafrash, M.; Wingfeld, M.J.; Wilson, J.R.U.; Hurley, B.P.; Slippers, B.; Paap, T. Botanical gardens as key resources and hazards for biosecurity. Biodivers. Conserv. 2021, 30, 1929–1946. [CrossRef] f y p y 10. Wondafrash, M.; Wingfeld, M.J.; Wilson, J.R.U.; Hurley, B.P.; Slippers, B.; Paap, T. Botanical gardens as k for biosecurity. Biodivers. Conserv. 2021, 30, 1929–1946. [CrossRef] y y, L.H. 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Biogeogr. 2021, 30, 1572–1582. [CrossRef] 16. Malcom, S.M. Education and Biodiversity; Levin, S.A., Ed.; Encyclopedia of Biodiversity; Elsevier: Amsterdam, The Netherlands, 2001; pp. 383–394. 17. Hulme, P.E.; Bacher, S.; Kenis, M.; Klotz, S.; Kühn, I.; Minchin, D.; Nentwig, W.; Olenin, S.; Panov, V.; Pergl, J.; et al. Grasping at the routes of biological invasions: A framework for integrating pathways into policy. Appendix A Woody NL - Rubiaceae Richardia brasiliensis Gomes Herb NL Invasive Salicaceae Populus canescens (Aiton) Sm. Woody 2 Invasive Salviniaceae Azolla ﬁliculoides Lam. Fern 1b Invasive Sapindaceae Cardiospermum grandiﬂorum Swartz Woody 1b Invasive Scrophulariaceae Verbascum chaixii Vill. Herb NL - Simaroubaceae Ailanthus altissima (Mill.) Swingle Woody 1b Invasive Solanaceae Cestrum aurantiacum Lindl. Woody 1b Invasive Cestrum laevigatum Schltdl. Woody 1b Invasive Cestrum parqui L’Her. Woody 1b Invasive Datura ferox L. Herb 1b Invasive Datura innoxia Mill. Herb 1b Invasive Datura stramonium L. Herb 1b Invasive Physalis angulata L. Herb NL Introduced but not naturalized Physalis peruviana L. Herb NL Invasive Physalis viscosa L. Herb NL Invasive Solanum elaeagnifolium Cav. Woody 1b Invasive Solanum mauritianum Scop. Woody 1b Invasive Solanum nigrum L. Herb NL - Solanum pseudocapsicum L. Herb 1b Invasive Solanum seaforthianum Andrews Woody 1b Invasive Solanum sisymbriifolium Lam. Herb 1b Invasive Tropaeolaceae Tropaeolum majus L. Herb NL Invasive Tropaeolum speciosum Poepp. & Endl. Herb 3 NA Verbenaceae Lantana camara L. Woody 1b Invasive Phyla nodiﬂora (L.) Greene Herb NL - Verbena aristigera S.Moore Herb NL - Verbena bonariensis L. Herb 1b Invasive Zingiberaceae Hedychium coronarium J.Koenig. Herb 1b Invasive Hedychium ﬂavescens Carey ex Roscoe. Herb 1b Invasive Table A1. Cont. Diversity 2023, 15, 407 18 of 20 18 of 20 References J. Appl. Ecol. 2008, 45, 403–414. [CrossRef] 18. Willis, K.C. 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https://openalex.org/W2221029565	https://europepmc.org/articles/pmc5456558?pdf=render	English	null	First-Principles Study of Mo Segregation in MoNi(111): Effects of Chemisorbed Atomic Oxygen	Materials	2,015	cc-by	7,070	First-Principles Study of Mo Segregation in MoNi(111): Effects of Chemisorbed Atomic Oxygen Yanlin Yu 1, Wei Xiao 1,2, Jianwei Wang 1 and Ligen Wang 1,3,* Yanlin Yu 1, Wei Xiao 1,2, Jianwei Wang 1 and Ligen Wang 1,3,* Received: 7 October 2015; Accepted: 17 December 2015; Published: 26 December 2015 Academic Editor: Federico Bella 1 General Research Institute for Nonferrous Metals, Beijing 100088, China; yuyanlin_121@163.com (Y.Y.); wxiao@ustb.edu.cn (W.X.); jswjw@sina.com (J.W.) 2 School of Materials Science and Engineering, University of Science and Technology Beijing, Beijing 100083, China 3 Power Environmental Energy Research Institute, Covina, CA 91722, USA 1 General Research Institute for Nonferrous Metals, Beijing 100088, China; yuyanlin_121@163.com (Y.Y.); wxiao@ustb.edu.cn (W.X.); jswjw@sina.com (J.W.) j g 3 Power Environmental Energy Research Institute, Covina, CA 91722, USA 3 Power Environmental Energy Research Institute, Covina, CA 91722, USA * Correspondence: lg_wang1@yahoo.com; Tel.: +86-10-8224-1124; Fax: +86-10-8224-0096 3 Power Environmental Energy Research Institute, Covina, CA 91722, USA * Correspondence: lg_wang1@yahoo.com; Tel.: +86-10-8224-1124; Fax: +86-10-8224-0096 * Correspondence: lg_wang1@yahoo.com; Tel.: +86-10-8224-1124; Fax: +86-10-8 Abstract: Segregation at metal alloy surfaces is an important issue because many electrochemical and catalytic properties are directly correlated to the surface composition. We have performed density functional theory calculations for Mo segregation in MoNi(111) in the presence of chemisorbed atomic oxygen. In particular, the coverage dependence and possible adsorption-induced segregation phenomena are addressed by investigating segregation energies of the Mo atom in MoNi(111). The theoretical calculated results show that the Mo atom prefers to be embedded in the bulk for the clean MoNi(111), while it segregates to the top-most layer when the oxygen coverage is thicker than 1/9 monolayer (ML). Furthermore, we analyze the densities of states for the clean and oxygen-chemisorbed MoNi(111), and see a strong covalent bonding between Mo d-band states and O p-states. The present study provides valuable insight for exploring practical applications of Ni-based alloys as hydrogen evolution electrodes. Keywords: density-functional theory calculation; surface segregation; hydrogen evolution electrode; oxygen chemisorptions; water electrolysis Materials 2016, 9, 5; doi:10.3390/ma9010005 www.mdpi.com/journal/materials 1. Introduction Adding a second metal into a pure metal catalyst can provide a great opportunity to tailor the properties of the catalyst [1–4]. Therefore, bimetallic systems have attracted considerable attention due to their importance in basic science and industry [5–8]. Bimetallic systems are much more complicated than pure metal catalysts since one element may segregate to the surface and lead to surface composition enrichment/depletion compared to the bulk. The adsorbate-induced surface segregation of metallic alloys under the reaction conditions and, thus, the changes in local atomic composition and surface structure have been predicted and demonstrated to occur for a number of bimetallic systems [9,10]. So for a given bimetallic conﬁguration which exhibits a desired property, it is crucial to know whether the particular conﬁguration is stable under the operating environment for a speciﬁc application. Many theoretical and experimental studies have been performed to investigate the surface segregation for various bimetallic systems [11–29]. The recent experimental and theoretical works focusing on the description of bimetallic and ternary, extended and nanosized, alloy surfaces under reactive gas phase environments were reviewed by Zafeiratos, et al. [19] and by Guesmi [20]. On the theory side, Guesmi and co-workers had performed extensive ﬁrst-principles calculations to investigate the transition metal segregation behaviors for the gold-based bimetallic systems in the presence of various gas adsorptions [11,12,21–23]. Speciﬁcally, the authors found that segregation of Pd on Materials 2016, 9, 5; doi:10.3390/ma9010005 www.mdpi.com/journal/materials 2 of 10 Materials 2016, 9, 5 PdAu(111) is oxygen coverage–dependent and that Pd atoms tend to be in the bulk for the clean surface while they segregate to the surface in the presence of more than 1/3 ML of oxygen [12]. The surface phase diagrams were investigated within the ﬁrst-principles atomistic thermodynamics framework by addressing the effect of the bulk alloy and the gas phase reservoir [13,24–26]. The ﬁrst-principles-based cluster expansion technique [27] had also been employed to model the surface ordering and segregation of alloys in a reactive environment [28,29]. This approach allows us to explore enormous different atomic conﬁgurations. Although the latter two methodologies are very useful for studying the alloy surface ordering and segregation in the presence of adsorbates, the direct ﬁrst-principles calculations [11,12,21–23] are certainly able to predict the segregation tendency of alloy systems. 1. Introduction The advantage of the direct ﬁrst-principles calculation method is that it is accurate and computationally more affordable, while its drawback is that it cannot predict what equilibrium phases and structures will form under given conditions. Among the bimetallic systems, nickel-based alloys are especially interesting because of their applications in a number of catalytic reactions including the hydrodeoxygenation of esters [30], methanation reaction [31], and propane reforming [32]. In the context of hydrogen evolution reaction (HER), recent experiments showed that adding some Mo element into nickel-based electrodes could improve the electro-catalytic activity [33]. Previously, the calculations without any gas adsorption and the experiments in a ultra high vacuum (UHV) chamber showed that it is difﬁcult for Mo atoms to segregate to the surfaces in NiMo alloys [34–36]. However, several previous experiments found that Mo atoms segregate to the NiMo alloy surfaces and induce an enrichment of the surface Mo composition under the reaction conditions [17,34]. The composition and structure of Ni-2 at % Mo(100), Ni-2 at % Mo(110) and Ni-6 at % Mo(110) surfaces were studied by Auger electron spectroscopy (AES) and low-energy electron diffraction (LEED) [34]. The authors obtained the molybdenum pre-enriched surfaces by annealing the samples in puriﬁed hydrogen. We also experimentally investigated the Mo surface segregation by energy-dispersive spectroscopy (EDS) in the NiMoCo electrolysis electrode materials [17]. In our experiments, the Ni at 20.8 wt % and Mo at 1.6 wt % Co foam electrodes were prepared by the electro-deposition method and decarbonized at atmospheric conditions to remove the residual carbon due to the polyurethane foams, followed by a reduction process in hydrogen atmosphere for removing the surface oxides. This Mo surface enrichment (40 wt % Mo for the surface vs. the nominal 20.8 wt % Mo in the bulk) could be because of the presence of chemisorbed atomic oxygen during the decarbonation of the MoNi electrodes [17]. Obviously, detailed theoretical calculations are desirable and helpful to understand the experimental observation. In this paper, we investigate the effects of chemisorbed atomic oxygen on the segregation behavior of the Mo element in MoNi(111) by performing ﬁrst-principles calculations. The (111) surface is likely the dominant facet for the NiMo electrolysis electrodes since, among various Ni surfaces, Ni(111) is the most stable one. Based on the previous studies [22,35] we believe that, if we consider other orientations, the segregation behaviors will not change. 1. Introduction This is because the surface segregation has a weak dependence on the orientation [22]. Mo segregation on NiMo(111) and NiMo(100) in vacuum has been calculated and the results show that the Mo atom has the same segregation tendency for the two surfaces [35,36]. In the work by Sansa, et al. [22], the authors found that the stronger adsorption energies of M impurities on the (100) alloy surfaces compared to the (111) surfaces do not induce a better segregation toward the (100) facet. The authors argued that the adsorption anisotropy is mostly generated by the matrix metal Au and slightly depends on the chemical nature of M. Inconsistent with previous studies [35,36], our calculations show that the surface segregation of the Mo element does not occur in the vacuum, namely for the clean MoNi(111) it is energetically unfavorable for Mo atoms to segregate onto the surface. In the presence of chemisorbed atomic oxygen, the Mo element is found to segregate to the alloy surface when the coverage is thicker than 1/9 ML. By carefully analyzing the densities of states, we see that the preference of Mo atoms to be embedded in the Ni bulk is mainly governed by surface energy considerations and atomic size effects. We also ﬁnd a strong covalent bonding between Mo d-states and O p-states. The remainder of the paper is organized as follows. 3 of 10 Materials 2016, 9, 5 In Section 2, the theoretical methods and computational details are described. Section 3 presents the calculated results of segregation energies, and electronic structure analyses. A brief summary is given in Section 4. 2. Computational Methods The ﬁrst-principles calculations were performed within a density functional theory using the Vienna Ab-initio Simulation Package (VASP) [37–39]. The electron-ion interaction was described using the projector augmented wave method (PAW) [40,41] and the exchange correlation potential using the Perdew–Burke–Ernzerhof (PBE) functional method [42]. The energy cutoff for the plane wave basis set was 450 eV for all the calculated systems. Spin polarization was taken into account in the calculations and the Methfessel and Paxton [43] was employed to determine electron occupancies with a smearing parameter σ of 0.14 eV. The convergence criteria for the electronic self-consistent iteration and the ionic relaxation loop were set to 10´5 eV and 0.02 eV/A, respectively. To simulate metallic surfaces, a slab supercell was employed. All the calculations presented in this work were based on slabs of 54 atoms, containing six atomic layers representing a 3 ˆ 3 supercell, separated by 15 A of vacuum space. MoNi(111) alloy systems corresponding to the substitution of one Ni atom by one Mo atom in the ﬁrst, second, third or fourth nickel layer are presented in Figure 1, respectively. The Mo atom plays a role as a prober to determine whether it prefers to stay on the surface or in the bulk. As pointed out above, the drawback of this method is that it cannot predict the equilibrium phases and structures. However, investigation of the equilibrium phases and structures under certain given conditions is beyond the scope of the present study. Chemisorbed atomic oxygen was located on only one side of the slab. The inﬂuence of the resulting electric dipole on the computed energy values was estimated to be very small according to standard methods [44], and had thus been neglected. The atoms in the top four layers and the chemisorbed atomic oxygen were allowed to relax, while the atoms in the bottom two layers were ﬁxed at the bulk geometry positions. Brillouin zone integrations were performed using Monkhorst–Pack grids [45] of 4 ˆ 4 ˆ 1 for slab calculations. The gas-phase oxygen molecule was simulated through a large supercell with dimensions of 12 ˆ 12 ˆ 12 A3. In order to characterize the segregation behavior of the Mo atom in nickel, the segregation energy (Esegr) was deﬁned as the energy difference between the states with the Mo atom located at the upper surface layer and in the bulk. Conflicts of Interest: The auth 3. Results and Discussion We have done an exhaus Materials 2016, 9, 352; doi:10.3390/ma9050352 www.mdpi.com/journal/materials Reference 1. Yu, Y.; Xiao, W.; Wang, J.; Wang, L. First-Principles Study of Mo Segregation in MoNi(111): Effects of Chemisorbed Atomic Oxygen. Materials 2016, 9, 5. © 2016 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/). We have done an exhaustive and extensive search on the lowest-energy O adsorption patterns (or arrangements) for each oxygen coverage with the Mo atom located at various atomic layers. The most stable O adsorption conﬁgurations for Mo located in the top-most atomic layer are shown in Figure 2. For the 1/9 ML oxygen coverage case, the O atom prefers to occupy a fcc site near the surface Mo atom. The two O atoms occupy a fcc site and an hcp site next to the Mo atom, respectively, for the oxygen coverage of 2/9 ML It is less stable by 0.16 eV for the two O atoms to occupy the two fcc sites. For the higher oxygen coverage cases (such as 3/9 ML and 4/9 ML) O atoms ﬁrst occupy the fcc sites next to the surface Mo atom and then those fcc sites far away from the Mo atom. We ﬁnd that the subsurface Mo atom prefers to stay away from the adsorbed O atoms; for instance, when the Mo atom is located at the second atomic layer, O atoms occupying the fcc sites far away from the top position of the Mo atom are energetically more favorable. When the Mo atom is located at the third or lower atomic layer, it imposes a negligible effect on the O adsorption, as we can see from Figure 3, that the segregation energies do not rely on the oxygen coverage. gy p p arrangements) for each oxygen coverage with the Mo atom located at various atomic layers. The mo ble O adsorption configurations for Mo located in the top‐most atomic layer are shown in Figure r the 1/9 ML oxygen coverage case, the O atom prefers to occupy a fcc site near the surface M m. 2. Computational Methods According to this deﬁnition, the segregation energies were calculated according to the following equation: Esegr “ EMoNipMo,x´layerq ´ EMoNipMo,4th´layerq (1) (1) where EMoNipMo,x´layer) represents the total energy of the MoNi alloy system with the Mo atom located in the upper x nickel layers (x = 1, 2 or 3), and EMoNipMo,4th-layerq represents the total energy of the MoNi alloy system with the Mo atom located in the fourth nickel layer, which corresponds to the presence of the Mo atom in the “bulk” nickel matrix. For the oxygen adsorption cases, EMoNipMo,x-layerq and EMoNipMo,4th-layerq are the total energies of the slabs with O atoms adsorbed on the top layer and the Mo atom located at the corresponding atomic layer. According to our calculations, a chemisorbed atomic oxygen prefers to occupy a three-fold fcc hollow site on MoNi(111) and Ni(111). For higher oxygen coverage cases (such as 2/9, 3/9, and 4/9 ML oxygen coverages corresponding to the adsorption of two, three and four oxygen atoms, respectively), we considered all possible oxygen adsorption conﬁgurations and calculated the segregation behavior by using the most stable adsorption conﬁgurations. 4 of 10 tanding 4 of 10 tanding Materials 2016, 9, 5 And the author caused by them (a) (a) (b) (b) (a) (b) (c) (d) Figure 1. MoNi(111) alloy systems showing one Mo monomer substituting one Ni atom in the (a) first; (b) second; (c) third; and (d) fourth nickel layer. Only the four top layers are shown. Gray and blue balls represent Ni and Mo atoms, respectively. Figure 1. MoNi(111) alloy systems showing one Mo monomer substituting one Ni atom in the (a) ﬁrst; (b) second; (c) third; and (d) fourth nickel layer. Only the four top layers are shown. Gray and blue balls represent Ni and Mo atoms, respectively. als 2016, 9, page–page sults and Discussion (c) (c) (d) (d) (d) (b) (c) (a) Figure 1. MoNi(111) alloy systems showing one Mo monomer substituting one Ni atom in the (a) first; (b) second; (c) third; and (d) fourth nickel layer. Only the four top layers are shown. Gray and blue balls represent Ni and Mo atoms, respectively. Figure 1. MoNi(111) alloy systems showing one Mo monomer substituting one Ni atom in the (a) ﬁrst; (b) second; (c) third; and (d) fourth nickel layer. Only the four top layers are shown. Gray and blue balls represent Ni and Mo atoms, respectively. Conflicts of Interest: The auth 3. Results and Discussion We have done an exhaus The two O atoms occupy a fcc site and an hcp site next to the Mo atom, respectively, for t ygen coverage of 2/9 ML It is less stable by 0.16 eV for the two O atoms to occupy the two fcc sit r the higher oxygen coverage cases (such as 3/9 ML and 4/9 ML) O atoms first occupy the fcc sit xt to the surface Mo atom and then those fcc sites far away from the Mo atom. We find that t bsurface Mo atom prefers to stay away from the adsorbed O atoms; for instance, when the Mo ato ocated at the second atomic layer, O atoms occupying the fcc sites far away from the top positi the Mo atom are energetically more favorable. When the Mo atom is located at the third or low mic layer, it imposes a negligible effect on the O adsorption, as we can see from Figure 3, that t gregation energies do not rely on the oxygen coverage. (a) (b) (c) (d) Figure 2. Top views of the most stable adsorption configurations for the oxygen coverage of (a) 1/9; (b) 2/9; (c) 3/9 and (d) 4/9 with Mo located at the top‐most atomic layer of the slab. Only the top‐most metal atomic layer and adsorbed O atoms are shown. Gray, blue and red balls represent Ni, Mo and O, respectively. Figure 2. Top views of the most stable adsorption conﬁgurations for the oxygen coverage of (a) 1/9; (b) 2/9; (c) 3/9 and (d) 4/9 with Mo located at the top-most atomic layer of the slab. Only the top-most metal atomic layer and adsorbed O atoms are shown. Gray, blue and red balls represent Ni, Mo and O, respectively. a) (b) (c) (d) (a) (c) (d) (a) (b) gure 2. Top views of the most stable adsorption configurations for the oxygen coverage of (a) 1 2/9; (c) 3/9 and (d) 4/9 with Mo located at the top‐most atomic layer of the slab. Only the top‐m tal atomic layer and adsorbed O atoms are shown. Gray, blue and red balls represent Ni, Mo a respectively. Figure 2. Top views of the most stable adsorption conﬁgurations for the oxygen coverage of (a) 1/9; (b) 2/9; (c) 3/9 and (d) 4/9 with Mo located at the top-most atomic layer of the slab. Only the top-most metal atomic layer and adsorbed O atoms are shown. 2. Computational Methods 2016, 9, page–page lt d Di i Conflicts of Interest: The auth 3. Results and Discussion We have done an exhaus Gray, blue and red balls represent Ni, Mo and O, respectively. The calculated segregation energies for clean and oxygen- adsorbed surfaces are given in Table 1 and also plotted in Figure 3. In absence of adsorbed gas, i.e., under vacuum conditions, the segregation energy for Mo located at the top-most layer is positive and has a value of 0.79 eV. This indicates that Mo does not segregate to the top-most surface layer. We can attribute this behavior to the smaller surface energy of nickel compared to that of molybdenum (experimental values are 2.45 eV/atom for Ni and 3.00 eV/atom for Mo [46]). Interestingly, the Mo atom prefers to occupy a site below the top-most surface layer. This oscillatory phenomenon is quite common in alloying systems [47–51]. That the Mo atoms located at the second layer are energetically favorable can be understood by two facts: (1) since it is not on the top-most layer it can avoid the higher surface energy of Mo; and (2) by locating at the second layer, the system can largely release the elastic energy due to the atomic size mismatch. 5 of 10 and Materials 2016, 9, 5 metal atomic O ti O, respectively. Figure 3. Evolution of the segregation energies (eV) of Mo atom from the nickel “bulk” (fourth layer) to upper layers toward the surface in the presence of different atomic oxygen coverage. Figure 3. Evolution of the segregation energies (eV) of Mo atom from the nickel “bulk” (fourth layer) to upper layers toward the surface in the presence of different atomic oxygen coverage. Figure 3. Evolution of the segregation energies (eV) of Mo atom from the nickel “bulk” (fourth layer) to upper layers toward the surface in the presence of different atomic oxygen coverage. Figure 3. Evolution of the segregation energies (eV) of Mo atom from the nickel “bulk” (fourth layer) to upper layers toward the surface in the presence of different atomic oxygen coverage. 4 The calculated segregation energies for clean and oxygen‐ adsorbed surfaces are given in Table 1 and also plotted in Figure 3. In absence of adsorbed gas, i.e., under vacuum conditions, the segregation energy for Mo located at the top‐most layer is positive and has a value of 0.79 eV. This indicates that Mo does not segregate to the top‐most surface layer. Conflicts of Interest: The auth 3. Results and Discussion We have done an exhaus We can attribute this behavior to the smaller surface energy of nickel compared to that of molybdenum (experimental values are 2.45 eV/atom for Ni and 3.00 eV/atom for Mo [46]). Interestingly, the Mo atom prefers to occupy a site below the top‐most surface layer. This oscillatory phenomenon is quite common in alloying At the oxygen coverage of 1/9 ML, although the segregation energy drops to 0.23 eV from the value of 0.79 eV for the clean surface, the Mo atom still prefers to locate inside of the bulk. With increasing the oxygen coverage to 2/9 ML or thicker, the Mo atom becomes more stable in the top-most layer than in the lower layer and in the bulk. This result indicates that the presence of chemisorbed oxygen on the surface of a MoNi alloy electrode, such as during the decarbonation of the electrode, may cause the surface to be Mo-rich, i.e., having a concentration higher than the nominal concentration in the alloy. This explains our recent experimental observation that the electrode surface is Mo-rich [17]. We can understand the O adsorption-driven segregation of Mo as follows. There are two factors that favor Mo to segregate onto the top-most layer. The ﬁrst one is that segregation of the Mo atom onto the top-most layer helps to release the elastic energy caused by the size mismatch. Another factor is that the Mo-O bond is stronger than the Ni-O bond (from the chemical rubber company (CRC) handbook [52], the bond strengths for diatomic Mo-O and Ni-O molecules are 145.1 kcal/mol and 93.6 kcal/mol at 298 K, respectively). There exists a factor that is against the segregation of Mo onto the ﬁrst surface layer. This unfavorable factor is that the Mo surfaces have a larger surface energy than the corresponding Ni surfaces. The ﬁnal segregation behavior is determined by the competition between the two favorable factors and the unfavorable factor. Table 1. Calculated segregation energies for MoNi alloy conﬁgurations with different adsorbed atomic oxygen coverage. The reported segregation energies of molybdenum in O-MoNi(111) systems are calculated by considering the most energetically stable adsorbed oxygen conﬁgurations. Conflicts of Interest: The auth 3. Results and Discussion We have done an exhaus (a) d-band DOS of Ni atom in the top-most atomic layer for the alloy and pure Ni surfaces; (b) d-band DOS of Mo atom in the top-most atomic layer for the alloy and pure Mo surfaces. Figure 4. Calculated density of states (DOS) of the Mo and Ni atoms in the alloy and pure metal surfaces. (a) d‐band DOS of Ni atom in the top‐most atomic layer for the alloy and pure Ni surfaces; (b) d‐band DOS of Mo atom in the top‐most atomic layer for the alloy and pure Mo surfaces. Figure 4. Calculated density of states (DOS) of the Mo and Ni atoms in the alloy and pure metal surfaces. (a) d-band DOS of Ni atom in the top-most atomic layer for the alloy and pure Ni surfaces; (b) d-band DOS of Mo atom in the top-most atomic layer for the alloy and pure Mo surfaces. The d‐band DOS for the Mo atom located at the top‐most surface layer, the second layer and the third layer without oxygen adsorption are presented in Figure 5a. From Figure 5a, we see the shape and position of the Mo d‐band DOS do not significantly change no matter which layer the Mo atom is located at. This indicates that the preference of Mo atoms to locate in the Ni bulk is mainly governed by surface energy considerations and atomic size effects (2.39 A for Mo and 2.22 A for Ni [54]) [12]. Figure 5b shows that the d‐band DOS of Mo in MoNi(111) interacting with oxygen are drastically modified compared to those for the clean surface (i.e., without oxygen adsorption). The d‐band DOS for the oxygen adsorption case are largely widened. The narrower DOS for the surface atoms are easy to understand, given they have a smaller coordination number relative to the bulk atoms. For the adsorption cases, it can be considered that as the adsorbate increases the coordination number for the surface atoms, it therefore broadens the DOS [55]. From Figure 5b, we can see that the broadening of the Mo d‐band DOS causes some d‐states to move to the lower energy region upon O adsorption. This effect lowers the system energy and leads to better stability of the system. Conflicts of Interest: The auth 3. Results and Discussion We have done an exhaus Position of the Mo Atom Atomic Oxygen Sub-Monolayer on MoNi(111) Alloy 0 1/9 ML 2/9 ML 3/9 ML 4/9 ML Etot (eV) First layer 0.79 0.23 ´1.11 ´2.52 ´3.01 Second layer ´0.16 ´0.13 ´0.19 ´0.21 ´0.27 Third layer 0.03 0.02 0.03 0.02 0.03 Fourth layer 0 0 0 0 0 The d-band densities of states (DOS) for Mo and its nearest neighboring nickel atoms in the top-most surface layer with and without oxygen adsorption were calculated. Figure 4 shows the d-band DOS for both molybdenum and nickel atoms in the MoNi alloy compared to their corresponding DOS 6 of 10 Materials 2016, 9, 5 in pure metals. The d-band DOS for Ni(111) and Mo(110) are presented because they are the most stable surfaces for the metals. Compared to the pure metal surface atom, the nickel d-band DOS center is slightly shifted up towards the Fermi level in the alloy case (Figure 4a), while the molybdenum d-band DOS center is shifted towards the lower energy region, away from the Fermi level (Figure 4b). According to the d-band center model developed by Hammer, et al. [53], these d-band center shifts closely correlate to oxygen adsorption–induced Mo surface segregation as we have observed above. There exists an additional peak at the higher edge of the d-band DOS for the alloy case (Figure 4a). This peak indicates the formation of a covalent Ni-Mo bond [12]. From Figure 4b we can see that the Mo d-band not only shifts to the lower energy region, it also becomes narrower than in the pure metal, and the empty states are fewer. This means that the Mo atom gains some electrons from its neighboring nickel atoms, which is consistent with the d-band DOS of Ni shifting up and having more empty states in the alloy case (Figure 4a). Materials 2016, 9, page–page Figure 4. Calculated density of states (DOS) of the Mo and Ni atoms in the alloy and pure metal surfaces. (a) d‐band DOS of Ni atom in the top‐most atomic layer for the alloy and pure Ni surfaces; (b) d‐band DOS of Mo atom in the top‐most atomic layer for the alloy and pure Mo surfaces. Figure 4. Calculated density of states (DOS) of the Mo and Ni atoms in the alloy and pure metal surfaces. Conflicts of Interest: The auth 3. Results and Discussion We have done an exhaus Furthermore, we also see that the O‐Mo anti‐bonding states, located above the Fermi level, are largely unoccupied and that the O p‐states and Mo d‐states have a large overlap, thus allowing a strong covalent bonding and hybridization interaction between molybdenum and the oxygen atom. The d-band DOS for the Mo atom located at the top-most surface layer, the second layer and the third layer without oxygen adsorption are presented in Figure 5a. From Figure 5a, we see the shape and position of the Mo d-band DOS do not signiﬁcantly change no matter which layer the Mo atom is located at. This indicates that the preference of Mo atoms to locate in the Ni bulk is mainly governed by surface energy considerations and atomic size effects (2.39 A for Mo and 2.22 A for Ni [54]) [12]. Figure 5b shows that the d-band DOS of Mo in MoNi(111) interacting with oxygen are drastically modiﬁed compared to those for the clean surface (i.e., without oxygen adsorption). The d-band DOS for the oxygen adsorption case are largely widened. The narrower DOS for the surface atoms are easy to understand, given they have a smaller coordination number relative to the bulk atoms. For the adsorption cases, it can be considered that as the adsorbate increases the coordination number for the surface atoms, it therefore broadens the DOS [55]. From Figure 5b, we can see that the broadening of the Mo d-band DOS causes some d-states to move to the lower energy region upon O adsorption. This effect lowers the system energy and leads to better stability of the system. Furthermore, we also see that the O-Mo anti-bonding states, located above the Fermi level, are largely unoccupied and that the O p-states and Mo d-states have a large overlap, thus allowing a strong covalent bonding and hybridization interaction between molybdenum and the oxygen atom. 7 of 10 rong Materials 2016, 9, 5 noccupied and th l t b di Figure 5. (a) d‐band DOS for the Mo atom located in the first, second and third MoNi(111) layers in absence of oxygen; (b) d‐band DOS for the Mo atom located in the top‐most atomic layer with and without oxygen adsorption and oxygen p‐band DOS. Figure 5. Conflicts of Interest: The auth 3. Results and Discussion We have done an exhaus (a) d-band DOS for the Mo atom located in the ﬁrst, second and third MoNi(111) layers in absence of oxygen; (b) d-band DOS for the Mo atom located in the top-most atomic layer with and without oxygen adsorption and oxygen p-band DOS. gure 5. (a) d‐band DOS for the Mo atom located in the first, second and third MoNi(111) layers in bsence of oxygen; (b) d‐band DOS for the Mo atom located in the top‐most atomic layer with and ithout oxygen adsorption and oxygen p‐band DOS. Figure 5. (a) d-band DOS for the Mo atom located in the ﬁrst, second and third MoNi(111) layers in absence of oxygen; (b) d-band DOS for the Mo atom located in the top-most atomic layer with and without oxygen adsorption and oxygen p-band DOS. 4. Conclusions 6 We have performed density functional theory calculations to investigate the effects of chemisorbed atomic oxygen on the segregation behavior of the Mo element in MoNi(111). In particular, the coverage dependence and possible adsorption-induced segregation phenomena are addressed by calculating the segregation energies of the Mo atom in the upper layers of MoNi(111). The theoretical calculated results show that the Mo atom prefers to be embedded in the bulk in the clean MoNi(111), while it segregates to the top-most layer when the oxygen coverage is thicker than 1/9 ML For the clean MoNi(111) we see that the d-band center of Ni atoms surrounding the Mo atom shifts up to the Fermi energy and the Mo d-band becomes narrower with its center shifted down away from the Fermi energy. The shape and position of the Mo d-band DOS do not signiﬁcantly change, no matter which layer the Mo atom is located at. This indicates that the preference of Mo atoms to be embedded in the Ni bulk is mainly governed by surface energy considerations and atomic size effects. The Mo d-band DOS for the oxygen adsorption cases are largely widened and lead to a strong covalent bonding between the molybdenum atom and the oxygen atom. The present study provides valuable insight for exploring practical applications of Ni-based alloys as hydrogen evolution electrodes. Acknowledgments: This work was supported by Special foundation for institute of technology research development under 2013EG115003 and 2014EG115002. Author Contributions: Ligen Wang and Yanlin Yu conceived and designed the experiments; Yanlin Yu perform he experiments; Wei Xiao and Jianwei Wang analyzed the data; Yanlin Yu and Ligen Wang wrote the paper Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. 4. Yarulin, A.; Berguerand, C.; Alonso, A.O.; Yuranov, I.; Kiwi-Minsker, L. Increasing Pt selectivity to vinylaniline by alloying with Zn via reactive metal-support interaction. Catal. Today 2015, 256, 241–249. 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[CrossRef] © 2015 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons by Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W2105272113	https://repub.eur.nl/pub/89201/REPUB_89201_OA.pdf	English	null	Required duration of mass ivermectin treatment for onchocerciasis elimination in Africa: a comparative modelling analysis	Parasites & vectors	2,015	cc-by	13,616	Required duration of mass ivermectin treatment for onchocerciasis elimination in Africa: a comparative modelling analysis Wilma A. Stolk1†, Martin Walker2†, Luc E. Coffeng1, María-Gloria Basáñez2† and Sake J. de Vlas1 Abstract Background: The World Health Organization (WHO) has set ambitious targets for the elimination of onchocerciasis by 2020–2025 through mass ivermectin treatment. Two different mathematical models have assessed the feasibility of reaching this goal for different settings and treatment scenarios, namely the individual-based microsimulation model ONCHOSIM and the population-based deterministic model EPIONCHO. In this study, we harmonize some crucial assumptions and compare model predictions on common outputs. Methods: Using a range of initial endemicity levels and treatment scenarios, we compared the models with respect to the following outcomes: 1) model-predicted trends in microfilarial (mf) prevalence and mean mf intensity during 25 years of (annual or biannual) mass ivermectin treatment; 2) treatment duration needed to bring mf prevalence below a provisional operational threshold for treatment interruption (pOTTIS, i.e. 1.4 %), and 3) treatment duration needed to drive the parasite population to local elimination, even in the absence of further interventions. Local elimination was judged by stochastic fade-out in ONCHOSIM and by reaching transmission breakpoints in EPIONCHO Results: ONCHOSIM and EPIONCHO both predicted that in mesoendemic areas the pOTTIS can be reached with annual treatment, but that this strategy may be insufficient in very highly hyperendemic areas or would require prolonged continuation of treatment. For the lower endemicity levels explored, ONCHOSIM predicted that the time needed to reach the pOTTIS is longer than that needed to drive the parasite population to elimination, whereas for the higher endemicity levels the opposite was true. In EPIONCHO, the pOTTIS was reached consistently sooner than the breakpoint. Conclusions: The operational thresholds proposed by APOC may have to be adjusted to adequately reflect differences in pre-control endemicities. Further comparative modelling work will be conducted to better understand the main causes of differences in model-predicted trends. This is a pre-requisite for guiding elimination programmes in Africa and refining operational criteria for stopping mass treatment. Keywords: Onchocerciasis, Onchocerca volvulus, Africa, Mathematical model, Mass treatment, Ivermectin, Elimination, Prevalence, Breakpoint, Forecasting Correspondence: w.stolk@erasmusmc.nl †Equal contributors 1Department of Public Health, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands Full list of author information is available at the end of the article © 2015 Stolk et al. * Correspondence: w.stolk@erasmusmc.nl †Equal contributors 1Department of Public Health, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands Full list of author information is available at the end of the article Stolk et al. Parasites & Vectors (2015) 8:552 DOI 10.1186/s13071-015-1159-9 Stolk et al. Parasites & Vectors (2015) 8:552 DOI 10.1186/s13071-015-1159-9 Background extent influence the duration of mass treatment required to achieve elimination. Human onchocerciasis, a neglected tropical disease (NTD), is a vector-borne filarial infection caused by Onchocerca volvulus. The infection can lead to skin disease, visual impairment and eventually blindness. It occurs primarily in tropical sub-Saharan Africa (99 % of cases), but some foci also exist in Yemen and Latin America. Over the past de- cades, the overall disease burden of onchocerciasis has been greatly reduced thanks to the implementation of large-scale control programmes, namely, the Onchocerciasis Con- trol Programme in West Africa (OCP, 1974–2002), the African Programme for Onchocerciasis Control (APOC, 1995–2015) and the Onchocerciasis Elimination Program for the Americas (OEPA, 1991-present). In the first dec- ade of the OCP, vector control interventions (aimed at the immature stages of the Simulium vectors) were used to interrupt transmission, but the current mainstay of con- trol is annual or biannual mass treatment with ivermectin. Mathematical models of onchocerciasis transmission and control provide useful tools with which to estimate the required duration of mass treatment in different settings. Two different models have been used to estimate the required duration for various endemic settings and treatment scenarios: the individual-based microsimulation model, ONCHOSIM [19, 20] and the population-based de- terministic model EPIONCHO [21–23]. Both models have predicted that the required duration increases with higher baseline endemicity and lower treatment coverage, and can be shortened by about 30–40 % when treating biannually instead of annually. Estimates of the required duration in absolute terms have been more difficult to compare due to a lack of harmonization of model assumptions, simu- lated scenarios, and presentation of types of output. In this paper, we present a comparative modelling study to explore the level of agreement between the ONCHO- SIM and EPIONCHO models in their projections of esti- mated programme duration to achieve elimination. A set of policy-relevant scenarios was simulated with both models, after harmonizing a number of critical input parameters. Congruent and disparate results are discussed to understand factors contributing to similarities and di- vergences. We also pinpoint areas where our knowledge base about the parasite population biology and drug activ- ity is insufficient and further research is needed. OEPA has successfully interrupted transmission in most foci in the Americas through 6- or 3-monthly ivermectin mass treatment [1–6]. Background Success was also reported in several African foci with annual or biannual ivermectin mass treatment [7, 8] and other areas also seem to move to- wards elimination [9], although there are also reports of ongoing transmission in spite of prolonged ivermectin mass treatment [10, 11]. In view of this evidence, APOC decided to target elimination where feasible [12]. The World Health Organization (WHO) set ambitious targets for the elimination of onchocerciasis, which is to be achieved by 2015 in the Americas and Yemen, by 2020 in selected African countries, and by 2025 in 80 % of African countries [13, 14]. There is broad international commit- ment towards these goals, expressed through the adoption of World Health Assembly Resolution on Neglected Trop- ical Diseases (WHA66.12) and the endorsement of the London Declaration on Neglected Tropical Diseases 2012 by pharmaceutical companies, donors, endemic country governments and non-governmental organizations in- volved in NTD control [15]. Abstract Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Stolk et al. Parasites & Vectors (2015) 8:552 Page 2 of 16 Mathematical models d ONCHOSIM and EPIONCHO, which were developed in- dependently, have been applied in several previous model- ling studies (ONCHOSIM [19, 20, 24–26]; EPIONCHO [21–23, 27–29]). A comparison of key features and key model parameters is presented in Table 1 and Table 2. There are many similarities, but the models also differ in some important aspects, e.g. on the extent to which het- erogeneities in the human population (e.g. in exposure to blackfly bites) and density dependencies in various pro- cesses are captured (e.g. in parasite establishment rate within humans and excess mortality of infected flies). The sections below provide a brief description of the models and their main characteristics. A detailed comparison of the two models and previously published predictions will also be presented elsewhere (Basáñez et al: River blind- ness: mathematical models for control and elimination, unpublished results). While past successes provide reason for optimism, an important question remains regarding where and when elimination can be achieved, and whether treatment strat- egies need to be adjusted to achieve the WHO targets. Work is ongoing to estimate when mass treatment can likely be stopped in different countries and sub-national regions. Important factors to consider when estimating elimination prospects include local transmission condi- tions (e.g. the endemicity level at baseline in the core of the transmission zone, vector competence, contiguity of a transmission zone), the start year of treatment, treatment frequency, achieved treatment coverage levels and compli- ance patterns, and complicating factors such as Loa loa co-endemicity, the occurrence of suboptimal responses, or lack of infrastructure [16–18]. All these factors to some ONCHOSIM d l b Model background ONCHOSIM is an individual-based model for simulating onchocerciasis transmission and con- trol in a dynamic human population, based on the tech- nique of stochastic microsimulation [30]. The underlying Page 3 of 16 Page 3 of 16 Stolk et al. Parasites & Vectors (2015) 8:552 Table 1 Overview of the main characteristics of the ONCHOSIM and EPIONCHO models Characteristics ONCHOSIM EPIONCHO Basic model structure Number and type of spatial locations modelled Single place Single place Population-based or individual- based Individual-based regarding humans and worms Population-based Way of representing infection in hosts Presence and density at individual level Mean density in population subgroups (e.g. age, sex, treatment compliance group). Prevalence as a function of mean density assuming an underlying negative binomial distribution Role of chance Stochastic Deterministic Interventions considered in previous publications Mass treatment, selective treatment (test and treat), vector control, Mass treatment, vector control Features included in the model Human population demographics Birth and death rate dynamically modelled; age and sex composition Birth and death rate, age and sex composition Heterogeneities in the human population Age, sex, life expectancy, level of exposure to blackflies, compliance with MDA, efficacy of treatment Age, sex, life expectancy, level of exposure to blackflies, compliance with MDA Blackfly population density Fixed input as annual biting rate (ABR); seasonal monthly biting rates Fixed input as ABR; seasonality in biting rates can be included Exposure to blackfly vectors Heterogeneous (dependent on age, sex, personal attractiveness to blackflies) Heterogeneous (dependent on age and sex) Uptake of infection by blackfly vectors Varying non-linearly (density-dependent) with infection intensity in human hosts Varying non-linearly (density-dependent) with infection intensity in human hosts Infection in blackfly vectors Density (average L3 load per fly) Density (average L3 load per fly) Excess mortality of infected flies No Yes Parasite acquisition in humans Proportional to mean number of L3 larvae inoculated, denoted by the success ratio Non-linearly (density-dependent) related to rate of exposure to L3 larvae Infection in humans Density (immature or mature worms, mf per skin snip) Density (non-fertile and fertile worms, mf per mg of skin) Diagnostic outcomes Mf count sampling to relate model predictions to data Sampling process and diagnostic performance of skin snipping not yet included Diagnostic outcomes generalised modelling framework has formed the basis for similar models for other helminthic diseases, including lymphatic filariasis [31], schistosomiasis [32] and soil- transmitted helminthiases (presented elsewhere in this collection [33]). ONCHOSIM d l b larvae that will develop, from L1 larvae, in flies after taking a blood meal. The biting rate varies between individuals, both randomly and as a function of host age and sex. Therefore, the rate of acquisition of new, incoming worms and the intensity of infection vary between individuals. The relative contribution of different individuals to infec- tion levels in the blackfly population varies in exactly the same way. Only a small, random proportion of the L3 lar- vae that are released during a bite will develop successfully into an adult worm, defined by a parameter named as the success ratio. The model simulates a dynamic human population, con- sisting of a discrete number of individuals. The population composition changes over time due to birth, aging and death of individuals. Through exposure to bites of Simu- lium damnosum vectors, humans are populated by worms and microfilariae (mf); transmission of infection between human individuals is simulated by means of one central population of blackflies. The fly density is expressed in terms of the average number of fly bites received per (adult) man per year, which is assumed to be constant over time with fixed seasonal variation during the year. At each fly bite, infection may be transferred from human to fly and vice versa. The model considers a non-linear rela- tionship between the mf intensity in human skin (microfi- laridermia) and the average number of infective stage (L3) Before introducing an intervention in the simulation, a burn-in period is included to allow infection levels to reach a dynamic, endemic equilibrium. The equilibrium infection levels can be adjusted by modifying assump- tions on the average biting rate and, if opportune, expos- ure heterogeneity among individuals. Mass ivermectin treatment programmes are simulated by specifying the timing of treatment and the therapeutic coverage (i.e. the proportion of the total population taking treatment). Page 4 of 16 Page 4 of 16 Stolk et al. ONCHOSIM d l b In this paper, we adopt a set of assumptions about ivermectin efficacy from a recent publication [20] (termed “assumption set 1” in the cited paper), which has been shown to fit well to trends in skin mf levels as observed in a community trial encompassing five consecutive annual ivermectin treatments in Ghana [35, 36]. According to this set of assumptions: i) the microfilaricidal efficacy of iver- mectin is 100 % and it acts instantaneously upon adminis- tration; ii) there is no macrofilaricidal effect; iii) the embryostatic effect causes all female worms to temporarily cease mf production, which then recovers gradually over time and reaches maximum production capacity after an average of 11 months; iv) the cumulative effect on female worm fertility amounts to an average 35 % reduction per treatment, with cumulative effects in worms repeatedly exposed to ivermectin. ONCHOSIM has been previously used to successfully mimic observed longitudinal epidemiological data from various locations [35–38], and has been used for policy making in the West-African Onchocerciasis Control Programme [19, 34]. Further, ONCHOSIM predictions fit reasonably well to longitudinal data from villages along the Gambia and Bakoye River basins in West Africa [20], where 15 to 17 years of annual and/or biannual ivermectin mass treatment have led to elimination of onchocerciasis [7, 8]. ONCHOSIM has been previously used to successfully mimic observed longitudinal epidemiological data from various locations [35–38], and has been used for policy making in the West-African Onchocerciasis Control Programme [19, 34]. Further, ONCHOSIM predictions fit reasonably well to longitudinal data from villages along the Gambia and Bakoye River basins in West Africa [20], where 15 to 17 years of annual and/or biannual ivermectin mass treatment have led to elimination of onchocerciasis [7, 8]. More information is provided in the additional files. Additional file 1 provides a formal mathematical descrip- tion of the model, instructions on installing and running the model, a complete overview of the probability distri- butions, functional relationships, and parameter values that are used for this study, and annotated input and out- put files. Additional file 2 contains a zip file, which in- cludes the computer simulation program itself (with the JAVA program code embedded in it), batch files used to run the model, PDF documentation of the XML input, and example input and output files. ONCHOSIM d l b Parasites & Vectors (2015) 8:552 Table 2 Parameter assumptions used for the comparisons presented in this paper Assumption ONCHOSIM EPIONCHO Life expectancy of adult worms 10 years [38] 10 years [38] Life expectancy of microfilariae 0.75 years [58] 1.25 years [59] Distribution of worm survival times Weibull Exponential Proportion of blood meals taken by vectors on humans 0.96 [30] and expert opinion 0.96 (matched to ONCHOSIM)a Macrofilaricidal effect of ivermectin Not included Not included Microfilaricidal effect of ivermectin 100 %, instantaneous upon administration [36] 98-99 % at 2 mo. post-treatment following [40] Embryostatic effect of ivermectin All female worms temporarily stop producing mf but resume production gradually, reaching maximum production capacity 11 months post-treatment on average [36] Fertile worms exposed to ivermectin decrease their mf production according to the dynamics presented in [40] and would fully recover if further untreated Cumulative effect on mf production by adult worms 35 % reduction in the rate of mf production per dose, on average [36]. 35 % reduction in the rate of mf production per dose [36]a aDifferent values were applied in previous publications, but for the current model comparison presented in this paper the assumptions were harmonized with those in ONCHOSIM Table 2 Parameter assumptions used for the comparisons presented in this paper Fertile worms exposed to ivermectin decrease their mf production according to the dynamics presented in [40] and would fully recover if further untreated The probability that a simulated individual participates in mass treatment with ivermectin is governed by age and sex (children under five years of age are not treated; a ran- dom proportion of women of reproductive age is not treated, assuming that they are pregnant or lactating), and a lifelong compliance factor (the higher the factor, the higher the probability that an individual participates in any given treatment round). Furthermore, some individ- uals never participate in treatment, because they are chronically ill or because they may refuse treatment (these individuals comprise the systematic non-compliers, 5 % of the population in this study). Regarding ivermectin effi- cacy, we assume the same working mechanism as in previ- ous simulation studies [19, 24, 34]. Drug effects include a microfilaricidal effect, a temporal embryostatic effect, and an anti-macrofilarial cumulative effect that reduces mf production by adult female worms with each treatment dose. ONCHOSIM d l b Model outputs ONCHOSIM keeps track of changes over time in the infection status (number of immature and ma- ture, male and female worms, and mf density per skin snip) in human individuals, and of the mean infective load in the blackfly populations. Output is obtained by simulating an epidemiological survey, in which mf intensity is measured for each individual as the mean mf count per skin snip (ss), assuming that two snips are taken of about 2 mg each. Measurement variation in mf counts is considered (de- scribed by a Poisson distribution around the true mf dens- ity) and mf counts may sometimes be false negative (with the probability of false negatives decreasing with higher mf Page 5 of 16 Stolk et al. Parasites & Vectors (2015) 8:552 loads). Individual outputs are aggregated to obtain informa- tion on the mf prevalence (proportion of all individuals with a positive mf count in either of the two snips), arith- metic mean of individuals’ mf counts per snip (per individ- ual calculated as the mean of two skin snips), and the geometric mean (calculated as exp [(Σ log (x +1))/n] - 1, with x being the an individual’s mean mf count per skin snip (as above) and n the number of individuals included). These outputs are provided for the population as a whole and stratified by age group and sex. In this paper, we always present the mf prevalence in the population aged 5 years and above. The community microfilarial load (CMFL) is equal to the geometric mean mf load per snip in adults aged ≥20 years [39]. reproducing observed pre-control age-mf (intensity) pro- files in Cameroon; patterns also reported in forest areas of Cameroon [42] and elsewhere in former OCP areas of West Africa [39]. EPIONCHO reflects pre-control infec- tion levels in a range of hypo-, meso-, hyper- and highly hyperendemic onchocerciasis foci by varying the annual biting rate (ABR, number of bites received per person per year) of the simuliid vectors. Model outputs The natural output of EPIONCHO is the per host mean number of mf per mg of skin. Microfilarial prevalence is determined by assuming a negative binomial distribution of mf among hosts with overdispersion par- ameter treated as a non-linear (hyperbolic) function of the (modelled) mean [43], and fitted to (pre-control) data on the prevalence and intensity of microfilaridermia in Cameroon [27]. EPIONCHO d l b Model background EPIONCHO is a deterministic on- chocerciasis transmission model that describes the rate of change with respect to time and host age (in both sexes) of the mean number of fertile and non-fertile female adult worms per host, the mean number of mf per milligram (mg) of skin, and the mean number of L3 larvae per simu- liid fly. Full mathematical details of EPIONCHO can be found in Turner et al. [21] and Basáñez et al: River blind- ness: mathematical models for control and elimination, unpublished results. Briefly, the model is based on a prototype presented by Basáñez and Boussinesq [27], ex- tended to include age and sex structure of the host popu- lation [28]; the population-level effects of a single [40] and multiple treatments with ivermectin, and increased pro- grammatic realism related to patterns of treatment cover- age and systematic non-compliance [21]. Aligning with ONCHOSIM and in accordance with empirical data [41], we have assumed that 5 % of the population is systematic- ally non-compliant with treatment. Additional files 3, 4 and 5 provide instructions for in- stalling and running EPIONCHO, and the source C code (EPIONCHO.c) and R script (EPIONCHO.R) needed to run the simulations presented in this paper. ONCHOSIM d l b In these data, the prevalence and intensity of microfilaridermia were measured by counting mf in two skin snips per person (from the right and left iliac crests), after 24 h incubation in saline. By assuming that this parameterization holds in all population age groups, EPIONCHO estimates: (a) mf prevalence in children aged ≥5 years and (b) by Monte Carlo simulation, and using an average weight of 1.7 mg per skin snip [44], the community microfilarial load (CMFL, the geometric mean intensity of mf per skin snip in people aged ≥20 years. Design of the model comparison study Simulated scenarios In this paper, we present a comparative modelling study to explore the level of agreement between the ONCHO- SIM and EPIONCHO models regarding three different outcomes. This was done for a range of pre-control en- demicity levels, varying from mesoendemic to very highly hyperendemic or holoendemic (mf prevalence in the population aged ≥5 years ranging from 51 % to 91 %). Treatment scenarios varied with respect to the achieved treatment coverage (50 %, 65 % or 80 %) and treatment frequency (annual, biannual). An overview of all scenarios The human demography reflects that of savannah areas of northern Cameroon, where the prevailing O. volvulus– Simulium damnosum sensu lato combinations (i.e. savannah parasites–S. damnosum sensu stricto / S. sirbanum) are re- sponsible for the most severe sequelae of onchocerciasis. The age distribution is assumed stationary and the popu- lation closed (i.e. no migration). The model captures age- and sex-specific host exposure to blackfly bites, Table 3 Setting characteristics and treatment scenarios for simulations Factors varied in the simulations: Values considered Setting characteristics Pre-control endemicity (mf prevalence in the population aged ≥5 years)a 51 %, 62 %, 81 %, 87 %, 91 % Treatment scenarios (treatment frequency and coverage constant over time) Population coverage of mass treatment Coverage low (50 %), intermediate (65 %), or high (80 %) Treatment frequency Annual or biannual Duration of mass treatment Up to 25 years aSee Table 4 for information regarding the corresponding biting rates and CMFL Table 3 Setting characteristics and treatment scenarios for simulations Factors varied in the simulations: Values considered Setting characteristics Pre-control endemicity (mf prevalence in the population aged ≥5 years)a 51 %, 62 %, 81 %, 87 %, 91 % Treatment scenarios (treatment frequency and coverage constant over time) Population coverage of mass treatment Coverage low (50 %), intermediate (65 %), or high (80 %) Treatment frequency Annual or biannual Duration of mass treatment Up to 25 years aSee Table 4 for information regarding the corresponding biting rates and CMFL Table 3 Setting characteristics and treatment scenarios for simulations aSee Table 4 for information regarding the corresponding biting rates and CMFL Table 3 Setting characteristics and treatment scenarios for simulations Pre-control endemicity (mf prevalence in the population aged ≥5 years)a Treatment scenarios (treatment frequency and coverage constant over time) Population coverage of mass treatment Treatment frequency Stolk et al. Parasites & Vectors (2015) 8:552 Page 6 of 16 is provided in Table 3. By tuning the assumed biting rates, both models were calibrated to the predefined levels of mf prevalence in the population aged ≥5 years (as this is the population group that typically participates in epidemio- logical surveys). For ONCHOSIM, the epidemiological settings are matched to the settings considered by Coffeng et al. [20], where the inter-individual variation in exposure to blackfly bites was low (see also Table 4 below). In this paper, we provide additional model output for the same simulated scenarios. EPIONCHO matched the pre-control levels of mf prevalence, whereas the assumed annual biting rates (partly influenced by the assumed proportion of hu- man blood meals taken by the vectors) and the resulting CMFL are not necessarily identical in the two models. arithmetic mean mf intensity in the whole population, for each of the five baseline mf prevalence levels consid- ered. The prevalence and intensity of mf were assessed annually at the moments of treatment, just before the scheduled treatment round. The dynamic changes in- between treatment rounds are therefore not visualized. For ONCHOSIM, we performed 150 repeated runs per scenario all with the exact same inputs. After exclusion of runs with extinction of infection during the burn-in period (only at the lowest endemicity level, where this occurs in about 10 % of simulation runs) we calculated the average trend in mf prevalence. For EPIONCHO, in accordance with the deterministic nature of the model, only a single simulation was needed per scenario. Outcome 2: treatment duration needed to achieve a provision operational threshold for treatment inter- ruption For each baseline mf prevalence and for the different treatment scenarios considered, we determined the minimum duration of mass treatment that would be required to bring the mf prevalence as measured just be- fore what would be the next treatment round below a provisional Operational Threshold for Treatment Inter- ruption followed by Surveillance (pOTTIS), as previously reported and defined in [22]. The pOTTIS is based on the working thresholds proposed by APOC in its conceptual and operational framework for onchocerciasis elimination with ivermectin treatment [12]. Table 3 Setting characteristics and treatment scenarios for simulations These thresholds are de- fined (by APOC) as an mf prevalence of <5 % in all sur- veyed villages and <1 % in 90 % of such villages, as well as fewer than 0.5 infective larvae per 1000 examined flies (which, given the probability that – near elimination – in- fective flies will carry only one L3 larva, translates into 0.05 % infective flies). The APOC criteria involve a dual threshold, to capture distribution of mf prevalence levels in multiple communities in an area. APOC’s first criterion (prevalence <5 % in all surveyed villages) suggests that bringing prevalence below 5 % should be sufficient for achieving elimination. The second criterion may serve to verify that mass treatment was effectively implemented throughout the area: if this 5 % threshold were reached Outcomes on which the models are being compared Outcomes on which the models are being compared In past publications, ONCHOSIM provided predictions of the treatment duration needed to drive the parasite popu- lation irreversibly to local elimination as evaluated many years post-treatment, while EPIONCHO focused on the time needed to bring mf prevalence below a critical threshold, measured just before what would be the next treatment round [19–21, 23, 43]. This was chosen to re- flect the provisional operational thresholds for treatment interruption and commencement of surveillance proposed by APOC in 2010. We now consider both outcomes, to allow comparison with previous work and to understand how the choice of endpoint influences the required dura- tions. In addition, we will compare the models’ predicted trends in infection indicators (prevalence and intensity of microfilaridermia) during mass ivermectin treatment. This is explained in more detail below. Outcome 1: predicted trends in infection with skin microfilariae during ivermectin mass treatment We compared the models with respect to their predicted trends in microfilarial infection over time during a 25- year programme of annual mass ivermectin treatment, assuming that 65 % of the total population is treated per round. In particular, we looked at predicted trends in mf prevalence among the population aged ≥5 years and the Table 4 Comparison of ONCHOSIM and EPIONCHO with respect to the annual biting rate and community microfilarial load (CMFL, the geometric mean no. of mf per skin snip in those aged 20 years and above) that correspond to the pre-set value of mf prevalence in the population aged ≥5 years matched by both models Pre-set value of mf prevalence in the 5+ population ONCHOSIM EPIONCHO ABR (bites / person / year) CMFL (mf/ss) ABR (bites / person / year) CMFL (mf/ss) 51 % 9,409 5.9 2,250 5.5 62 % 10,150 10.5 3,375 9.8 81 % 14,098 33.6 18,906 30.5 87 % 18,078 56.7 34,219 55.0 91 % 22,212 79.4 46,875 83.6 ONCHOSIM and EPIONCHO with respect to the annual biting rate and community microfilarial load (CMFL, the mf per skin snip in those aged 20 years and above) that correspond to the pre-set value of mf prevalence in the s matched by both models Stolk et al. Parasites & Vectors (2015) 8:552 Page 7 of 16 even in the communities closest to breeding sites, then considerably lower levels would be expected in most other communities with less intense transmission. Outcomes on which the models are being compared This defin- ition has been rendered compatible with the closed popu- lation structure of the two models under comparison by defining a single threshold. Rather than using the upper threshold of 5 %, which is still subject to uncertainty and may lead to misinterpretation of the criteria, we have chosen to use the weighted average of the upper and lower thresholds: when the modelled mf prevalence falls to <1.4 %, measured just before the next treatment round, the pOTTIS has been achieved [22]. The pOTTIS is as- sumed to refer to the mf prevalence in the population aged ≥5 years rather than in the total population, because children under 5 are generally excluded from field surveys or strongly underrepresented. as the fraction of 1000 repeated simulations that result in elimination. Elimination was defined as absence of infec- tion 50 years after the last mass treatment, where infection diagnosis was based on two skin snips per person (assum- ing that the chance of finding zero mf-positive individuals among all simulated individuals (~400) is negligible during sustainable transmission). As in previous ONCHOSIM publications, the required duration is the minimum num- ber of treatment rounds that result in a probability of elim- ination of ≥99 %. Deterministic models sometimes allow analytical explor- ation of breakpoints, e.g. in the absence of interventions or by applying simplifying assumptions on the dynamical responses elicited by interventions [45]. This is not feasible with relatively more complex models such as EPIONCHO. Therefore, for EPIONCHO we evaluated numerically whether the breakpoint was reached by tracking the para- site population long after cessation of the simulated inter- vention. The implicit breakpoint and hence required treatment duration to drive the parasite to elimination de- pend on assumptions concerning the mating probability (the probability that female worms are mated), which in turn is influenced by the worm sex ratio, the sexual system (monogamous or polygamous), and the distribution of adult worms in the host population [47]. For the purposes of this paper we have assumed a balanced sex ratio (1:1), a system of polygamy [48], and a Poisson distribution of adult worms in the human host population (assumed to follow a negative binomial distribution in previous papers), with male and female worms distributed together. To estimate the number of treatment rounds required for achieving the pOTTIS, we simulated the respective treatment scenarios (see below) for a maximum duration of 25 years. Availability of data and materials Data and simulation software (EPIONCHO and ONCH- OSIM) are made available or can be reproduced via the additional files included in this paper. See the descrip- tion of additional files below. Outcomes on which the models are being compared Trends in mf prevalence were simulated as de- scribed above for outcome 1, with mf prevalence mea- sured at the moments of treatment (either annually or biannually, always just before treatment). Treatment was assumed to be no longer needed if the average mf preva- lence dropped below the pOTTIS threshold. The required duration in years is then either the minimum number of annual treatments needed to reach the pOTTIS or the number of biannual treatments multiplied by 0.5. Outcome 3: the treatment duration that is needed to drive the parasite population irreversibly to local elimination The third outcome considered is the mini- mum required treatment duration that is needed to drive the parasite population irreversibly to local elimination, as previously done with ONCHOSIM and described by Coffeng et al. [20]. As laid out by the transmission break- point theory for dioecious parasite species [45, 46], the prevalence (or intensity) of infection does not need to be reduced exactly to zero for mass treatment to be able to stop. Below some epidemiological threshold, which de- pends on transmission conditions, the probability that a worm successfully reproduces and brings forth at least one new reproducing worm falls below 1 so that transmis- sion becomes unsustainable and the worm population will gradually disappear for the scenario analysed. Results The two models were calibrated to match the required pre-control mf prevalence levels in the population aged ≥5 years by adjusting the annual biting rate. Table 4 shows the biting rates that were used as well as the correspond- ing mf prevalence and CMFL levels. The relationship between annual biting rate and mf prevalence differs somewhat between the models (Fig. 1). The biting rates in ONCHOSIM varied from about 9 to 22 thousand to simu- late the required levels of mf prevalence (50–90% in the population aged ≥5 years), whereas in EPIONCHO the biting rates covered a wider range, from about 2 to 47 thousand bites per person per year. The corresponding predicted CMFL values (which were not matched by de- sign) are comparable for the two models (Table 4). With ONCHOSIM, the required duration of mass treat- ment was estimated based on the eventual occurrence of elimination in a simulation, 50 years after the last treat- ment, allowing for stochastic fade-out or natural disappear- ance. Because many processes simulated in ONCHOSIM involve probabilities, repeated model simulations based on the same assumptions will result in slightly different pre- dictions because of stochastic variation. Therefore, with ONCHOSIM, we estimated the probability of elimination Figure 2 compares the predicted trends in mf prevalence in the population aged ≥5 years during a 25-year mass Stolk et al. Parasites & Vectors (2015) 8:552 Page 8 of 16 40% 50% 60% 70% 80% 90% 100% 0 10,000 20,000 30,000 40,000 50,000 Mf prevalence (in 5+ population) Annual biting rate ONCHOSIM EPIONCHO Fig. 1 Relationship between the annual biting rate (bites per person per year) and microfilarial (mf) prevalence in the population aged 5 years and above in the two models treatment programme where 65 % of the population is treated annually with a single dose of ivermectin. Similarly, parasite population to local elimination for all settings and treatment scenarios. The same data are graphically repre- sented in Fig. 4 to visualize the patterns in the results. The EPIONCHO- and ONCHOSIM-predicted treatment dura- tions for reaching the pOTTIS are pretty close for settings with moderate baseline prevalence (51 or 62 % mf preva- lence). Yet, EPIONCHO predicts a greater lengthening in required treatment duration with increasing baseline en- demicity than ONCHOSIM; also predictions for areas with higher baseline endemicity levels (≥81 % mf prevalence) are more divergent. Results ONCHOSIM predicts that pOTTIS can still be reached by 20–25 rounds of annual mass treat- ment, if coverage is high enough (80 % required in the highest transmission settings) and that the required treat- ment duration can be reduced by ~35 % if mass treatment is provided biannually. EPIONCHO is more pessimistic, Figure 3 compares predicted trends in the arithmetic mean intensity of mf in the population (all ages) relative to the pre-control (endemic equilibrium) level. EPIONCHO predicts a fast initial decline in both mf prevalence and mean mf count for all 5 endemic settings, but the decline levels off and the two infection indicators tend to move towards a new equilibrium. In ONCHOSIM, the initial de- cline is less pronounced, but it does not level off as much. Eventually, the infection indicators reach zero faster in ONCHOSIM than in EPIONCHO. The difference be- tween the two models is more pronounced for the mf prevalence than for the mean mf intensity. Figure 3 compares predicted trends in the arithmetic mean intensity of mf in the population (all ages) relative to the pre-control (endemic equilibrium) level. EPIONCHO predicts a fast initial decline in both mf prevalence and mean mf count for all 5 endemic settings, but the decline levels off and the two infection indicators tend to move towards a new equilibrium. In ONCHOSIM, the initial de- cline is less pronounced, but it does not level off as much. Eventually, the infection indicators reach zero faster in ONCHOSIM than in EPIONCHO. The difference be- tween the two models is more pronounced for the mf prevalence than for the mean mf intensity. Table 5 summarises for both models the estimated re- quired durations to achieve the pOTTIS and to drive the Fig. 2 Comparison of expected trends in microfilarial (mf) prevalence during mass treatment, as predicted by ONCHOSIM and EPIONCHO, for settings with different baseline endemicity (mf prevalence in the population aged ≥5 years) assuming a coverage of 65 % Fig. 2 Comparison of expected trends in microfilarial (mf) prevalence during mass treatment, as predicted by ONCHOSIM and EPIONCHO, for settings with different baseline endemicity (mf prevalence in the population aged ≥5 years) assuming a coverage of 65 % Stolk et al. Parasites & Vectors (2015) 8:552 Page 9 of 16 Page 9 of 16 Fig. Results 3 Comparison of expected trends in arithmetic mean mf intensity during mass treatment, as predicted by ONCHOSIM and EPIONCHO, for settings with different baseline endemicity (mf prevalence in the population aged 5 years and above) assuming a coverage of 65 % Fig. 3 Comparison of expected trends in arithmetic mean mf intensity during mass treatment, as predicted by ONCHOSIM and EPIONCHO, for settings with different baseline endemicity (mf prevalence in the population aged 5 years and above) assuming a coverage of 65 % females. By fitting ONCHOSIM to data on mf loads ob- tained during an early community trial of annual ivermec- tin treatment in Asubende, Ghana [35], Plaisier et al. [36] had estimated a loss of mf production ranging from 22 to 40% per treatment round. A value of 35 % was recently used in ONCHOSIM by Coffeng et al. [20], but a more conservative value of 7 % (varied in a sensitivity analysis from 1 to 30 %) had been used in EPIONCHO by Turner et al. [22]. In this paper we have used the value of 35 %, which has yielded a good qualitative match for both models to the longitudinal parasitological data on mf loads from the feasibility of elimination study conducted by Diawara et al. [7] in some foci of Mali and Senegal [20]. suggesting that the pOTTIS cannot be achieved in settings with baseline mf prevalence of 81 % or higher, not even with 25 years of biannual treatment and 80 % coverage. EPIONCHO is also more pessimistic than ONCHOSIM about the possibility of driving the parasite population to local elimination. EPIONCHO suggests that this will only be achievable within 25 years for the setting with 51 % baseline mf prevalence, and that this would require longer continuation of mass treatment than required to achieve the pOTTIS. ONCHOSIM suggests that local extinction is achievable everywhere, although in settings with very high baseline endemicity this might require biannual treat- ment and/or high treatment coverage (80 %). For areas with moderate baseline endemicity (51 % or 62% mf prevalence), ONCHOSIM suggests that the required treat- ment duration for driving the parasite population to local elimination is shorter than that needed for achieving the pOTTIS. The reverse was found in settings with the high- est baseline mf prevalence. A previous modelling study by Bottomley et al. Discussion This paper presents for the first time a vis-à-vis compari- son of the ONCHOSIM and EPIONCHO models. We found that whilst EPIONCHO predicts a faster initial decline in mf prevalence and intensity than ONCHO- SIM, EPIONCHO is more pessimistic about the long- term prospects of achieving the pOTTIS and local elimination. Results [49]—who fitted a model to data from a community trial of biannual ivermectin treatment in Guatemala [44]—had reached the conclusion that the effect of repeated ivermectin treatments on mf production by adult worms was not cumulative. Other studies, e.g. [50, 51], have reported that repeated ivermectin doses may have deleterious effects on adult worms, but the mechanisms and magnitude of such effects remain poorly understood. Model predictions on required treatment duration are also highly sensitive to this param- eter, and both models therefore assumed a cumulative effect. It remains, however, critical to better understand the impact of ivermectin on the survival and reproduction (the components of fitness) of O. volvulus, to improve our abil- ity to accurately project the outcome of interventions and to appreciate the potential evolutionary implications of such interventions (e.g. selection pressure due to treatment [16]). Harmonized input assumptions For the purpose of the presented comparisons, we harmo- nized some key assumptions which have previously been identified as very influential on the duration of ivermectin MDA programmes [21]. One critical assumption is the magnitude and irreversibility of the effect of ivermectin on fertility (production of live mf) by adult O. volvulus The fraction of bites that a blackfly takes on humans (assumed to be 0.96) is also a key parameter. By aligning it between the two models, we brought together the annual biting rates necessary to reproduce initial mf prevalence values (Fig. 1). However, field studies on blood host choice Page 10 of 16 Stolk et al. Harmonized input assumptions Parasites & Vectors (2015) 8:552 by onchocerciasis vectors [52] have indicated that the human blood index may be variable among compo biting rates needed to produce different infection en- demicity levels Table 5 Comparison of ONCHOSIM and EPIONCHO with respect to estimated duration of treatment that is needed to bring mf prevalence below the provisional operational threshold for treatment interruption followed by commencement of surveillance (pOTTIS) of 1.4 %, measured just before what would be the next treatment round, and the estimated duration of treatment needed to drive the parasite population to local elimination in the absence of further treatment (allowing for the slow natural extinction in the absence of further interventions) Approximate initial mf prevalence (%) in the population aged ≥ 5 years Coverage (%) Treatment duration needed to bring the 12-month or 6-month post-treatment mf prevalence below pOTTIS (years) Treatment duration needed to drive the parasite population irreversibly to extinction in the absence of further treatment (years) ONCHOSIM EPIONCHO ONCHOSIM EPIONCHO Annual treatment 51 50 18 17 12 >25 65 14 15 8 23 80 12 12 6 21 62 50 21 24 14 >25 65 16 20 10 >25 80 14 17 8 >25 81 50 >25 >25 >25 >25 65 21 >25 18 >25 80 17 >25 15 >25 87 50 >25 >25 >25 >25 65 25 >25 >25 >25 80 20 >25 20 >25 91 50 >25 >25 >25 >25 65 >25 >25 >25 >25 80 23 >25 >25 >25 Biannual treatment 51 50 12.5 12 6 21 65 10 11 4.5 20 80 8 10 4 19.5 62 50 14 17 8.5 >25 65 11 16 6 >25 80 9.5 10 5 >25 81 50 18.5 >25 17 >25 65 13.5 >25 12 >25 80 12 >25 10 >25 87 50 22.5 >25 24 >25 65 15.5 >25 16.5 >25 80 13.5 >25 14 >25 91 50 >25 >25 >25 >25 65 17 >25 21 >25 80 14.5 >25 18 >25 Results are shown for different settings, varying with respect to the pre-control mf prevalence in the population aged ≥5 years, and for several treatment scenarios, varying with respect to the treatment frequency and achieved coverage (defined as the percentage of people who receive treatment in the total population) Results are shown for different settings, varying with respect to the pre-control mf prevalence in the population aged ≥5 years, and for several treatment scenarios, varying with respect to the treatment frequency and achieved coverage (defined as the percentage of people who receive treatment in the total population) biting rates needed to produce different infection en- demicity levels. Harmonized input assumptions by onchocerciasis vectors [52] have indicated that the human blood index may be variable among compo- nent species of the S. damnosum s.l. complex, and this information remains important when modelling trans- mission in different epidemiological settings across Africa, in particular to get an accurate reflection of by onchocerciasis vectors [52] have indicated that the human blood index may be variable among compo- nent species of the S. damnosum s.l. complex, and this information remains important when modelling trans- mission in different epidemiological settings across Africa, in particular to get an accurate reflection of We also harmonized assumptions on the proportion of the population that is systematically non-compliant with treatment, a common parameter in both models. This was done, because a core group of individuals who are Stolk et al. Parasites & Vectors (2015) 8:552 Page 11 of 16 Fig. 4 Duration of mass ivermectin treatment in years that is needed to bring mf prevalence below the pOTTIS (red lines and symbols) or to eventually reach local elimination (blue lines and symbols), for ONCHOSIM (left) and EPIONCHO (right) and for annual (top) and biannual treatment (bottom). Dashed lines in each graph connect estimates obtained for different endemicity levels under the assumption that 65 % of the total population is treated per round (coverage). The vertical bars indicate how the duration would change if the coverage was 50 % per round (triangles) or 80 % (circles). To be able to differentiate the prediction intervals obtained for the different endpoints, the results are displayed slightly to the left or right of the actual simulated baseline prevalence (+/−0.6 %) Fig. 4 Duration of mass ivermectin treatment in years that is needed to bring mf prevalence below the pOTTIS (red lines and symbols) or to eventually reach local elimination (blue lines and symbols), for ONCHOSIM (left) and EPIONCHO (right) and for annual (top) and biannual treatment (bottom). Dashed lines in each graph connect estimates obtained for different endemicity levels under the assumption that 65 % of the total population is treated per round (coverage). The vertical bars indicate how the duration would change if the coverage was 50 % per round (triangles) or 80 % (circles). Harmonized input assumptions To be able to differentiate the prediction intervals obtained for the different endpoints, the results are displayed slightly to the left or right of the actual simulated baseline prevalence (+/−0.6 %) untreated and remain infected, potentially provides a source of onward transmission in the human host popula- tion, as was also indicated by epidemiological observations of lymphatic filariasis in Haiti, where continuing transmis- sion was related to rates of systematic noncompliance [53]. Harmonization of assumptions on systematic non- compliance does not make the models completely compar- able; differences remain in the distribution of treatments over the remainder of the population because of the differ- ent approaches to modelling compliance patterns. We need to understand better how treatment compliance pat- terns can best be modelled. More programmatic data on patterns of individual compliance to inform the mathemat- ical constructs used to model compliance are therefore essential [54]. The factors contributing to differences in long-term predictions are discussed below. Here we discuss the fac- tors that contribute to differences in the shorter-term predictions. The differences in the initial decline in mf intensity may be explained by somewhat different assumptions regard- ing the temporal dynamics of the microfilaricidal effect of ivermectin as well as the rate of mf production by female worms and mf lifespan, leading to different mf repopula- tion rates in the period between treatments. This, how- ever, does not fully explain the more marked differences in predicted mf prevalence trends. The individual-based model ONCHOSIM always predicts a relatively slow ini- tial decline in prevalence, because treated individuals are expected to remain mf positive for some time, albeit with considerably lower mf loads. This is in line with observa- tions from a study in Ghana, which showed that mf preva- lence rapidly bounced back in the interval between treatment rounds, nearly to pre-treatment levels, while the bounce back in mean mf intensity is less pronounced [35]. In EPIONCHO, mf prevalence is indirectly derived from the predicted mean mf load, through a non-linear prevalence–intensity relationship fitted to pre-control data Predicted trends in infection during mass treatment In spite of harmonized treatment efficacy assumptions, EPIONCHO predicted a faster initial decline in mean mf intensity and mf prevalence than ONCHOSIM. In the lon- ger term, ONCHOSIM predicts that infection intensity will decline to zero everywhere, while EPIONCHO sug- gests that mf intensity may stabilizes at a level above zero. Stolk et al. Parasites & Vectors (2015) 8:552 Page 12 of 16 [43]. In this relationship, low mf loads are associated with similarly low mf prevalence levels. The relationship be- tween the two indicators was assumed to remain un- changed during mass treatment, for consistency with previous EPIONCHO publications. This assumption will have to be adjusted in future work, as the mf prevalence- intensity is likely to be altered by mass treatment, due to the direct microfilaricidal effect of treatment and the rela- tively slow rate of mf repopulation. Quantification of the post-treatment relationship, ideally using parasitological data obtained during MDA programmes, is therefore an imperative area of further investigation for EPIONCHO. predictions for areas with higher baseline endemicity levels became more pessimistic and divergent. ONCHO- SIM suggests that reaching the pOTTIS would often still be feasible, albeit with longer continuation of treatment, higher coverage, or more frequent treatment. EPIONCHO, however, suggests that even 25 years of biannual treatment with 80 % coverage is not sufficient to achieve the pOTTIS. This is reflected in the EPIONCHO-predicted trends in mf intensity and prevalence, which tend to stabilize at a new non-zero equilibrium after long-term mass treatment (Figs. 2 and 3). ONCHOSIM is also more optimistic than EPIONCHO about the possibility of driving the parasite population to local extinction. EPIONCHO suggests that the parasite can only be driven to elimination in settings with moder- ate baseline mf prevalence, although this would require longer continuation of treatment than needed to achieve the pOTTIS. ONCHOSIM suggests that the parasite population would be driven to elimination even before the pOTTIS is reached in settings with moderate baseline en- demicity; elimination can also be achieved in settings with higher baseline mf prevalence, although treatment will have to be continued longer than needed for achieving the pOTTIS. This suggests that the fixed operational elimin- ation thresholds proposed by APOC may overestimate the required duration for elimination in the former settings, but underestimate it in the latter. Predicted trends in infection during mass treatment The model-predicted trends in infection prevalence and intensity, as well as corresponding frequency distributions of mf counts, should be compared against epidemiological data on trends in mf prevalence and intensity during mass ivermectin treatment. Such data are available from the previously mentioned 5-year community intervention trial on the impact of ivermectin mass treatment that was carried out in a highly endemic setting in Ghana [35]. ONCHOSIM has been fitted to these data [36], and the validity of EPIONCHO-predicted trends can be tested against the same data. However, models should also be tested with similar data from other endemic settings, cov- ering a range of pre-control endemicity levels. Required duration to reach the pOTTIS or to drive the parasite population to local elimination Long-term predictions on the time needed to reach the pOTTIS or drive the parasite population to local elimin- ation should be interpreted with caution for both models. It will be difficult to validate the models’ predictions regarding the time needed to drive the parasite locally to elimination. Yet, empirical data may help to validate pre- dicted durations for reaching the pOTTIS. In this respect, useful data are available from a study performed in Mali and Senegal, which provided the first evidence that oncho- cerciasis can be eliminated in Africa through ivermectin mass treatment [7, 8]. Baseline endemicity levels of these regions reflect the lower range of values considered in this study. Data from epidemiological monitoring of ongoing elimination programmes in Africa (such as [9]) will also be informative, in particular if baseline data are available and the area is highlyendemic. Whether or not elimination will really be feasible in very highly endemic areas, with ei- ther annual or biannual treatment, remains an important question. An important question for ongoing onchocerciasis elim- ination programmes concerns the required duration of mass treatment. We explored this on the basis of two endpoints, namely 1) the duration of ivermectin mass treatment required to reach a defined threshold of mf prevalence below which treatment can be stopped (the pOTTIS), and 2) the duration required to drive the para- site locally to elimination, even without further interven- tions. The first reflects operational criteria for deciding when to stop interventions, although the critical threshold remains to be validated. A limitation of the pOTTIS ap- proach is the focal nature of onchocerciasis, whereby communities with ongoing transmission may act as a source of new infections for those communities where the infection has been eliminated. It is noteworthy that nei- ther EPIONCHO nor ONCHOSIM currently capture spatial transmission processes that may couple transmis- sion among geographically distinct foci. Hence, the elim- ination projections should be interpreted as capturing the likely outcome of interventions undertaken in circum- scribed foci with negligible influx of extraneous infections. Al h h di d d i i f i d i Possible explanations for differences in required durations for elimination elimination of the parasite population (stochastic fade- out), which becomes increasingly likely at very low inten- sities of infection, especially for small settings (villages) with a couple of hundred inhabitants (as assumed by ONCHOSIM). Secondly, the models differ with respect to assumptions about density dependence in the various pro- cesses involved in transmission dynamics (as indicated in Table 1), which may also be important for elimination pros- pects [45, 55]. In particular, EPIONCHO includes a (nega- tive) density-dependent relationship between the annual transmission potential and the parasite establishment rate; ONCHOSIM does not capture this mechanism, which makes the model more optimistic. Thirdly, the assumed distribution of adult worm and microfilarial survival times and assumptions regarding mf productivity in relation to worm-age may play a role. EPIONCHO assumes an expo- nential distribution of worm survival times with a long right tail, implying that worm mortality rates are independent of worm age (an implicit assumption of the exponential model). ONCHOSIM assumes a Weibull distribution [38], a more symmetrical distribution with the same mean sur- vival time but a shorter right tail, implying age-dependency of worm-mortality rates. Therefore, it takes considerably longer for the parasite population to die out naturally in EPIONCHO than in ONCHOSIM. In addition to this, ONCHOSIM assumes that the mf production rate declines in older worms, so that the relatively old worm population remaining after long-term ivermectin mass treatment has a relatively low mf production. Such a process is not consid- ered by EPIONCHO. Lastly, the distribution of adult worms among the human population will play a role again through its influence on the mating probability. This as- sumed distribution is explicit in EPIONCHO (in this paper by using a Poisson distribution) and implicit in ONCHO- SIM, driven by between-host heterogeneities in exposure and compliance with treatment. establishment rate at low levels of transmission intensity, a lower biting rate is initially required to produce the same prevalence and (approximate) intensity (CMFL, Table 4) of infection as ONCHOSIM. However, for higher ende- micities, and due to the action of the density-dependent establishment of adult worms that is modelled in EPIONCHO but not in ONCHOSIM (Table 1), a higher biting rate is required by EPIONCHO to arrive at the same levels of endemic infection prevalence (and inten- sity) as ONCHOSIM. Possible explanations for differences in required durations for elimination Disentangling the relative importance of different as- sumptions for various outcomes would require in-depth theoretical research, which is beyond the scope of this paper. This can be done through the development and stepwise comparison of structurally different models of increasing complexity and realism, similar to a previous study on HIV elimination models [56]. To understand which level of complexity is required to address policy questions on control and elimination, it would also be useful to consider the predicted frequency distributions of mf among the host population. Possible explanations for differences in required durations for elimination Although predicted trends in infection during mass treatment differ between the two models, estimates of the required duration of annual treatment for achieving the pOTTIS were comparable for settings with moderate baseline mf prevalence (51–62 % mf prevalence). The Several factors contribute to the longer treatment duration required for achieving elimination in EPIONCHO com- pared to ONCHOSIM, in spite of the faster initial drop in mf prevalence and to a lesser extent intensity. Firstly, EPIONCHO does not account for the possibility of chance Stolk et al. Parasites & Vectors (2015) 8:552 Page 13 of 16 elimination of the parasite population (stochastic fade- out), which becomes increasingly likely at very low inten- sities of infection, especially for small settings (villages) with a couple of hundred inhabitants (as assumed by ONCHOSIM). Secondly, the models differ with respect to assumptions about density dependence in the various pro- cesses involved in transmission dynamics (as indicated in Table 1), which may also be important for elimination pros- pects [45, 55]. In particular, EPIONCHO includes a (nega- tive) density-dependent relationship between the annual transmission potential and the parasite establishment rate; ONCHOSIM does not capture this mechanism, which makes the model more optimistic. Thirdly, the assumed distribution of adult worm and microfilarial survival times and assumptions regarding mf productivity in relation to worm-age may play a role. EPIONCHO assumes an expo- nential distribution of worm survival times with a long right tail, implying that worm mortality rates are independent of worm age (an implicit assumption of the exponential model). ONCHOSIM assumes a Weibull distribution [38], a more symmetrical distribution with the same mean sur- vival time but a shorter right tail, implying age-dependency of worm-mortality rates. Therefore, it takes considerably longer for the parasite population to die out naturally in EPIONCHO than in ONCHOSIM. In addition to this, ONCHOSIM assumes that the mf production rate declines in older worms, so that the relatively old worm population remaining after long-term ivermectin mass treatment has a relatively low mf production. Such a process is not consid- ered by EPIONCHO. Lastly, the distribution of adult worms among the human population will play a role again through its influence on the mating probability. This as- sumed distribution is explicit in EPIONCHO (in this paper by using a Poisson distribution) and implicit in ONCHO- SIM, driven by between-host heterogeneities in exposure and compliance with treatment. Additional files Additional file 1: A PDF file, providing a formal mathematical description of the model, instructions on installing and running the model, a complete overview of the probability distributions, functional relationships, and parameter values that are used for this study, and annotated input and output files (Documentation ONCHOSIM v2.58Ap9.pdf). (PDF 1360 kb) Additional file 1: A PDF file, providing a formal mathematical description of the model, instructions on installing and running the model, a complete overview of the probability distributions, functional relationships, and parameter values that are used for this study, and annotated input and output files (Documentation ONCHOSIM v2.58Ap9.pdf). (PDF 1360 kb) Additional file 2: A zip-file, which includes the computer simulation program itself (with the JAVA program code embedded in it), batch files used to run the model, PDF documentation of the XML input, and example input and output files. Instructions on how to run the model are provided in Additional file 1 (ONCHOSIM simulation program.zip). (DOCX 15 kb) Additional file 3: A Word document containing instructions for installing and running EPIONCHO (Instructions for installing & running EPIONCHO.docx). (DOCX 71 kb) Additional file 4: Source EPIONCHO code, written in programming language C (EPIONCHO.c). (C 30 kb) Additional file 5: R script needed to run the simulations presented in this paper (EPIONCHO.R). (R 5 kb) Additional file 1: A PDF file, providing a formal mathematical description of the model, instructions on installing and running the model, a complete overview of the probability distributions, functional relationships, and parameter values that are used for this study, and annotated input and output files (Documentation ONCHOSIM v2.58Ap9.pdf). (PDF 1360 kb) Additional file 2: A zip-file, which includes the computer simulation program itself (with the JAVA program code embedded in it), batch files used to run the model, PDF documentation of the XML input, and example input and output files. Instructions on how to run the model are provided in Additional file 1 (ONCHOSIM simulation program.zip). (DOCX 15 kb) Additional file 3: A Word document containing instructions for installing and running EPIONCHO (Instructions for installing & running EPIONCHO.docx). (DOCX 71 kb) Additional file 4: Source EPIONCHO code, written in programming language C (EPIONCHO.c). (C 30 kb) Additional file 5: R script needed to run the simulations presented in this paper (EPIONCHO.R). (R 5 kb) 2. Convit J, Schuler H, Borges R, Olivero V, Dominguez-Vazquez A, Frontado H, et al. Author details 1 1Department of Public Health, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands. 2London Centre for Neglected Tropical Disease Research, Department of Infectious Disease Epidemiology, School of Public Health, Imperial College London, London, UK. Funding The authors of this paper gratefully acknowledge funding of the NTD Modelling Consortium by the Bill and Melinda Gates Foundation in partnership with the Task Force for Global Health. MW and MGB also acknowledge support from the Wellcome Trust (http://www.wellcome.ac.uk; grant number 092677/Z/10/Z); MGB thanks the Royal Society-Leverhulme Trust (http://royalsociety.org/) for an Africa Award. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The views, opinions, assumptions or any other information set out in this article are solely those of the authors. Our next steps using EPIONCHO and ONCHOSIM will include testing model-predicted trends with observed trends in infection during mass treatment, elucidating the differences between the pOTTIS and the transmission breakpoints, refining operational guidance to programme managers based on these results, and identifying APOC projects where elimination goals can be achieved with current strategies and where adjusted, alternative, or complementary interventions are required. Acknowledgements We thank Dr Hugo Turner at the London Centre for Neglected Tropical Disease Research, Imperial College London, for helpful discussions on the technical details of EPIONCHO. We thank dr. Roel Bakker for his help in preparing the documentation about ONCHOSIM. Competing interests The authors declare that they have no competing interest. 11. Katabarwa MN, Lakwo T, Habomugisha P, Agunyo S, Byamukama E, Oguttu D, et al. Transmission of Onchocerca volvulus continues in Nyagak-Bondo focus of northwestern Uganda after 18 years of a single dose of annual treatment with ivermectin. Am J Trop Med Hyg. 2013;89(2):293–300. 11. Katabarwa MN, Lakwo T, Habomugisha P, Agunyo S, Byamukama E, Oguttu D, et al. Transmission of Onchocerca volvulus continues in Nyagak-Bondo focus of northwestern Uganda after 18 years of a single dose of annual treatment with ivermectin. Am J Trop Med Hyg. 2013;89(2):293–300. 12. WHO/APOC. Conceptual and operational framework of onchocerciasis References 1 R d i Not applicable. 1. Rodriguez-Pérez MA, Lutzow-Steiner MA, Segura-Cabrera A, Lizarazo-Ortega C, Dominguez-Vazquez A, Sauerbrey M, et al. Rapid suppression of Onchocerca volvulus transmission in two communities of the Southern Chiapas focus, Mexico, achieved by quarterly treatments with Mectizan. Am J Trop Med Hyg. 2008;79(2):239–44. 1. Rodriguez-Pérez MA, Lutzow-Steiner MA, Segura-Cabrera A, Lizarazo-Ortega C, Dominguez-Vazquez A, Sauerbrey M, et al. Rapid suppression of Onchocerca volvulus transmission in two communities of the Southern Chiapas focus, Mexico, achieved by quarterly treatments with Mectizan. Am J Trop Med Hyg. 2008;79(2):239–44. Conclusion As was to be expected, this revealed several differences in model predictions, in spite of harmonization Stolk et al. Parasites & Vectors (2015) 8:552 Page 14 of 16 Page 14 of 16 of some key parameters. We identified several explanations for the differences, which will be further explored to help to understand strengths and weaknesses of the different modelling approaches and to help to reach consensus on predicted timeframes and optimum interventions for the elimination of onchocerciasis in Africa. Additional files Traore MO, Sarr MD, Badji A, Bissan Y, Diawara L, Doumbia K, et al. Proof-of- principle of onchocerciasis elimination with ivermectin treatment in endemic foci in Africa: final results of a study in Mali and Senegal. PLoS Negl Trop Dis. 2012;6(9):e1825. 8. Traore MO, Sarr MD, Badji A, Bissan Y, Diawara L, Doumbia K, et al. Proof-of- principle of onchocerciasis elimination with ivermectin treatment in endemic foci in Africa: final results of a study in Mali and Senegal. PLoS Negl Trop Dis. 2012;6(9):e1825. Conclusion With the eventual aim to improve the predictive accuracy of simulation models for onchocerciasis transmission and control, and shed more light on whether current interven- tions are on track to achieve the time-bound elimination goals, two modelling groups working from different meth- odological traditions have joined forces to harmonize their models and examine the level of agreement in their pre- dictions. This paper focused on comparing, contrasting and understanding the similarities and differences in projected elimination outcomes by two independently developed, well-established models for onchocerciasis transmission, ONCHOSIM and EPIONCHO. Predicting eventual achievement of elimination is a challenge in infec- tious disease modelling, and possibly even more so when it concerns neglected tropical diseases, because of a general lack of long-term empirical data on the outcome of interest and gaps in knowledge on influential key population- biological parameters. This makes cross-validation between models particularly relevant: converging results help to build trust in predictions, while deviations trigger investi- gation into the causes and re-evaluation of available evi- dence which helps to improve model quality. Transparency is required and following “good modelling practice” [57] we provide complete access to the models, with the neces- sary documentation. EPIONCHO and ONCHOSIM also differ considerably in their assumptions regarding the life expectancy of microfilariae, being 0.75 years in ONCHOSIM and 1.25 years in EPIONCHO (Table 2). This is unlikely to have a strong influence on the projected programme dura- tions, because (a) the potency of ivermectin against mf is such that their natural life-span becomes much less rele- vant and (b) the transmission breakpoint (and the chance of stochastic fade-out) is much more influenced by the life span of adult worms that have a life-expectancy an order of magnitude greater than that of mf (about 10 years versus 1 year). Yet, this difference may explain at least partly—and in combination with the different modelled density-dependent population processes—the markedly different shapes in the relationship between the fitted an- nual biting rate and the pre-set endemic mf prevalence presented in Fig. 1. In EPIONCHO, on account of the lon- ger life expectancy of mf, and the greater parasite For this first model comparison, we have used a limited set of hypothetical scenarios regarding epidemiological fea- tures (initial endemicity, mf prevalence, CMFL and vector biting rates), ranging from mesoendemic to holoendemic onchocerciasis. Abbreviations ABR: Annual biting rate; APOC: African Programme for Onchocerciasis Control; CMFL: Community microfilarial load; mf: Microfilariae/microfilarial; mg: Milligram; OEPA: Onchocerciasis Elimination Program for the Americas; OCP: Onchocerciasis Control Programme in West Africa; pOTTIS: Provisional operational thresholds for treatment interruption followed by surveillance; ss: Skin snip. 9. Tekle AH, Elhassan E, Isiyaku S, Amazigo UV, Bush S, Noma M, et al. Impact of long-term treatment of onchocerciasis with ivermectin in Kaduna State. Nigeria: first evidence of the potential for elimination in the operational area of the African Programme for Onchocerciasis Control Parasit Vectors. 2012;5:28. 9. Tekle AH, Elhassan E, Isiyaku S, Amazigo UV, Bush S, Noma M, et al. Impact of long-term treatment of onchocerciasis with ivermectin in Kaduna State. Nigeria: first evidence of the potential for elimination in the operational area of the African Programme for Onchocerciasis Control Parasit Vectors. 2012;5:28. 10. Katabarwa MN, Eyamba A, Nwane P, Enyong P, Yaya S, Baldiagai J, et al. Seventeen years of annual distribution of ivermectin has not interrupted onchocerciasis transmission in north region, cameroon. Am J Trop Med Hyg. 2011;85(6):1041–9. 10. Katabarwa MN, Eyamba A, Nwane P, Enyong P, Yaya S, Baldiagai J, et al. Seventeen years of annual distribution of ivermectin has not interrupted onchocerciasis transmission in north region, cameroon. Am J Trop Med Hyg. 2011;85(6):1041–9. Additional files Interruption of Onchocerca volvulus transmission in Northern Venezuela. Parasit Vectors. 2013;6(1):289. 3. Rodriguez-Pérez MA, Fernandez-Santos NA, Orozco-Algarra ME, Rodriguez- Atanacio JA, Dominguez-Vazquez A, Rodriguez-Morales KB, et al. Elimination of onchocerciasis from Mexico. PLoS Negl Trop Dis. 2015;9(7):e0003922. Additional file 2: A zip-file, which includes the computer simulation program itself (with the JAVA program code embedded in it), batch files used to run the model, PDF documentation of the XML input, and example input and output files. Instructions on how to run the model are provided in Additional file 1 (ONCHOSIM simulation program.zip). (DOCX 15 kb) 4. Progress towards eliminating onchocerciasis in the WHO Region of the Americas. verification by WHO of elimination of transmission in Colombia. Wkly Epidemiol Rec. 2013;88(36):381–5. y p 5. West S, Munoz B, Sommer A. River blindness eliminated in Colombia. Ophthalmic Epidemiol. 2013;20(5):258–9. 6. Lovato R, Guevara A, Guderian R, Proano R, Unnasch T, Criollo H, et al. Interruption of infection transmission in the onchocerciasis focus of Ecuador leading to the cessation of ivermectin distribution. PLoS Negl Trop Dis. 2014;8(5):e2821. Additional file 3: A Word document containing instructions for installing and running EPIONCHO (Instructions for installing & running EPIONCHO.docx). (DOCX 71 kb) Additional file 3: A Word document containing instructions for installing and running EPIONCHO (Instructions for installing & running EPIONCHO.docx). (DOCX 71 kb) 7. Diawara L, Traore MO, Badji A, Bissan Y, Doumbia K, Goita SF, et al. Feasibility of onchocerciasis elimination with ivermectin treatment in endemic foci in Africa: first evidence from studies in Mali and Senegal. PLoS Negl Trop Dis. 2009;3(7):e497. 7. Diawara L, Traore MO, Badji A, Bissan Y, Doumbia K, Goita SF, et al. Feasibility of onchocerciasis elimination with ivermectin treatment in endemic foci in Africa: first evidence from studies in Mali and Senegal. PLoS Negl Trop Dis. 2009;3(7):e497. Additional file 4: Source EPIONCHO code, written in programming language C (EPIONCHO.c). (C 30 kb) Additional file 4: Source EPIONCHO code, written in programming language C (EPIONCHO.c). (C 30 kb) Additional file 4: Source EPIONCHO code, written in programming language C (EPIONCHO.c). (C 30 kb) Additional file 5: R script needed to run the simulations presented in this paper (EPIONCHO.R). (R 5 kb) Additional file 5: R script needed to run the simulations presented in this paper (EPIONCHO.R). (R 5 kb) Additional file 5: R script needed to run the simulations presented in this paper (EPIONCHO.R). (R 5 kb) 8. Ethics approval and consent to participate Not applicable. pp Not applicable. Received: 31 August 2015 Accepted: 8 October 2015 Received: 31 August 2015 Accepted: 8 October 2015 Authors' contributions Plaisier AP, van Oortmarssen GJ, Remme J, Alley ES, Habbema JD. The risk and dynamics of onchocerciasis recrudescence after cessation of vector control. Bull World Health Organ. 1991;69(2):169–78. 38. Plaisier AP, van Oortmarssen GJ, Remme J, Habbema JD. The reproductive lifespan of Onchocerca volvulus in West African savanna. Acta Trop. 1991;48(4):271–84. 18. Kim YE, Remme JH, Steinmann P, Stolk WA, Roungou JB, Tediosi F. Control, elimination, and eradication of river blindness: scenarios, timelines, and ivermectin treatment needs in Africa. PLoS Negl Trop Dis. 2015;9(4):e0003664. 39. Remme J, Ba O, Dadzie KY, Karam M. A force-of-infection model for onchocerciasis and its applications in the epidemiological evaluation of the Onchocerciasis Control Programme in the Volta River basin area. Bull World Health Organ. 1986;64(5):667–81. 19. 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Comput Methods Programs Biomed. 1990;31(1):43–56. 46. Anderson RM, May RM. Infectious diseases of humans: dynamics and control. Oxford: Oxford University Press; 1991. 47. May RM. Togetherness among schistosomes: its effects on the dynamics of the infection. Math Biosci. 1977;35:301–43. 26. Coffeng LE, Stolk WA, Zoure HG, Veerman JL, Agblewonu KB, Murdoch ME, et al. African Programme for Onchocerciasis Control 1995–2015: updated health impact estimates based on new disability weights. PLoS Negl Trop Dis. 2014;8(6):e2759. 48. Schulz-Key H, Karam M. Periodic reproduction of Onchocerca volvulus. Parasitol Today. 1986;2(10):284–6. 49. Bottomley C, Isham V, Collins RC, Basáñez MG. Rates of microfilarial production by Onchocerca volvulus are not cumulatively reduced by multiple ivermectin treatments. Parasitology. 2008;135(13):1571–81. 27. Basáñez MG, Boussinesq M. Population biology of human onchocerciasis. Philos Trans R Soc Lond B Biol Sci. 1999;354(1384):809–26. 28. Filipe JAN, Boussinesq M, Renz A, Collins RC, Vivas-Martinez S, Grillet ME, et al. Human infection patterns and heterogeneous exposure in river blindness. Proc Natl Acad Sci U S A. 2005;102(42):15265–70. 50. Gardon J, Boussinesq M, Kamgno J, Gardon-Wendel N, Demanga N, Duke BO. Effects of standard and high doses of ivermectin on adult worms of Onchocerca volvulus: a randomised controlled trial. Lancet. 2002;360(9328):203–10. 29. Turner HC, Walker M, Lustigman S, Taylor DW, Basáñez MG. Human onchocerciasis: modelling the potential long-term consequences of a vaccination programme. PLoS Negl Trop Dis. 2015;9(7):e0003938. 51. Cupp EW, Cupp MS. Authors' contributions WAS, SJV, MW and MGB conceived and designed the analytical approach. WAS, MW and LEC ran the model simulations. WAS, MW, LEC and MGB prepared the model code and documentation for public release. WAS, MW and MGB wrote the initial draft of the manuscript. All authors read, commented on and approved the final version of the manuscript. p yg 12. WHO/APOC. Conceptual and operational framework of onchocerciasis elimination with ivermectin treatment. http://www.who.int/apoc/ oncho_elimination_report_english.pdf. Accessed 30 Aug 2015. 2010. p yg 12. WHO/APOC. Conceptual and operational framework of onchocerciasis elimination with ivermectin treatment. http://www.who.int/apoc/ oncho_elimination_report_english.pdf. Accessed 30 Aug 2015. 2010. Page 15 of 16 Page 15 of 16 Stolk et al. Parasites & Vectors (2015) 8:552 13. World Health Organization. Accelerating work to overcome the global impact of neglected tropical diseases: a roadmap for implementation. Geneva, Switzerland: World Health Organization; 2012. using the individual-based WORMSIM modelling framework. 2015 (in press Parasit Vectors). using the individual-based WORMSIM modelling framework. 2015 (in press Parasit Vectors). 34. Plaisier AP, Alley ES, van Oortmarssen GJ, Boatin BA, Habbema JDF. Required duration of combined annual ivermectin treatment and vector control in the Onchocerciasis Control Programme in west Africa. Bull World Health Organ. 1997;75(3):237–45. 14. World Health Organization. Sustaining the drive to overcome the global impact of neglected tropical diseases. Second WHO report on neglected tropical diseases. Geneva: World Health Organization; 2013. 15. London Declaration on Neglected Tropical Diseases. 2012. http://unitingtocombatntds.org/resource/london-declaration. Accessed 30 August 2015. 35. Alley ES, Plaisier AP, Boatin BA, Dadzie KY, Remme J, Zerbo G, et al. The impact of five years of annual ivermectin treatment on skin microfilarial loads in the onchocerciasis focus of Asubende, Ghana. Trans R Soc Trop Med Hyg. 1994;88(5):581–4. 16. Osei-Atweneboana MY, Eng JK, Boakye DA, Gyapong JO, Prichard RK. Prevalence and intensity of Onchocerca volvulus infection and efficacy of ivermectin in endemic communities in Ghana: a two-phase epidemiologica study. Lancet. 2007;369(9578):2021–9. 36. Plaisier AP, Alley ES, Boatin BA, Van Oortmarssen GJ, Remme H, De Vlas SJ, et al. Irreversible effects of ivermectin on adult parasites in onchocerciasis patients in the Onchocerciasis Control Programme in West Africa. J Infect Dis. 1995;172(1):204–10. 17. Wanji S, Kengne-Ouafo JA, Esum ME, Chounna PW, Tendongfor N, Adzemye BF, et al. Situation analysis of parasitological and entomological indices of onchocerciasis transmission in three drainage basins of the rain forest of South West Cameroon after a decade of ivermectin treatment. Parasit Vectors. 2015;8:202. 37. Authors' contributions Short report: impact of ivermectin community-level treatments on elimination of adult Onchocerca volvulus when individuals receive multiple treatments per year. Am J Trop Med Hyg. 2005;73(6):1159–61. 30. Habbema JDF, De Vlas SJ, Plaisier AP, Van Oortmarssen GJ. The microsimulation approach to epidemiologic modeling of helminthic infections, with special reference to schistosomiasis. Am J Trop Med Hyg. 1996;55(5 Suppl):165–9. 52. Lamberton PH, Cheke RA, Osei-Atweneboana MY, Winskill P, Shew KJ, Wilson MD, et al. Host choice by onchocerciasis vectors and ongoing transmission in areas under ivermectin control. Am J Trop Med Hyg. 2012;87(5 suppl):143. 31. Plaisier AP, Subramanian S, Das PK, Souza W, Lapa T, Furtado AF, et al. The LYMFASIM simulation program for modeling lymphatic filariasis and its control. Methods Inf Med. 1998;37:97–108. 53. Boyd A, Won KY, McClintock SK, Donovan CV, Laney SJ, Williams SA, et al. A community-based study of factors associated with continuing transmission of lymphatic filariasis in Leogane. Haiti PLoS Negl Trop Dis. 2010;4(3):e640. 32. De Vlas SJ, Van Oortmarssen GJ, Gryseels B, Polderman AM, Plaisier AP, Habbema JD. SCHISTOSIM: a microsimulation model for the epidemiology and control of schistosomiasis. Am J Trop Med Hyg. 1996;55(5 Suppl):170–5. 54. Brieger WR, Okeibunor JC, Abiose AO, Ndyomugyenyi R, Wanji S, Elhassan E, et al. Characteristics of persons who complied with and failed to comply with annual ivermectin treatment. Trop Med Int Health. 2012;17(7):920–30. 33. Coffeng LE, Bakker R, Montresor A, De Vlas SJ. Feasibility of controlling hookworm infection through preventive chemotherapy: a simulation study Page 16 of 16 Stolk et al. Parasites & Vectors (2015) 8:552 55. Duerr HP, Dietz K, Eichner M. Determinants of the eradicability of filarial infections: a conceptual approach. Trends Parasitol. 2005;21(2):88–96. 56. Hontelez JA, Lurie MN, Barnighausen T, Bakker R, Baltussen R, Tanser F, et al. Elimination of HIV in South Africa through expanded access to antiretroviral therapy: a model comparison study. PLoS Med. 2013;10(10):e1001534. 57. 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Modelling onchocerciasis transmission and control [PhD Thesis]. Rotterdam, the Netherlands: Erasmus University Rotterdam; 1996. 59. Duke BO. The population dynamics of Onchocerca volvulus in the human host. Trop Med Parasitol. 1993;44(2):61–8. 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https://openalex.org/W4289711090	https://digibug.ugr.es/bitstream/10481/76751/1/ijerph-19-09518-v2.pdf	English	null	Effect of Intensity and Duration of Exercise on Gut Microbiota in Humans: A Systematic Review	International journal of environmental research and public health/International journal of environmental research and public health	2,022	cc-by	12,032	1 Escuela de Nutrición y Dietética, Facultad de Ciencias, Universidad Mayor, Santiago 8580745, Chile; rominabonomini@gmail.com 1 Escuela de Nutrición y Dietética, Facultad de Ciencias, Universidad Mayor, Santiago 8580745, Chile rominabonomini@gmail.com 2 Children’s Hospital of Eastern Ontario Research Institute, Ottawa, ON K1H 8L1, Canada 3 2 Children’s Hospital of Eastern Ontario Research Institute, Ottawa, ON K1H 8L1, Canada 3 3 Department of Biochemistry and Molecular Biology II, School of Pharmacy, University of Granada, 18071 Granada, Spain 4 Instituto de Investigación Biosanitaria IBS.GRANADA, Complejo Hospitalario Universitario de Granada, 18014 Granada, Spain 5 Gastric Cancer Research Group—Laboratory of Oncology, UC Center for Investigational Oncology (CITO), Pontiﬁcia Universidad Católica de Chile, Santiago 8331150, Chile; airodriguez1@uc.cl 6 IRyS Group, Physical Education School, Pontiﬁcia Universidad Católica de Valparaíso, V l í 2374631 Chil f d d i @ l Gastric Cancer Research Group—Laboratory of Oncology, UC Center for Investigational O Pontiﬁcia Universidad Católica de Chile, Santiago 8331150, Chile; airodriguez1@uc.cl 6 IRyS Group, Physical Education School, Pontiﬁcia Universidad Católica de Valparaíso, Valparaíso 2374631, Chile; fernando.rodriguez@pucv.cl 6 IRyS Group, Physical Education School, Pontiﬁcia Univers Valparaíso 2374631, Chile; fernando.rodriguez@pucv.cl IRyS Group, Physical Education School, Pontiﬁcia Unive Valparaíso 2374631, Chile; fernando.rodriguez@pucv.cl * Correspondence: jrplaza@ugr.es (J.P.-D.); carlos.jorquera@mayor.cl (C.J.-A.); Tel.: +34-958241599 (J.P.-D.); +569-95791706 (C.J.-A.) Abstract: (1) Background: The gut microbiota might play a part in affecting athletic performance and is of considerable importance to athletes. The aim of this study was to search the recent knowledge of the protagonist played by high-intensity and high-duration aerobic exercise on gut microbiota composition in athletes and how these effects could provide disadvantages in sports performance. (2) Methods: This systematic review follows the PRISMA guidelines. An exhaustive bibliographic search in Web of Science, PubMed, and Scopus was conducted considering the articles published in the last 5 years. The selected articles were categorized according to the type of study. The risk of bias was assessed using the Joanna Briggs Institute’s Critical Appraisal Tool for Systematic Reviews. (3) Results: Thirteen studies had negative effects of aerobic exercise on intestinal microbiota such as an upsurge in I-FABP, intestinal distress, and changes in the gut microbiota, such as an increase in Prevotella, intestinal permeability and zonulin. In contrast, seven studies observed positive effects of endurance exercise, including an increase in the level of bacteria such as increased microbial diversity and increased intestinal metabolites. (4) Conclusions: A large part of the studies found reported adverse effects on the intestinal microbiota when performing endurance exercises. Citation: Bonomini-Gnutzmann, R.; Plaza-Díaz, J.; Jorquera-Aguilera, C.; Rodríguez-Rodríguez, A.; Rodríguez-Rodríguez, F. Effect of Intensity and Duration of Exercise on Gut Microbiota in Humans: A Systematic Review. Int. J. Environ. Res. Public Health 2022, 19, 9518. https://doi.org/10.3390/ijerph 19159518 Academic Editor: Paul B. Tchounwou Received: 15 June 2022 Accepted: 25 July 2022 Published: 3 August 2022 Published: 3 August 2022 Keywords: aerobic exercise; adults; elite athletes; large intestine; gut microbiota Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. International Journal of Environmental Research and Public Health International Journal of Environmental Research and Public Health International Journal of Environmental Research and Public Health 1 Escuela de Nutrición y Dietética, Facultad de Ciencias, Universidad Mayor, Santiago 8580745, Chile; rominabonomini@gmail.com In studies carried out on athletes, more negative effects on the microbiota were found than in those carried out on non-athletic subjects. Romina Bonomini-Gnutzmann 1, Julio Plaza-Díaz 2,3,4,* , Carlos Jorquera-Aguilera 1,, Andrés Rodríguez-Rodríguez 5 and Fernando Rodríguez-Rodríguez 6 Romina Bonomini-Gnutzmann 1, Julio Plaza-Díaz 2,3,4, , Carlos Jorquera-Aguilera 1,*, Andrés Rodríguez-Rodríguez 5 and Fernando Rodríguez-Rodríguez 6 1. Introduction The intestinal or gut microbiota is “the set of microbes that colonize our digestive tract that interact with each other and with the host” [1–3]. Currently, more than one thousand different microbial species have been found that can reside in the human gastrointestinal tract [4]. Approximately one hundred sixty species are found in the large intestine [3], developing a biomass of more than 1.5 kg [5]. The microbiota contain bacteria, as well as fungi, viruses, and protists [1]. The most abundant and diverse families of bacteria in the adult gastrointestinal tract are Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria, and less diverse are Verrucomicrobia, Lentisphaerae, Sinergistetes, Planctomycetes, Tenericutes, and Deinococcus-Thermus [2,3]. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/ijerph Int. J. Environ. Res. Public Health 2022, 19, 9518. https://doi.org/10.3390/ijerph19159518 Int. J. Environ. Res. Public Health 2022, 19, 9518 2 of 17 The composition of the gut microbiota is formed throughout early childhood, induced by genetic and environmental factors [1–3]. The maturation of the intestinal microbiota in the adult type is gone at the age of three years [1,6]. Factors such as age, lifestyle, diet, and genetics can alter the gut microbiota, creating a dynamic ecosystem [7]. Other related factors could include mode of delivery, geography, breastfeeding, weaning, and exposure to environmental bacteria [1]. Some researchers proposed that the intestinal microbiota can act as an endocrine organ [8] and can have an enormous impact on human health, including the immune function, physiology, metabolism, and nutrition of the host [9]. In the same line, the gut microbiota performs a series of protective, structural, and metabolic functions essential to the health of the host, including food handling, the ingestion of complex polysaccharides not digestible by the host, the movement of pathogens, and the synthesis of vitamins among others [10]. Observational studies have found that intestinal microbiota may contribute either to the pathogenesis of various common metabolic disorders including type 2 diabetes, obesity, cardio-metabolic diseases, malnutrition, and non-alcoholic liver disease, as well as to the metabolic health of the human host [11]. 1. Introduction Healthy gut microbiota shows an essential role in the conﬁguration of the local and systemic immune function of intestinal bacteria throughout life, favoring the maintenance of tolerance toward antigens of the commensals and activation against antigens of commensal pathogens [12]. The intestinal microbiota plays an important role in the regulation of host energy metabolism, hydration status, systemic inﬂammatory responses, and oxidative stress [13]. Physical exercise is described as the implementation of some activity in order to improve or preserve overall health and physical ﬁtness [14,15]. Currently, physical exercise is recognized as a formidable preventive and treatment mediation that is recognized to be efﬁcient in causing beneﬁts for immune and metabolic health [15,16]. Endurance exercise is described as cardiovascular activity, for example: cycling, running, swimming, skiing, and rowing that is performed for a long time, four to six hours per day, six days per week [17]. This intense exercise includes processes that involve physiological, affective, cognitive–behavioral, and biochemical responses in an effort to recover homeostasis [9]. g p Some professional athletes experience immunosuppression or gastrointestinal symp- toms, such as abdominal pain, diarrhea, or leaky gut syndrome [8]. Alterations in the intestinal microbiota produced by strenuous exercise can also produce exercise-induced gastrointestinal disorders [18]. Some symptoms that are stated during the performance of endurance exercise include bloating, nausea, cramps, and diarrhea [19]. It has been studied that exercising to exhaustion can disrupt the balance among the gut microbiota and the immune system [12]. Exercise-induced gastrointestinal damage or inﬂammation could adversely affect athletic routine and, in some cases, have competition dropout [20]. An- other study mentions that intense exercise creates increased gastrointestinal damage, mild endotoxemia, and intestinal permeability [21]. The main ﬁnding in post-exercise gastroin- testinal problems is possible ischemia–reperfusion injury developing from a momentary interruption of splanchnic blood ﬂow. When the intense physical exercise ﬁnishes, we ob- served triggering reactive oxygen species (ROS) production, damage to the gastrointestinal mucosa, and inﬂammation [22]. Likewise, it is little known thus far how high-intensity exercise inﬂuences the intestinal microbiota [21]. The importance of knowing the mechanisms in which the intestinal microbiota might have an important role in affecting athletic routine is of signiﬁcant attention to athletes working to expand their competitive performance and diminish recuperation time during training [12]. Such information could have an advantage in the comprehension of gut microbiota inﬂuences on athlete health [12]. 1. Introduction Therefore, the main aim of this systematic review is to elucidate the knowledge of the function played by high-intensity and high-duration aerobic exercise on gut micro- biota composition in athletes and how these effects could provide disadvantages in their sports performance. Int. J. Environ. Res. Public Health 2022, 19, 9518 3 of 17 3 of 17 2.2. Selection and Exclusion Criteria The selection criteria were: (i) articles written in English, (ii) databases aforementioned, (iii) human studies, (iv) original articles: clinical trials, randomized controlled trials (RCTs) quasi-experimental, long-term, prospective, and cross-sectional studies, (v) articles from January 2015 to August 2021. The exclusion criteria were: (i) studies that include people with pathologies, (ii) studies that include animals, (iii) studies that comprise children under 18 years of age and older adults (+65 years), (iv) studies that intervened with supplements or some diet, (v) case studies, case reports, letters to the editor, systematic review and meta-analyses and narrative review. No restrictions were placed on the body composition of the trained subject. j After removing repeated documents, suitability was measured by evaluation of the manuscript title and abstract and later evaluation of the full text. 2. Materials and Methods 2.1. Search Strategy This systematic review follows the PRISMA guidelines [23]. An exhaustive biblio- graphic search of three databases (Web of Science, PubMed, and Scopus) was conducted considering the articles published in the last 7 years (from 1 January 2015 to 31 August 2021). Table 1 shows the search strategy in the Web of Science, PubMed, and Scopus databases. This systematic review was listed on the PROSPERO (International prospective register of systematic reviews) website on 5 May 2022, with the following record CRD42022323300. Available from: https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD420 22323300 (accessed on 14 June 2022). Table 1. Search strategy in databases. Database Search Strategy Limits Filters Web of Science (ALL (Physical activity AND gut microbiota OR Physical activity AND intestinal barrier OR Physical activity AND intestinal permeability OR Physical exercise AND gut microbiota OR Physical exercise AND intestinal barrier OR Physical exercise AND intestinal permeability)) Title Articles English 238 items ﬁltered PubMed (Physical activity OR physical exercise) AND (gut microbiota OR intestinal barrier OR intestinal permeability) Title Articles English Humans 104 items ﬁltered Scopus TITLE-ABS-KEY (physical AND activity AND gut AND microbiota) OR (physical AND activity AND intestinal AND barrier) OR (physical AND activity AND intestinal AND permeability) OR (physical AND exercise AND gut AND microbiota) OR (physical AND exercise AND intestinal AND barrier) OR (physical AND exercise AND intestinal AND permeability) AND (LIMIT-TO (OA, “all”)) AND (LIMIT-TO (PUBYEAR, 2022) OR LIMIT-TO (PUBYEAR, 2021) OR LIMIT-TO (PUBYEAR, 2020) OR LIMIT-TO (PUBYEAR, 2019) OR LIMIT-TO (PUBYEAR, 2018) OR LIMIT-TO (PUBYEAR, 2017) OR LIMIT-TO (PUBYEAR, 2016) OR LIMIT-TO (PUBYEAR, 2015)) AND (LIMIT-TO (DOCTYPE, “ar”)) AND (LIMIT-TO (LANGUAGE, “English”)) Title Articles English 5934 items ﬁltered Table 1. Search strategy in databases. 2.4. Assessment of the Quality and Level of Evidence The risk of bias was assessed using the Joanna Briggs Institute’s Critical Appraisal Tool for Systematic Reviews. In summary, this tool includes four speciﬁc checklists depending on the study design (i.e., cross-sectional, quasi-experimental, cohort, RCTs studies). The answers for each of them had four possible categories: “yes” (criterion met) and “no” (criterion not met). Speciﬁc tools included: eight items for cross-sectional studies, nine items for quasi-experimental, and thirteen items for RCTs. According to the above, the studies were considered as “low quality” evidence when ≤49% of the items were classiﬁed as “yes” (criterion met). Following, the articles were considered as “medium quality” evidence between 50–74% of the items were scored as “yes” and “high quality” evidence when ≥75% of the items were classiﬁed as “yes”. The answers “not applicable” and “non- clear” were excluded by percentage [24–26]. The ﬁve reviewers assessed the studies’ quality separately. A consensus meeting was organized to resolve possible differences between the reviewers. 2.3. Data Extraction and Reliability The search was carried out by ﬁve independent reviewers (R.B.-G., F.R.-R., J.P.-D., C.J.-A. and A.R.-R.). They read the titles and abstracts of all retrieved articles. A meeting was held to resolve disagreements about eligibility. The following information was collected from each included study: the ﬁrst author, year of publication, type of study, objective, the number of subjects, body mass index (BMI), maximum oxygen consumption (VO2max), Int. J. Environ. Res. Public Health 2022, 19, 9518 4 of 17 gender, and age when it was available, type of exercise, how the exercise was carried out, the molecular analysis used for the detection of the gut microbiota, the main results obtained, and conclusions. The selected articles were categorized according to the type of study (low-, medium-, or high-intensity or long-term exercise interventions). The results of the studies that met the selection criteria for their recovery were examined. igure 1. Flowchart of articles through the search process Figure 1. Flowchart of articles through the search process. In this regard, one study fulﬁlled 100% of the criteria [27], and seven studies fulﬁlled ≥75% of the quality criteria [7,9,13,27,28,31,33], classifying themselves as high quality. The rest of the studies [5,16,18,19,22,29,30,32,34] were classiﬁed as medium quality because they obtained a value between >50% and <75% of the criteria (among 50–69.2%). No studies with low quality were found (<50% of the criteria). Table 2 shows the summary of the studies included. This review is focused on d om 513 participants, and the sample size ranged from 4 to 86 subjects. Two of the sixt udies involved only women [18,27], six involved only men [5,13,16,28–30], five invol oth men and women [7,9,22,31,32] and three studies did not report the sex of the subj 9,33,34]. The age of the subjects ranged from 18 and 49 years; two studies did not rep he age of the subjects [28,33]. The samples were from 10 different countries: three stud were led in Poland, two in Spain, two in the United States, two in China, two in Irela ne in the United Kingdom, one in Australia, one in Belgium, one in Germany and om Japan The results revealed that nine studies showed negative effects of aerobic exercise on the intestinal microbiota. Among these adverse effects, three studies found an increase in I-FABP [5,16,29], one study presented intestinal distress [5], three studies observed negative changes in the microbiome [30–32], two studies found an increase in Prevotella [7,33], three studies observed an increase in intestinal permeability [16,19,29], and two studies reported an increase in zonulin [19,29]. In contrast, seven studies observed positive effects of en- durance exercise, including an increase in the level of bacteria such as Roseburia hominis, Biﬁdobacterium spp., Akkermansia muciniphila, Faecalibacterium prausnitzii [18,27], Coriobacteri- aceae [22], increased microbial diversity [9,13,33] and increased intestinal metabolites [28]. om Japan. Regarding the characteristics of the sample, eleven studies of the sixteen were c ucted on endurance athletes (runners, cyclists, and triathletes). Among these nine stud onsidering medium- and long-distance runners [5,13,16,18,22,29,30,32,33], three stud ncorporated physically active subjects [27,31,34], one study on triathletes [19], a study yclists [7], a study included martial arts professionals [9], and a study on rugby play 28]. Most of the studies incorporated in this systematic review (81%) used fecal samp o determine changes in the intestinal microbiota. igure 1. Flowchart of articles through the search process Figure 1. Flowchart of articles through the search process. gure 1. Flowchart of articles through the search process Figure 1. Flowchart of articles through the search process. Table 2 shows the summary of the studies included. This review is focused on d rom 513 participants, and the sample size ranged from 4 to 86 subjects. Two of the sixt tudies involved only women [18,27], six involved only men [5,13,16,28–30], five invol oth men and women [7,9,22,31,32] and three studies did not report the sex of the subj 19,33,34]. The age of the subjects ranged from 18 and 49 years; two studies did not rep he age of the subjects [28,33]. The samples were from 10 different countries: three stud were led in Poland, two in Spain, two in the United States, two in China, two in Irela ne in the United Kingdom, one in Australia, one in Belgium, one in Germany and rom Japan. Regarding the characteristics of the sample, eleven studies of the sixteen were c ducted on endurance athletes (runners, cyclists, and triathletes). Among these nine stud onsidering medium- and long-distance runners [5,13,16,18,22,29,30,32,33], three stud ncorporated physically active subjects [27,31,34], one study on triathletes [19], a study yclists [7], a study included martial arts professionals [9], and a study on rugby play 28]. Most of the studies incorporated in this systematic review (81%) used fecal samp o determine changes in the intestinal microbiota. Of these, mainly 75% determined The results revealed that nine studies showed negative effects of aerobic exercise on the intestinal microbiota. Among these adverse effects, three studies found an increase in I-FABP [5,16,29], one study presented intestinal distress [5], three studies observed negative changes in the microbiome [30–32], two studies found an increase in Prevotella [7,33], three studies observed an increase in intestinal permeability [16,19,29], and two studies reported an increase in zonulin [19,29]. In contrast, seven studies observed positive effects of en- durance exercise, including an increase in the level of bacteria such as Roseburia hominis, Biﬁdobacterium spp., Akkermansia muciniphila, Faecalibacterium prausnitzii [18,27], Coriobacteri- aceae [22], increased microbial diversity [9,13,33] and increased intestinal metabolites [28]. Additionally, an analysis has been performed to determine the study qualities included in this review. For this, the Joanna Briggs Institute’s criterium checklist (Table 3) was used. Different criteria were used according to the characteristics of the studies. 3. Results Figure 1 displays the chosen reporting elements for systematic reviews and the ﬂow chart for the search strategy. A total of 6277 studies were located in the three databases assessed. Then, 95 studies were excluded for duplicates and 6136 studies were excluded after reading the title and abstract that were outside the topic of the review. A total of 45 studies were assessed for eligibility. After analyzing the exclusion criteria, sixteen studies were included. Seven studies had an observational design (i.e., ﬁve cross-sectional and two long-term designs), and nine studies had an experimental design (eight were quasi-experimental, and one was RCT). Table 2 shows the summary of the studies included. This review is focused on data from 513 participants, and the sample size ranged from 4 to 86 subjects. Two of the sixteen studies involved only women [18,27], six involved only men [5,13,16,28–30], ﬁve involved both men and women [7,9,22,31,32] and three studies did not report the sex of the sub- jects [19,33,34]. The age of the subjects ranged from 18 and 49 years; two studies did not report the age of the subjects [28,33]. The samples were from 10 different countries: three studies were led in Poland, two in Spain, two in the United States, two in China, two in Ireland, one in the United Kingdom, one in Australia, one in Belgium, one in Germany and one from Japan. p Regarding the characteristics of the sample, eleven studies of the sixteen were con- ducted on endurance athletes (runners, cyclists, and triathletes). Among these nine studies considering medium- and long-distance runners [5,13,16,18,22,29,30,32,33], three studies incorporated physically active subjects [27,31,34], one study on triathletes [19], a study on cyclists [7], a study included martial arts professionals [9], and a study on rugby play- ers [28]. Most of the studies incorporated in this systematic review (81%) used fecal samples to determine changes in the intestinal microbiota. Of these, mainly 75% determined the 16S ribosomal RNA genetic sequence that is commonly used for identiﬁcation, classiﬁcation, and quantitation of microbes within complex biological mixtures such as environmental samples and intestinal samples. A minority of four studies (25%) used plasma samples through the protein enzyme-linked immunosorbent assay test to determine markers such as intestinal fatty-acid binding protein (I-FABP) related to mucosal damage, zonulin associ- ated with intestinal permeability, and cortisol, c-reactive protein, and TNF-α related to a proinﬂammatory status. 3. Results 5 of 175 o 5 of 175 o Int. J. Environ. Res. Public Health 2022, 19, 9518 . Environ. Res. Public Health 2022, 19, x igure 1. Flowchart of articles through the search process Figure 1. Flowchart of articles through the search process. Of these, mainly 75% determined y Additionally, an analysis has been performed to determine the study qualities included in this review. For this, the Joanna Briggs Institute’s criterium checklist (Table 3) was used. Different criteria were used according to the characteristics of the studies. In this regard, one study fulﬁlled 100% of the criteria [27], and seven studies fulﬁlled ≥75% of the quality criteria [7,9,13,27,28,31,33], classifying themselves as high quality. The rest of the studies [5,16,18,19,22,29,30,32,34] were classiﬁed as medium quality because they obtained a value between >50% and <75% of the criteria (among 50–69.2%). No studies with low quality were found (<50% of the criteria). 6 of 17 6 of 17 Int. J. Environ. Res. Public Health 2022, 19, 9518 Table 2. Characteristics as the type of study, aim, sample, design, and mean results of the studies. Author, Year Type of Study AIM Sample Study Design Results Pugh et al. (2017) [5] Quasi-Experimental Characterize the HIIT effects on small intestinal damage markers n = 11 (men runners trained) Aged 33.1 ± 10.4; VO2max 60.0 ± 3.2 mL/kg/min Acute HIIT episode markers of intestinal permeability and damage were evaluated and compared with resting conditions. Minimum running performance of 10 km (39 min) and a minimum of 5 workout sessions per week, using serum sampling, pre-exercise, after each set of exercises, and 2 h post-baseline HIIT signiﬁcantly increased the serum lactulose: rhamnose ratio and sucrose concentrations compared with rest. In contrast, urinary lactulose: rhamnose or sucrose concentrations did not vary between study groups. Plasma I-FABP augmented in the recuperation period from HIIT only. After 24 h of HIIT, the researchers found mild symptoms of GI distress Liang et al. (2019) [9] Cross-sectional Whether the intestinal microbiota is distinctive between higher-level and lower-level athletes n = 31 (professional martial arts athletes). 15 women and 16 men; aged 20–24 Martial arts athletes; Wushu routine, vigorous, fast and dynamic sports. The researchers used 16S rRNA gene sequencing to determine the intestinal changes Higher-level athletes have augmented metabolic capacity and diversity in the intestinal microbiota compared with lower-level athletes. Petersen et al. igure 1. Flowchart of articles through the search process Figure 1. Flowchart of articles through the search process. (2017) [7] Cross-sectional Determine the presence of distinctive organisms in professional and amateur level competitive cyclists n = 33 (professional and amateur level competitive cyclists); 11 women and 22 men; aged 19–49 The study used metatranscriptomic (RNA-Seq) sequencing and mWGS The increase in Prevotella was associated with time reported exercising during an average week. Several professional cyclists have augmented levels of Methanobrevibacter smithii transcripts compared with amateur cyclists. Bressa et al. (2017) [27] Cross-sectional Compare intestinal composition among two groups divided by physical exercise levels n = 40 (premenopausal women). 19 active and 21 sedentary Aged 18–40; BMI 20–25 kg/m2 The researchers used 16S rRNA gene sequencing to determine the intestinal changes Performance of physical activity was associated with the presence of health-promoting bacteria (R. hominis, A. muciniphila, Biﬁdobacterium spp., and F. prausnitzii). Decreased levels of diversity were correlated with sedentary parameters Results HIIT signiﬁcantly increased the serum lactulose: rhamnose ratio and sucrose concentrations compared with rest. In contrast, urinary lactulose: rhamnose or sucrose concentrations did not vary between study groups. Plasma I-FABP augmented in the recuperation period from HIIT only. After 24 h of HIIT, the researchers found mild symptoms of GI distress Acute HIIT episode markers of intestinal permeability and damage were evaluated and compared with resting conditions. Minimum running performance of 10 km (39 min) and a minimum of 5 workout sessions per week, using serum sampling, pre-exercise, after each set of exercises, and 2 h post-baseline n = 11 (men runners trained) Aged 33.1 ± 10.4; VO2max 60.0 ± 3.2 mL/kg/min n = 11 (men runners trained) Aged 33.1 ± 10.4; VO2max 60.0 ± 3.2 mL/kg/min Characterize the HIIT effects on small intestinal damage markers Whether the intestinal microbiota is distinctive between higher-level and lower-level athletes n = 31 (professional martial arts athletes). 15 women and 16 men; aged 20–24 n = 31 (professional martial arts athletes). 15 women and 16 men; aged 20–24 Higher-level athletes have augmented metabolic capacity and diversity in the intestinal microbiota compared with lower-level athletes. n = 33 (professional and amateur level competitive cyclists); 11 women and 22 men; aged 19–49 Determine the presence of distinctive organisms in professional and amateur level competitive cyclists The study used metatranscriptomic (RNA-Seq) sequencing and mWGS n = 40 (premenopausal women). 19 active and 21 sedentary Aged 18–40; BMI 20–25 kg/m2 n = 40 (premenopausal women). 19 active and 21 sedentary Aged 18–40; BMI 20–25 kg/m2 Compare intestinal composition among two groups divided by physical exercise levels Compare intestinal composition among two groups divided by physical exercise levels The researchers used 16S rRNA gene sequencing to determine the intestinal changes 7 of 17 7 of 17 Int. J. Environ. Res. Public Health 2022, 19, 9518 thor, Year Type of Study AIM Sample Study Design Results t al. (2017) [16] Quasi-experimental Evaluate the effect of running on GI function markers n = 17 (active runners); 8 women and 9 men; aged 18–45 The researchers measured secondary variables, such as zonulin, levels of serum intestinal I-FABP, and bacterial LPS, among others Both, serum I-FABP and intestinal permeability increased after running, without differences amongst groups. No changes were observed in the bacterial LPS in serum ohane et al. The observed changes were associated only with the CRE group, resulting in disturbance of the intestinal microbiota Results 019) [13] Long-term Analyze the changes in the intestinal microbiota of four well-trained male athletes to prolonged, high-intensity trans-oceanic rowing n = 4 (men athletes transatlantic rowing). Aged 25–27; BMI 23–25 kg/m2; VO2Max 46–50 mL/kg/min Metagenomic whole-genome shotgun sequencing was used Intense exercise clearly impacts the diversity of the intestinal microbiota, with changes in speciﬁc bacteria related to metabolic pathways et al. (2021) [31] Quasi-experimental Impact of CRE or RTE on intestinal microbiota n = 56 n = 28 CRE group (21 women; Aged 20.7; BMI 24.5 kg/m2 and 7 men; aged 20; BMI 24.0 kg/m2. n = 28 RTE group (17 women; aged 20.4; BMI 23.2 kg/m2 and 11 men; aged 22.6; BMI 24.59 kg/m2 Intestinal microbiota was measured using 16S rRNA gene sequencing The observed changes were associated only with the CRE group, resulting in disturbance of the intestinal microbiota ishima et al. 020) [18] Cross-sectional Effects of highly intensive endurance exercise on the intestinal microbiota and its relationship with the onset of the exercise-induced GI disorders n = 29 (15 women Japanese endurance runners and 14 nonathletic but healthy women). Aged 20–21; BMI 20.7–21.9 kg/m2 Fecal microbiota was tested using 16S rRNA metagenomics, and other variables such as moisture content, organic acids, and putrefactive metabolites concentrations were examined Female elite endurance runners have more abundance of Faecalibacterium, and these changes could be associated with the succinate concentration in this group al. (2019) [19] Long-term Evaluate intestinal and muscle damage in triathletes n = 15 (triathletes). Aged 6–14; VO2max 58.8 ± 4.5 mL/kg/min Variables used for the analysis were: cortisol, c-reactive protein, zonulin, and TNF-α Zonulin and variables of permeability were augmented after the race Study Design Both, serum I-FABP and intestinal permeability increased after running, without differences amongst groups. No changes were observed in the bacterial LPS in serum n = 4 (men athletes transatlantic rowing). Aged 25–27; BMI 23–25 kg/m2; VO2Max 46–50 mL/kg/min n = 4 (men athletes transatlantic rowing). Results Aged 25–27; BMI 23–25 kg/m2; VO2Max 46–50 mL/kg/min Intense exercise clearly impacts the diversity of the intestinal microbiota, with changes in speciﬁc bacteria related to metabolic pathways Intense exercise clearly impacts the diversity of the intestinal microbiota, with changes in speciﬁc bacteria related to metabolic pathways Metagenomic whole-genome shotgun sequencing was used Intestinal microbiota was measured using 16S rRNA gene sequencing Intestinal microbiota was measured using 16S rRNA gene sequencing n = 29 (15 women Japanese endurance runners and 14 nonathletic but healthy women). Aged 20–21; BMI 20.7–21.9 kg/m2 Fecal microbiota was tested using 16S rRNA metagenomics, and other variables such as moisture content, organic acids, and putrefactive metabolites concentrations were examined Female elite endurance runners have more abundance of Faecalibacterium, and these changes could be associated with the succinate concentration in this group n = 15 (triathletes). Aged 6–14; VO2max 58.8 ± 4.5 mL/kg/min Zonulin and variables of permeability were augmented after the race Variables used for the analysis were: cortisol, c-reactive protein, zonulin, and TNF-α Variables used for the analysis were: cortisol, c-reactive protein, zonulin, and TNF-α 8 of 17 8 of 17 Int. J. Environ. Res. Public Health 2022, 19, 9518 Table 2. Cont. Author, Year Type of Study AIM Sample Study Design Results Zhao et al. (2018) [22] Quasi-experimental The gut microbiota immediately responds to the enteric changes in amateur half-marathon runners n = 20 (4 women and 16 men amateur half-marathon runners). Aged 31.3; BMI 22.6 kg/m2 Fecal samples were analyzed before and after the marathon using 16 rDNA sequencing analyses Coriobacteriaceae changes were related to the exercise role in avoiding disease and reﬁning health outcomes. Moitinho-Silva et al. (2021) [34] Randomized controlled trial Analyze the changes in the intestinal microbiota on previously physically inactive, healthy adults in comparison to controls that did not perform regular exercise n = 36 (11 controls; 13 endurance group; 12 strength group). Aged 22–41.3; BMI 19.7–32.5 kg/m2 Fecal microbiota was tested using 16S rRNA metagenomics Mucosal damage and inﬂammation were found after short-term resistance training. No changes were observed in intestinal microbiota Sadowska-Krepa et al. (2021) [29] Quasi-experimental Evaluate intestinal damage in middle-aged male subjects n = 10 (amateur long-distance runners). Aged 21–35 Variables used for the analysis were: TAS, TOS/TOC, hs-CRP, I-FABP, and zonulin After the exercise, the levels of intestinal permeability biomarkers as, hs-CRP, I-FABP, zonulin, and inﬂammation were augmented Kulecka et al. Results (2020) [33] Quasi-experimental Evaluate differences in intestinal microbiota amongst healthy controls and endurance athletes n = 71 n = 14 marathon runners; n = 11 cross-country skiers; n = 46 healthy control individuals Fecal microbiota was tested using 16S rRNA metagenomics Excessive training is associated with changes in Bacteroides and Prevotella and bacterial diversity Tabone et al. (2021) [30] Quasi-experimental Determine whether the changes are driven by exercise on the gut microbiota (with 16S rRNA gene) and the serum and fecal metabolome n = 40 (men endurance cross-country runners). Aged 35.8 ± 8.0; BMI 22.8 ± 2.1 kg/m2; VO2max 58.8 ± 3.24 mL/kg/min Fecal microbiota was tested using 16S rRNA metagenomics The changes in gut microbiota could be related to physiological changes in ammonia, uric acid, and lactate n = 71 n = 14 marathon runners; n = 11 cross-country skiers; n = 46 healthy control individuals Fecal microbiota was tested using 16S rRNA metagenomics The changes in gut microbiota could be related to physiological changes in ammonia, uric acid, and lactate Fecal microbiota was tested using 16S rRNA metagenomics Fecal microbiota was tested using 16S rRNA metagenomics 9 of 17 9 of 17 Int. J. Environ. Res. Public Health 2022, 19, 9518 Table 2. Cont. Author, Year Type of Study AIM Sample Study Design Results Barton et al. (2017) [28] Cross-Sectional Evaluate differences in intestinal microbiota amongst exercise and a more sedentary state n = 86 (40 men professional international rugby union players and 46 men controls) Fecal microbiota was tested using 16S rRNA metagenomics Professional international rugby union players had more favorable effects in metabolic pathways than the control group Craven et al. (2021) [32] Quasi-experimental Evaluate differences in intestinal microbiota according to training volume n = 14 (highly trained middle-distance runners). n = 6 women; aged 22.0 ± 3.4; VO2max 59.0 ± 3.2 mL/kg/min n = 8 men; aged 20.7 ± 3.2; VO2max 70.1 ± 4.3 mL/kg/min Fecal microbiota was tested using 16S rRNA metagenomics No changes were observed in intestinal microbiota according to training volume in upper taxons. Results Changes in family, genus, and species were observed, these changes did not return to pre-levels Abbreviations: AFT, after fecal; BEF, before fecal; BMI, body mass index; CRE, cardiorespiratory exercise; DS, standard deviation; FCCS, female cross-country skiers; FDR, false discovery rate; FMR, female marathon runners; GI, gastrointestinal; HIIT, high-intensity interval training; hs-CRP, High-sensitivity C-reactive protein; HvolTr, high-volume training; I-FABP, intestinal fatty acid-binding protein; kg/m2, kilogram per square meter; LPS, lipopolysaccharide; MCCS, male cross-country skiers; MCHC, mean corpuscular hemoglobin concentration; mL/kg/min, milliliters per minute per kilogram; MMR, male marathon runners; mWGS, metagenomic whole genome shotgun; NormTr normal training; PGM, personal genome machine; PWC, physical working capacity; rRNA, ribosomal ribonucleic acid; RTE, resistance training exercise; TaperTr, exponential reduction in training; TAS, total antioxidant status; TOC, total oxidant capacity; TOS, total oxidant status; VO2max, the maximum amount of oxygen; WHO, World Health Organization; WSER, Western States Endurance Run. Study Design No changes were observed in intestinal microbiota according to training volume in upper taxons. Changes in family, genus, and species were observed, these changes did not return to pre-levels Results Professional international rugby union players had more favorable effects in metabolic pathways than the control group Fecal microbiota was tested using 16S rRNA metagenomics n = 14 (highly trained middle-distance runners). n = 6 women; aged 22.0 ± 3.4; VO2max 59.0 ± 3.2 mL/kg/min n = 8 men; aged 20.7 ± 3.2; VO2max 70.1 ± 4.3 mL/kg/min No changes were observed in intestinal microbiota according to training volume in upper taxons. Changes in family, genus, and species were observed, these changes did not return to pre-levels Fecal microbiota was tested using 16S rRNA metagenomics Abbreviations: AFT, after fecal; BEF, before fecal; BMI, body mass index; CRE, cardiorespiratory exercise; DS, standard deviation; FCCS, female cross-country skiers; FDR, false discovery rate; FMR, female marathon runners; GI, gastrointestinal; HIIT, high-intensity interval training; hs-CRP, High-sensitivity C-reactive protein; HvolTr, high-volume training; I-FABP, intestinal fatty acid-binding protein; kg/m2, kilogram per square meter; LPS, lipopolysaccharide; MCCS, male cross-country skiers; MCHC, mean corpuscular hemoglobin concentration; mL/kg/min, milliliters per minute per kilogram; MMR, male marathon runners; mWGS, metagenomic whole genome shotgun; NormTr normal training; PGM, personal genome machine; PWC, physical working capacity; rRNA, ribosomal ribonucleic acid; RTE, resistance training exercise; TaperTr, exponential reduction in training; TAS, total antioxidant status; TOC, total oxidant capacity; TOS, total oxidant status; VO2max, the maximum amount of oxygen; WHO, World Health Organization; WSER, Western States Endurance Run. Int. J. Environ. Res. Public Health 2022, 19, 9518 10 of 17 Table 3. Checklist from Joanna Briggs Institute’s criterium according to kind of study, percentage of criterium reached, and quality level of evidence. Criteriums According to Kind of Study Authors 1 2 3 4 5 6 7 8 9 10 11 12 13 Percentage Reached Quality Level Pugh et al. [5] 1 0 0 0 1 1 1 1 1 66.7 MQ Liang et al. [9] 0 1 1 1 1 0 1 1 75.0 HQ Petersen et al. [7] 0 1 1 1 1 0 1 1 75.0 HQ Bressa et al. [27] 1 1 1 1 1 1 1 1 100.0 HQ Karhu et al. [16] 1 0 0 0 1 1 1 1 0 55.6 MQ Keohane et al. [13] 0 1 1 1 1 1 1 1 87.5 HQ Bycura et al. [31] 1 1 1 0 1 1 1 1 1 88.9 HQ Morishima et al. [18] 0 0 1 1 1 0 0 1 50.0 MQ Tota et al. 4. Discussion The main aim of this study was to elucidate the recent knowledge of the function played by high-intensity and high-duration aerobic exercise on gut microbiota composition in athletes and how these effects could provide disadvantages in sports performance. Since the beginning of the metagenomic era, microbial communities have been associ- ated with human health [35]. We have described that the microbiota, the full collection of microbes, are important in the development of several diseases [36]. However, the presence of these microbes is not the only factor that affects the host. The genetics of all the microbes (bacteria, fungi, protozoa, and viruses), deﬁned as the microbiome, and the metabolic products that they produce are other sources of important changes [37]. Centenarians have shown speciﬁc intestinal microbiota that are improved in microbes that are efﬁcient in generating exclusive secondary bile acids comprising different lithocholic acid isoforms: isoallolithocholic acid, iso-, 3-oxo, allo-, and 3-oxoallo- [38]. The metabolic catalog of the intestinal microbiome is immense, but the well-being associations of these bacterial pathways is weakly comprehended. In the case of physical exercise, Veillonella atypica increases run time in humans via its metabolic conversion of exercise-induced lactate into propionate, thus recognizing a natural, microbiome-encoded enzymatic process that enhances athletic performance [39]. Following these lines, the main topics in the present systematic review were: (i) the strategies of analysis, (ii) the effects of exercise duration on microbiota, and ﬁnally, (iii) the effects of exercise intensity on the microbiota. Results [19] 0 1 1 1 0 0 1 0 50.0 MQ Zhao et al. [22] 0 0 0 0 1 1 1 1 1 55.6 MQ Moitinho-Silva et al. [34] 1 0 1 0 0 0 1 1 1 1 1 1 1 69.2 MQ Sadowska-Krepa et al. [29] 1 0 0 0 1 1 0 1 1 55.6 MQ Kulecka et al. [33] 0 1 1 1 1 1 1 1 1 88.9 HQ Tabone et al. [30] 1 0 0 0 1 1 1 1 1 66.7 MQ Barton et al. [28] 1 0 1 1 0 1 1 1 75.0 HQ Craven et al. [32] 0 0 0 0 1 1 1 1 1 55.6 MQ HQ: high quality; MQ: medium quality. Table 3. Checklist from Joanna Briggs Institute’s criterium according to kind of study, percentage of criterium reached, and quality level of evidence. HQ: high quality; MQ: medium quality. 4.2. Effects of Exercise Duration on Microbiota Physical activity, exercise, or physical ﬁtness are considered advantageous therapies to decrease inﬂammatory pathways [47]. Recent evidence proposes that exercise can positively modify the intestinal microbiota composition in healthy adults [47,48]. These changes can also be discovered in patients with inﬂammatory bowel disease. Exercise programs of at least 12 weeks produce modiﬁcations in the gut microbiota composition through immunometabolic pathways associated with anti-inﬂammatory effects [49]. A recent systematic review has found that Prevotella relative abundance looks to be associated with training duration [50]. In our systematic review, we found a similar result with a high Prevotella abundance associated with time-reported exercising during an average week. In addition, the authors have found that Methanobrevibacter smithii transcripts were more predominant in professional cyclists in comparison to amateur cyclists [7]. In the same line, a higher Faecalibacterium abundance was found in the intestinal microbiota of female elite endurance runners related to the accumulation of succinate [18]. Marathon runners have shown increased levels of Prevotella and bacterial diversity [33] and alterations in the intestinal microbiota related to Coriobacteriaceae [22]. Both Faecalibacterium and Prevotella are related to human health beneﬁts [51,52] and to plant-rich diets characterized by high levels of complex carbohydrates and vegetable and fruit intake [51,52]. The family Coriobacteriaceae may partly mediate the positive effects of Roux-en-Y gastric bypass on type 2 diabetes [53]. yp According to the secondary variables related to the gut microbiota, the studies have shown that running induced an upsurge in serum I-FABP concentration and intestinal permeability, but there were no differences between asymptomatic and symptomatic run- ners [16]. Serum LPS activity did not change from baseline following the running test, but the symptomatic group exhibited higher LPS activity at baseline compared to the asymptomatic runners [16]. Twelve-hour runs would provoke metabolic stress in middle- aged amateur runners and elevated levels of biomarkers such as zonulin, hs-CRP, and I-FABP [29]. Endurance-sport athletes have excessive gastrointestinal disorders prevalence and bargaining performance, possibly affecting general health status. Ultramarathoners and triathlon athletes have shown an increase in several proinﬂammatory cytokines and proteins [54]. The analyzed studies have shown a pro-inﬂammatory status related to in- testinal microbiota, similar to the recent systematic review that states that exercise duration could be related to a higher pro-inﬂammatory bacteria abundance [50,55,56]. Exercise is a powerful intervention to ﬁght obesity that is also related to a proinﬂam- matory status and poorer vascular function [57]. 4.1. Strategies of Analysis These microbes could be analyzed by several molecular techniques, for example using probes that detected a long range of bacterial strains [40]. This approach is more general, in which microbes could be presented in the sample, and is less effective in an individual and precise identiﬁcation. In addition, another alternative is using the conserved 16S ribosomal RNA gene [41], which allows us better sensibility and precision in identiﬁcation. These methods are joined with plenty of bioinformatic tools and processes [42]. Finally, other biochemical techniques such as assessment of intestinal permeability and gastrointestinal discomfort were used. 11 of 17 Int. J. Environ. Res. Public Health 2022, 19, 9518 11 of 17 Since the initiation of high-throughput sequencing, PCR-ampliﬁed 16S sequences have habitually been gathered based on similarity to produce operational taxonomic units (OTUs), amplicon sequence variants (ASVs), and these descriptive sequences compared with reference databases such as Silva [43] or Greengenes [44], and Ribosomal Database Project (RDP) [45] to extrapolate the taxonomy [46]. In the total of 16 studies, we found that the majority of the studies used the 16S rRNA technique and metagenomic approaches (12/16; 75.0%), which ensure strong identiﬁcation of the microbes that are presented in the different samples. The remaining studies (4/16; 25.0%) have reported secondary values, such as intestinal permeability or the release of different proteins, which could show a general perspective about gut microbiota. p g p p g The conclusions that were based on 16S and metagenomic approaches were more consistent and allowed the investigators to attribute the effects or not to individual genera or singular strains. Another important issue in the strategies of analysis was the type of study. Here, we have eight quasi-experimental studies, ﬁve cross-sectional studies, two long-term studies, and one RCT. 4.3. Effects of Exercise Intensity on Microbiota Several studies mention that aerobic exercise can be a beneﬁcial strategy for modulat- ing the microbiota composition in the presence of metabolic diseases, speciﬁcal exercises of moderate or vigorous intensity [59]. It has also been reported that stress induced by intense exercise increases intestinal inﬂammation and an increase in Ruminococcus gnavus, as well as Butyrivibrio, Coprococcus, and Oscillospira, and a decrease in Turicibacter spp. [17]. In athletes who practice intense and prolonged exercises, they report a particular microbiota composition, described by a bigger abundance of bacteria involved in inﬂamma- tory processes, such as Haemophilus and Rothia [60], Mucispirillum [61,62], and Ruminococcus gnavus [63]. Faecalibacterium abundance, generally recognized as favorable to human health [64–66], has been detected concurrently with an excessive pro-inﬂammatory abun- dance of bacteria in endurance runners whose abnormal gut environment may cause it to act as an opportunistic bacterium [18]. Finally, in a recent RCT [67], a reduction in microbial heterogeneity was observed in the intense-exercise group versus the control group. On the contrary, it has been studied that intense exercise can reduce intestinal inﬂam- mation by changing the microbial proﬁle [68]. An increase in Bacteroidetes has also been observed, which might be helpful to athletes by having a crucial role in the metabolic conversion of protein, complex sugar polymers degradation [4,68–71], improvements in glucose metabolism, and branched-chain amino acid degradation [72–75]. Another study deﬁned that high-intensity exercise increases mitochondrial function and grows essential bacteria in urease production and lactate metabolism [76]. Likewise, a study showed that high-intensity interval training and resistance work modiﬁed the composition of the intestinal microbiota [77]. Regarding resistance work, it is capable of modestly modifying the microbiota composition and function compared to other types of exercise [78]. Regarding moderate-intensity exercise, a study in obese mice showed alterations in the gut microbiota of the colon and effective activation of the AMPK/CDX2 signaling pathway to improve the intestinal barrier [79]. In this sense, this has deﬁned a correlation between cardiorespiratory ﬁtness and greater microbial diversity in healthy subjects; therefore, enhanced cardiovascular ﬁtness and oxygen consumption correlate positively with a more diverse microbial proﬁle [80]. In accordance with the above, it has also been studied that subjects with low aerobic capacity display a greater Eubacterium rectale-Clostridium coccoides presence, related to metabolic disorders [81,82]. 4.2. Effects of Exercise Duration on Microbiota Studies relating to intestinal microbiota changes have stated that exercise could raise the microbial variance and enhance the Firmicutes/Bacteroidetes ratio, and both actions could neutralize obesity progression and diminish body weight [47]. Finally, the duration of the exercise should change the intestinal Int. J. Environ. Res. Public Health 2022, 19, 9518 12 of 17 microbiota composition, especially in beneﬁcial-related bacteria, and as a consequence, permeability intestinal [58]. There is no detailed information regarding the ideal duration of the exercise, how the exercise could interact with the diet, and how other microbes (Archaea, viruses, and fungi) could be inﬂuenced by the duration of the exercise. Those issues remain unclear, and further studies are required. 4.3. Effects of Exercise Intensity on Microbiota However, moderate-low intensity training has also been indicated to produce limited gut microbiota changes, and higher intensity appears to be essential in provoking changes in obese and overweight subjects [50]. Some speciﬁc microbial genera were related to speciﬁc diets and exercise-induced regulation of cardiometabolic health [83]. Figure 2 summarizes the main ﬁndings in the present systematic review. 4.4. Limitations o This study 5. Conclusions This study has found some limitations. The first of these is that not all the studies were controlled trials. Furthermore, only 7 of the 16 studies are of high quality. This makes interpretation difficult and increases bias. Therefore, caution should be exercised in inter- preting the data obtained in this review. In addition, a combination of methods of meas- uring the quality of the articles had to be carried out based on the type of study. That is, one type of criterion was used for a cross-sectional study, another criterion for controlled trials, another criterion for quasi-experimental, etc. Finally, the studies used different methods to obtain a microbiota marker. Therefore, the state of the microbiota can be dif- ferent depending on the method used. 5. Conclusions Using the main findings of the present systematic review, it can be established that a large part of the studies found reported adverse effects on the intestinal microbiota when performing endurance exercises, such as an increase in distress, bacteria, a decrease in ic obial di e sity a d a i testi al pe eability dec ease Ho e e the est of the stud Using the main ﬁndings of the present systematic review, it can be established that a large part of the studies found reported adverse effects on the intestinal microbiota when performing endurance exercises, such as an increase in distress, bacteria, a decrease in microbial diversity, and an intestinal permeability decrease. However, the rest of the studies found positive effects with aerobic exercise. In addition, in the studies carried out on athletes, more negative effects on the microbiota were found than in those carried out on non-athletic subjects. It was observed that strength training obtains the lowest beneﬁts to the microbiota. In general, it is appreciated that the studies obtain molecules that favor the microbiota and other pro-inﬂammatory elements at the same time. This leads us to think that there is no absolute clarity of the mechanisms and personal and environmental factors that inﬂuence an improvement or worsening of the microbiota as a function of exercise. Future studies should propose what is the amount of exercise that must be achieved in order to have favorable effects on the microbiota and what is the cut-off point in the dose of exercise that begins to worsen the conditions of the intestinal microbiome. ies found positive effects with aerobic exercise. 4.4. Limitations of the Study This study has found some limitations. The ﬁrst of these is that not all the studies were controlled trials. Furthermore, only 7 of the 16 studies are of high quality. This makes interpretation difﬁcult and increases bias. Therefore, caution should be exercised in interpreting the data obtained in this review. In addition, a combination of methods of measuring the quality of the articles had to be carried out based on the type of study. That is, one type of criterion was used for a cross-sectional study, another criterion for controlled trials, another criterion for quasi-experimental, etc. Finally, the studies used 13 of 17 s, and ei ht Int. J. Environ. Res. Public Health 2022, 19, 9518 sity tra higher 13 of 17 s, and i ht different methods to obtain a microbiota marker. Therefore, the state of the microbiota can be different depending on the method used. induced regulation of cardiometabolic health [83]. Figure 2 summarizes the main findings in the present systematic review. Figure 2. Interaction of different intensities and duration of exercise on the intestinal microbiota. Abbreviations: CRP, C-reactive protein; hs-CRP, high-sensitivity C-reactive protein; I-FABP, intes- tinal fatty-acid binding protein; IL, interleukin. Figure 2. Interaction of different intensities and duration of exercise on the intestinal microbiota. Abbreviations: CRP, C-reactive protein; hs-CRP, high-sensitivity C-reactive protein; I-FABP, intestinal fatty-acid binding protein; IL, interleukin. Figure 2. Interaction of different intensities and duration of exercise on the intestinal microbiota. Abbreviations: CRP, C-reactive protein; hs-CRP, high-sensitivity C-reactive protein; I-FABP, intes- tinal fatty-acid binding protein; IL, interleukin. Figure 2. Interaction of different intensities and duration of exercise on the intestinal microbiota. Abbreviations: CRP, C-reactive protein; hs-CRP, high-sensitivity C-reactive protein; I-FABP, intestinal fatty-acid binding protein; IL, interleukin. 4.4. Limitations o This study 5. Conclusions In addition, in the studies carried out on athletes, more negative effects on the microbiota were found than in those carried out on Author Contributions: R.B.-G., J.P.-D., C.J.-A., A.R.-R. and F.R.-R. participated in the bibliographic search, discussion, and writing of the manuscript. R.B.-G., J.P.-D., C.J.-A., A.R.-R. and F.R.-R. designed the work. R.B.-G., J.P.-D., C.J.-A., A.R.-R. and F.R.-R. revised the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: J.P.-D. is part of the “UGR Plan Propio de Investigación 2016” and the “Excellence actions: Unit of Excellence on Exercise and Health (UCEES), University of Granada”. J.P.-D. is supported by a fellowship awarded to postdoctoral researchers at foreign universities and research centers from the “Fundación Ramón Areces”, Madrid, Spain. Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Data Availability Statement: Not applicable. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. 14 of 17 Int. J. Environ. Res. Public Health 2022, 19, 9518 References Keohane, D.M.; Woods, T.; O’Connor, P.; Underwood, S.; Cronin, O.; Whiston, R.; O’Sullivan, O.; Cotter, P.; Shanahan, F.; Molloy, M.G. Four men in a boat: Ultra-endurance exercise alters the gut microbiome. J. Sci. Med. Sport 2019, 22, 1059–1064. [CrossRef] 14. Caspersen, C.J.; Powell, K.E.; Christenson, G.M. Physical activity, exercise, and physical ﬁtness: Deﬁnitions and distinctions for h l h l d h bl l h p g yp 13. Keohane, D.M.; Woods, T.; O’Connor, P.; Underwood, S.; Cronin, O.; Whiston, R.; O’Sullivan, O.; Cotter, P.; Shanahan, F.; Molloy, M.G. 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https://openalex.org/W1568754763	https://www.sciendo.com/pdf/10.5334/csci.2	English	null	Notes for a Presentation: Historical Social Science as a Science of Culture	Cultural science	2,008	cc-by	16,846	http://doi.org/10.5334/csci.2 http://doi.org/10.5334/csci.2 The Modern World System and Its Structures of Knowledge World-systems analysis emerged in the 1970s in articulation with the medium-term historical conjuncture that was marked by the end of the world economic expansion that had been operative over the preceding quarter century and by the decline in the hegemony in the interstate system that had been enjoyed by the United States over the same period. On the one hand, world- systems analysis was a protest or resistance movement, in articulation with the social movements associated with the upheavals of 1968, concerned with the ways the world and its functioning had been portrayed and thus what actions, in whose interests, were deemed possible, and legitimate. It was a product of the system that it sought to understand. On the other hand, world- systems analysis was also an outgrowth of the secular crisis of the processes reproducing historical capitalism in the long term. In that sense it has been a forward-looking movement 1 during a period of transition. during a period of transition. The basic premise of world-systems analysis is that historical social systems have lives. They come into being as a unique set of singular, longue durée structures; the secular trends and cyclical rhythms of their reproduction may be observed over the life of the system; but eventually the processes reproducing these structures run up against asymptotes, or limitations, in overcoming the contradictions of the system and the system ceases to exist. The structures of the modern-world system, or capitalist world-economy, came into being in Europe at the beginning of the long sixteenth century, the period known as the transition from feudalism to capitalism. By the end of the Hundred Years’ War an axial division of labor was developing between a western European core where high-wage, skilled workers produced low- bulk, high value-added manufactures and an eastern European periphery where high-bulk, low value-added necessities were produced by a lower cost work force. The long-distance trade in these commodities resulted in the accumulation (concentration and centralization) of capital in the core. The processes reproducing this relationship over the long term—the “accumulation of accumulation” or profit making for reinvestment and thus more profit making—underwent periodic fluctuations, and the expansion of the system to incorporate new pools of low-cost labor provided the solutions that turned periods of world economic downturn into periods of upturn. The Modern World System and Its Structures of Knowledge A principle characteristic of the world today is that there no longer exist significant pools of labor outside the system to be incorporated at the bottom of the wage hierarchy as previously incorporated workers have militated and succeeded in negotiating higher remuneration, thus lowering the rate of profit. incorporated workers have militated and succeeded in negotiating higher remuneration, thus lowering the rate of profit. The “endless” accumulation resulting from the extraction and appropriation of surplus The “endless” accumulation resulting from the extraction and appropriation of surplus The “endless” accumulation resulting from the extraction and appropriation of surplus 2 2 2 2 produced by labor could only take place within the context of what developed as an interstate system. Unlike the “parcellized sovereignty” (Anderson 1974), or overlapping geographic jurisdictions of feudal “realms,” the multiple states of which this new system was composed were sovereign, with reciprocal rights and obligations, to the extent that their territorial extensions, and the monopoly on the use of force within them, were recognized by other states. Fluctuating flows of goods, capital, and labor could thus be controlled across semi-permeable borders throughout the system. Strong states worked to loosen controls during periods of world economic upturn and tighten controls during periods of downturn to favor accumulation and contain and defuse class conflict. Like its economic processes, the geopolitics of this system also underwent periodic fluctuations. Competition among elites resulted in “world wars,” the outcomes of which were short-lived states of “hegemony,” a status of the system itself, as a whole, during which one strong state exercised military, commercial, financial, and cultural dominance, before other parts of the world-system “caught up” to become once more competitive and the cycle repeated. Three such periods may be observed: Dutch hegemony after the Thirty Years’ War, British hegemony after the French Revolutionary/Napoleonic Wars, and U.S. hegemony after the thirty-years-long World War I-World War II. Significantly, over the past five hundred years, no power has been able to totally dominate the system and thus to turn it into a world-empire and today no seemingly credible scenario for establishing a new state of hegemony has emerged. There was a third set of structures that were just as constitutive of the modern world- system as those in the arenas of production and distribution, the economic, and coercion and decision making, the political. 1 See Lee (1996, 2003b), Lee and Wallerstein (2000, 2004) 1 See Lee (1996, 2003b), Lee and Wallerstein (2000, 2004) The Modern World System and Its Structures of Knowledge This third arena has come to be conceptualized as that of 3 3 3 3 cognition and intentionality, the structures of knowledge.1 The fundamental conceptualization of the structures of knowledge has proven to offer particularly thorny difficulties. It was not as though no one had been working on the problem; indeed, not only had questions in the cultural realm long offered rich subjects for study even at the macro level, culture had long been a central explanatory category of social analysis as well and had, in fact, given rise to an important knowledge movement beginning in the mid-1950s (see Lee 2003a). However, if the broadly “cultural” aspect of the world-system were just as constitutive as the economic and political realms, then ad-hoc, particularistic conceptualizations had to give way to a specification of the longue durée structures of this “third arena,” and include the cyclical rhythms and secular trends of their reproduction. These would have to be recognizable over the entire life of the system—in other words, a conceptualization analogous to those of the economic and political arenas (see Lee 2003b). From the beginning of the long sixteenth century, the practices of knowledge production took the form of a complex of processes that produced over time an intellectual and institutional hierarchy, a set of structures, within which authoritative knowledge was progressively defined as the “other” of societal/moral values. These processes of knowledge formation, in articulation with those sets of processes associated with the “economic” and “political” spheres, account for the dominant relational setting “disciplining” human cognition and intentionality, the “cultural” parameters of possible action. Determining micro-fluctuations indicating the direction of the transformation of medieval modes of knowing were the rise of visual representation and quantification dissociated from any value components and especially the emergence of the “modern fact” as the primary epistemological unit of valid knowledge and cultural authority 4 4 (Poovey 1998; Crosby 1997).. The medieval structures of knowledge recognized diverse fields or subject-matters; rhetoric was certainly not astronomy. What was not recognized was differing bodies of knowledge that were based on contradictory visions of the way the world worked. It was this new epistemological divide, and the hierarchy that privileged as legitimate and authoritative “factual knowledge”, that would become the norm. The Modern World System and Its Structures of Knowledge The possibility of such a double identity drove the processes of rationalization, the secular trend in the arena of the structures of knowledge, which, depending on circumstances, might be labeled “scientization” or “secularization.” The pursuit of objectivity—the view from nowhere; the erasure of agency and 5 5 5 5 history, in short, of subjectivity in whatever form (see Megill 1994)— embodies the progressive privileging of formal rationality, disinterested calculation as a generalized means of instrumental action, over substantive rationality, the normatively-oriented pursuit of specifically situated ends. The structures of knowledge of the modern world-system are, then, unique, like its economic history, in short, of subjectivity in whatever form (see Megill 1994)— embodies the progressive privileging of formal rationality, disinterested calculation as a generalized means of instrumental action, over substantive rationality, the normatively-oriented pursuit of specifically situated ends. The structures of knowledge of the modern world-system are, then, unique, like its economic structures (those of production and distribution) and political structures (those of coercion and decision making); indeed, no other historical system has created two antithetical, contradictory epistemological bases for the production of knowledge, one excluding human values a priori and one in which human values are an inseparable component. The long-term trend deepening this structure underwent two great conjunctural adjustments or "logistics" analogous to the waves of expansion and contraction in the economic arena and the cycles of relative concentration of power in the geopolitical realm (see Lee 2003b). The first consisted of the seventeenth-century Newtonian synthesis between Baconian induction and empiricism and Cartesian deduction and rationalism, which created the foundation for the dominant theoretical approaches and methodological practices in the sciences and led to the solidification of the separation of the sciences from the humanities. The long-term trend deepening this structure underwent two great conjunctural With the common purpose of mastering nature, two avenues in the search for truth independent of received values were charted in empiricist appeals to the senses and an inductive method and rationalist espousals of reason and a deductive method. During the eighteenth century, the Newtonian fusion of these two modes produced a synthesis of experimental and empirical approaches incorporating hypothesis construction and mathematical demonstrations. Classical science henceforth would be concerned with the discovery of universal laws governing a regular and constant nature that would lead to the prediction of change, both future and past. The Modern World System and Its Structures of Knowledge The mechanisms of this transformation, such as the establishment of double entry bookkeeping (see Poovey 1998) and the renewal of the authority of the principle of the excluded middle, were clearly articulated with the political and economic developments we generally consider the markers of what is termed the transition from feudalism to capitalism. These markers included the rise in the status of merchants and the legitimation of profit making (the virtù of the balance in the “bottom line”), the development and integration of the world market, and the transformation of the basic political entities of “realms” based on “parcellized sovereignty” into “states” with borders. The creation of the modern fact enabled the metamorphosis of the merchant into the capitalist by establishing the legitimacy of profit rooted in the virtues of “balance” inherent in the system of double-entry bookkeeping. With profit distinguished from usury, the accumulation of accumulation could take off. At the same time, however, there were concomitant effects which redefined the structures of knowledge. The modern fact could be affiliated with both specifics (of commerce) and their generalization (within a system which ordained the individual creditworthiness of merchants and their credibility as a group). The possibility of such a double identity drove the processes of rationalization, the secular trend in the arena of the structures of knowledge, which, depending on circumstances, might be labeled “scientization” or “secularization.” The pursuit of objectivity—the view from nowhere; the erasure of agency and The creation of the modern fact enabled the metamorphosis of the merchant into the capitalist by establishing the legitimacy of profit rooted in the virtues of “balance” inherent in the system of double-entry bookkeeping. With profit distinguished from usury, the accumulation of accumulation could take off. At the same time, however, there were concomitant effects which redefined the structures of knowledge. The modern fact could be affiliated with both specifics (of commerce) and their generalization (within a system which ordained the individual creditworthiness of merchants and their credibility as a group). The Modern World System and Its Structures of Knowledge With the displacement of the divine viewpoint to man, the humanities, not concerned with the 6 6 6 6 ordered certitude of regularities in the world of nature but with the chaotic finitude of the unique and unpredictable in the human world of conflicting values, could appeal to individual creativity for a “rational” understanding of emergence and change. Along these two lines, the long-term intellectual and institutional opposition of the sciences and the humanities, what has come to be called the “Two Cultures”, reached a clear delineation over the course of the nineteenth century (see Lee and Wallerstein 2004). Within this basic structure, the social sciences emerged in the nineteenth century as a medium-term solution to the tensions internal to the structures of knowledge that no longer offered practical ways of addressing the evolving geopolitics of the world-system. In the aftermath of the French Revolution it was no longer possible to imagine a static world; however, modes of interpreting social change in the human world, as marked off from the natural world, made contradictory appeals to values. The mutually exclusive alternatives were either order achieved through the authority of tradition or chaos arising from unfettered democracy. Neither offered a solution, on which any consensus seemed possible, to the political confrontations between conservatism and radicalism that threatened capital accumulation. The medium-term resolution of this dilemma was the late nineteenth-century creation of the social sciences, situated between the sciences and the humanities. The putatively value- neutral social sciences, which seemed to offer the possibility of a “scientific” or non-value- oriented policy-making process in the service of “progress”, came to occupy a tension-charged space in the wake of the irresolvable contest between the equally value-laden but mutually exclusive positions taken by conservatives (tradition, order) and radicals (democracy, anarchy) in the humanities on the political future of the world following the French Revolution. The key controversies in this process were the late-nineteenth-century Methodenstreit in the German- 7 7 7 7 speaking world (in both philosophy and economic history/economics: Dilthey and the Baden neo-Kantians, Schmoller, Menger) and the English order and anarchy debates (Carlyle, Arnold, Mill)—the one taking place in the context of state-formation and development in the Germanies, the other in the context of British political unrest at home and uprisings in the colonies. The Modern World System and Its Structures of Knowledge Instead of value sets, on which the politics of both the right and the left had been founded, the social sciences increasingly ordered collective decision-making along the lines of Mill's suggestion that from the "science of society" come "guidance" (Mill 1843: 64). T. H. Huxley called on the objective, value-neutral, problem-solving spirit of science to realize progress without moralism. On the occasion of the opening of Sir Josiah Mason's Science College, Birmingham, in 1880, Huxley delivered a lecture in which he asserted that if the evils which are inseparable from the good of political liberty are to be checked, if the perpetual oscillation of nations between anarchy and despotism is to be replaced by the steady march of self-restraining freedom; it will be because men will gradually bring themselves to deal with political, as they now deal with scientific questions (Huxley 1881: 158-9). Instead of value sets, on which the politics of both the right and the left had been founded, the social sciences increasingly ordered collective decision-making along the lines of Mill's suggestion that from the "science of society" come "guidance" (Mill 1843: 64). T. H. Huxley called on the objective, value-neutral, problem-solving spirit of science to realize progress without moralism. On the occasion of the opening of Sir Josiah Mason's Science College, Birmingham, in 1880, Huxley delivered a lecture in which he asserted that The political consequences of this medium-term solution were the “scientific” legitimation of the association of particular “nations” or “peoples” with individual states, the hierarchical placement of groups on a racialized and gendered world division of labor, and the rise of the “new liberalism” effectively eliminating clear alternative political agendas on the right and on the left, but holding out the fig leaf of, at least generational, progress to the exploited. Thus, this division of labor found on one side the factual, universal, positivistic, empirical, objective, fact-producing, and quantitative disciplines of the sciences engaged in explaining order in a world where past determined a predictable future via universal laws. On the 8 8 8 8 other side was to be found the particularistic (for instance, with regard to social contexts, locales, or time frames), chaotic, value-oriented, and qualitative disciplines of the humanities where scholars dealt with an unpredictable and relativistic world of free human agency. The Modern World System and Its Structures of Knowledge In their quest for legitimacy after 1945, the social sciences deepened their efforts to emulate the putative universalism of the natural sciences. They were, nonetheless, divided on questions of both theory and method. On the one hand, universalism was expressed in quantification and the comparative method in economics (econometrics), sociology (structural-functionalism), and political science (behaviorism), while, on the other hand, universalism was expressed additively in the more narrative bent of history and anthropology. Although all the disciplines exhibited to some extent both tendencies, scientism seemed to be gaining throughout. Similarly, even the humanities, in their effort to retain a credible voice, sought to echo the decontextualization, atemporality, and presumptive objectivity of the sciences with approaches such as “new criticism” and “close reading”. The result was the institutionalization of a set of disciplines, the social sciences, which would function to guarantee ordered change in the name of “progress” through “scientific” control, exercised by “experts” and based on “hard facts”; in practice, this amounted to liberal incrementalism maximizing accumulation and minimizing class struggle. The social sciences divided the study of the human world into isolated domains separated intellectually in disciplines and institutionally in university departments. Oriental studies and anthropology were concerned with the great civilizations and the “tribes” of the non-modern world respectively; history handled the past of the modern world; the present of the modern world was further divided among economics, political science and sociology, which treated the market, the state, and civil society as isolated fields. Although economics, political science and 9 9 9 9 sociology leaned more toward the sciences while history, Oriental studies and anthropology tended to be more humanistic, even within the disciplines there was no consensus on the composition of their data (quantitative, qualitative), or the appropriateness of their methods (statistical, narrative), or the nature of their “scientific” universality (discovery of laws, elaboration of exhaustive descriptions) on which they based the legitimacy of their claims. However, from the moment of the greatest intellectual and institutional success of this structur in the period immediately after 1945, the scholarly legitimacy of the premises underlying the partitions separating the disciplines and the practical usefulness of the distinctions became les and less self-evident, and after 1968 were overtly contested. The Modern World System and Its Structures of Knowledge sociology leaned more toward the sciences while history, Oriental studies and anthropology tended to be more humanistic, even within the disciplines there was no consensus on the composition of their data (quantitative, qualitative), or the appropriateness of their methods (statistical, narrative), or the nature of their “scientific” universality (discovery of laws, elaboration of exhaustive descriptions) on which they based the legitimacy of their claims. These then are the three analytically distinct but functionally, and existentially, inseparable structural arenas of the modern world system: the axial division of labor, the interstate system, and the structures of knowledge. They define a singular “world.” And that world is unique in human history in that from the time of its emergence it has expanded to incorporate the entire globe. It is this world, then, that constitutes the unit of analysis of the world-systems perspective. A persistent question for both analysts and activists has always been why the exploited majority, although successful in agitating for improved conditions in the medium term, has never been able to entirely change the rules of the system. Historically, world-economies have been unstable and have generally transformed into world-empires or disintegrated. The modern world- system, in contrast, has not (yet) met either fate. “[T]he secret of its strength ... is the political side of the form of economic organization called capitalism ... [which] as an economic mode is based on the fact that the economic factors operate within an arena larger than that which any political entity can totally control. This gives capitalists a freedom of maneuver that is 10 10 structurally based” (Wallerstein 1974: 348). What matters for the system as a whole is not where state borders are drawn (they have, in fact, changed greatly over time), but rather that there exists a fragmenting mechanism per se (the process of state formation) defining a hierarchical ordering of multiple centers of power that can unilaterally impose resolutions to struggles among competing interests, but, with maximum legitimacy, only within their exclusive geographic perimeters. Classes, as economic phenomena, are formed at the level of the division of labor, at the level of the world-economy; the answer to the political question why the exploited majority simply does not rise up is of course that it does, periodically. The Modern World System and Its Structures of Knowledge Indeed, this is what has happened historically as the world-economy has expanded (a fundamental process of 11 11 reproduction of the system) to incorporate fresh pools of cheap labor at the bottom of the wage scale to make up at the system level what was conceded locally. The “globalization” model acknowledges implicitly that the economic processes of historical capitalism have not changed over the past five centuries. Nonetheless, the rhetoric of globalization ignores the long-term trends of those processes and suggests that the contemporary crisis is cyclical (a downturn in comparison with the upturn of the post-1945 period) and is thus reversible. In the contemporary world, the perceived openness of the international economy and the ease with which it slips the bonds of state regulation, which globalization critics decry, expresses the recognition that the cycles of endless accumulation—expansion, incorporation, exploitation and appropriation over long distances for reinvestment—take precedence over regulative policies any state or states might try to impose. This is exactly what “world-economy” means; a world-economy functions within and over the entirety of the “world” defined by the spatio-temporal extent of its processes. On the one hand, globalization has both correctly identified and recognized as important a fundamental change relating to the politics of the modern world-system: the “external” geographic boundaries of the world-economy have disappeared. On the other hand, the reason this is important has not to do with the cyclical downturn in the perceived capacity of the states to regulate “international” capital. The significance lies rather with the long-term trend of the process of expansion having reached its asymptotic limit and the political consequences of the fact that there is no longer an “outside” available for incorporation to replenish the lowest strata of the world division of labor and produce the surplus necessary to stave off class struggle while maintaining the endless accumulation of capital. But there is a second point concerning the periodic settlements between capital and labor, The “globalization” model acknowledges implicitly that the economic processes of historical capitalism have not changed over the past five centuries. Nonetheless, the rhetoric of globalization ignores the long-term trends of those processes and suggests that the contemporary crisis is cyclical (a downturn in comparison with the upturn of the post-1945 period) and is thus reversible. The Modern World System and Its Structures of Knowledge Actual class struggle, however, always remains fragmented since political movements organize to effect change where the primary organs of power and decision-making are located, in the states. structurally based” (Wallerstein 1974: 348). What matters for the system as a whole is not where state borders are drawn (they have, in fact, changed greatly over time), but rather that there exists a fragmenting mechanism per se (the process of state formation) defining a hierarchical ordering of multiple centers of power that can unilaterally impose resolutions to struggles among competing interests, but, with maximum legitimacy, only within their exclusive geographic perimeters. Classes, as economic phenomena, are formed at the level of the division of labor, at the level of the world-economy; the answer to the political question why the exploited majority simply does not rise up is of course that it does, periodically. Actual class struggle, however, always remains fragmented since political movements organize to effect change where the primary organs of power and decision-making are located, in the states. Now given the structure of the world-economy, capital has always held the advantage over labor in the long term. Nonetheless, individual, competing capitalists figure their bottom lines in the short term and workers have to satisfy their needs every day. Considering the cost of active struggle to profits and wages, any deployment of force over a significant period of time is decidedly unattractive. In the medium term, the least costly outcome is the reestablishment of consensus, even though it entails the expense to local capitalists of granting some material gains to workers. These gains are kept to a minimum, for workers too absorb an expense in accepting less than they would like, by the addition of a codicil promising further progress at some unspecified time in the future. Of course, the distribution of aggregate surplus at any point in time is a zero-sum game. Acceding to some, even minimal, demands of labor in one locale has to be made up in another if ceaseless accumulation is to continue, or, to avoid a vicious circle wiping out accumulation altogether, new sources of surplus have to be found. The Modern World System and Its Structures of Knowledge In the contemporary world, the perceived openness of the international economy and the ease with which it slips the bonds of state regulation, which globalization critics decry, the ease with which it slips the bonds of state regulation, which globalization critics decry, expresses the recognition that the cycles of endless accumulation—expansion, incorporation, exploitation and appropriation over long distances for reinvestment—take precedence over regulative policies any state or states might try to impose. This is exactly what “world-economy” means; a world-economy functions within and over the entirety of the “world” defined by the spatio-temporal extent of its processes. On the one hand, globalization has both correctly identified and recognized as important a fundamental change relating to the politics of the expresses the recognition that the cycles of endless accumulation—expansion, incorporation, exploitation and appropriation over long distances for reinvestment—take precedence over regulative policies any state or states might try to impose. This is exactly what “world-economy” means; a world-economy functions within and over the entirety of the “world” defined by the spatio-temporal extent of its processes. On the one hand, globalization has both correctly identified and recognized as important a fundamental change relating to the politics of the modern world-system: the “external” geographic boundaries of the world-economy have disappeared. On the other hand, the reason this is important has not to do with the cyclical downturn in the perceived capacity of the states to regulate “international” capital. The significance lies rather with the long-term trend of the process of expansion having reached its asymptotic limit and the political consequences of the fact that there is no longer an “outside” available for incorporation to replenish the lowest strata of the world division of labor and produce the surplus necessary to stave off class struggle while maintaining the endless accumulation of capital. But there is a second point concerning the periodic settlements between capital and labor, But there is a second point concerning the periodic settlements between capital and labor, But there is a second point concerning the periodic settlements between capital and labor, 12 12 the promise of progress. The Modern World System and Its Structures of Knowledge As world-scale class conflict played out in localized struggles over the eighteenth and nineteenth centuries, the contradictory demands of radicals for freedom and democracy (echoing the voices of working class victims of variously coercive modes of labor exploitation), of conservatives for order over anarchy, and of capital for assured pools of cheap labor resulted in the collapse of clear ideological alternatives on the left and the right and the emergence of the “new liberalism” at the end of the nineteenth century (see Lee 2003a, ch. 2). Coming into its own with the incorporation of the last of the regions external to the capitalist world-economy, the new liberal “consensus” inscribed some groups into subordinate positions on socially constructed but politically functional status hierarchies of race and gender. These hierarchies were translated and naturalized into “nations” of cultural/historical peoples and the dominant, politically responsible social subjects, the “citizens” of which they were made up, and the excluded “others” relegated to a secondary station legitimating their exploitation. During the first half of the twentieth century, this new liberal consensus, the geoculture of the world-system, was extrapolated worldwide in the form of Wilsonian “self-determination of nations” and Rooseveltian “economic development,” the structural equivalents of universal suffrage and the welfare state at the national level within the core. This world-liberal compact relied on strengthened state structures and piecemeal reform to insure order, that is, keep democratic tendencies in check. Its unstable equilibrium pledging progress prevailed for upwards of a century, but the promise wore thin, especially for women, ethnic and racial “minorities” and the young in the core and (ex-)colonial peoples in the periphery on whose marginalization it had depended. By the 1960s the note had come due but there was no one left (“outside” the system) to whom the promise of progress had not been made to bring on-line to pay for its (partial) fulfillment for those to whom it had been made. Even the 13 13 modernizers could see that the sequential model did not describe development in the real world and all of the social, national, and Old Left movements that had bought into the promise by targeting state power found themselves targets, along with the powerful institutions guaranteeing the processes of endless accumulation, in the world revolution of 1968. The Modern World System and Its Structures of Knowledge Over the past three decades, the crisis in the processes reproducing the organizational patterns of the modern world-system in all three structural arenas, despite neoliberal efforts bolstered by the rhetoric of globalization (the idea that there is no other choice) to extend them, has become apparent. The major mechanisms through which accumulation has been guaranteed over the past five centuries by keeping costs of production down—the incorporation of new pools of lowest cost labor, the externalization of the costs of infrastructure and ecological degradation, and control over transfer payments resulting in higher taxes—have all run up against their limits resulting in rising costs of production at the world level that can no longer be offset locally. Within the structures of knowledge, too, rationalization has entered into crisis and the attendant transformation is already changing the way we view the world, and it will eventually alter the possibilities for human action that we are able to imagine. The structuralisms spelled the demise of European humanism and positivism alike, and from the late 1960s, developments at the level of theory were mirrored on the ground of practice. Those groups which had theretofore lacked a “voice” gained admittance to the academy and began to transform it from the inside by applying their differently situated knowledge of the workings of the social world. Since then, multiple, not always harmonious, varieties of feminism have contested received premises of knowledge formation through a conception of values expressed in hierarchies of difference and power and have directly undermined the (male) universalism and objectivity by which science 14 14 14 laid claim to a distinctive mode of knowledge production. Their work disputed essentialist categories of man and woman and situated the female body as a pivotal site of positioning women in society through scientific discourse. In a similar fashion, scholars and activists working in the area of race and ethnicity have, as they produced their own empirical studies, built up theories of difference that challenged (Western) universalism and objectivity. Their work likewise unveiled how the essentialism of received categories of difference functioned to inscribe whole groups into subordinate positions. Over the same period, the very premises of science have been undermined from the inside (see Lee 1992, 2004a). It took the better part of four centuries for what we now think of as the scientific model to dominate our common sense view. The Modern World System and Its Structures of Knowledge That model included the discrimination between true and false, in a world of independent, “objective” elements. It included the idea that explanations should be brief and simple and at their best couched in laws admitting predictability. These are exactly the notions which have lost their unquestioned intellectual legitimacy. They continue, however, to regulate our everyday thinking. Their great force resided in their naturalized, universal and trans-historical character, but they are not genetically encoded; they were constructed and may be, indeed are in the process of being, changed. Over the same period, the very premises of science have been undermined from the inside (see Lee 1992, 2004a). It took the better part of four centuries for what we now think of as the scientific model to dominate our common sense view. That model included the Thus, the structure of the superdisciplines of knowledge production are collapsing. Contingency, context-dependency, the collapse of essentialisms, and multiple, overlapping temporal and spatial frameworks are moving the humanities in the direction of the historical social sciences. That chance and necessity are indivisible and give rise to irreversibility and creativity in natural systems (see Prigogine 1996) are moving the sciences in the same direction. Coinciding with these developments, the intellectual sanctions and practical justifications for 15 15 independent disciplines in the social sciences is disintegrating. This is a world at the “end of certainty,” argues Ilya Prigogine (1996); one in which the direction of fundamental change is unpredictable, but intimately dependent on our choices among the real historical alternatives that we can imagine for a more egalitarian world, “utopistics,” as Immanuel Wallerstein argues (1998). So, what conclusions are we to draw from the simultaneous exhaustion of the processes insuring endless accumulation and containing class struggle, and the collapse of their intellectual foundations? The upper bound of the trajectory of historical capitalism is not a point of arrival. It is a frontier of transition implying an ethical imperative to make profoundly political choices. The real story of the post cold-war world is not the “victory of the West” but the disintegration of the liberal compact which began in 1968 and was completed in 1989 (see Wallerstein 1995). The liberal consensus, the geoculture of the world-system, was a politics of medium-term increments of reformist change adding up, putatively, to endless (long-term), linear, progress. The Modern World System and Its Structures of Knowledge This vision depicted a golden, extrapolatable, “now” with no allusion to the ideologies of either a future transformation (socialism) or an idyllic past (conservatism). The parallel to Newtonian dynamics is clear. Science itself offered the linear development model, based empirically and epistemologically on independent units. But in the post cold-war world, liberalism has proven unable to deliver on its universalist message of progress. In its neoliberal guise it has failed in its bid to reinstate the geopolitical realities of the world characterized by US hegemony in the post-1945 period, or through structural adjustment to reproduce the conditions of the Kondratieff A-phase economic expansion of the same period. So, what conclusions are we to draw from the simultaneous exhaustion of the processes insuring endless accumulation and containing class struggle, and the collapse of their intellectual foundations? The upper bound of the trajectory of historical capitalism is not a point of arrival. It is a frontier of transition implying an ethical imperative to make profoundly political choices. The real story of the post cold-war world is not the “victory of the West” but the disintegration of the liberal compact which began in 1968 and was completed in 1989 (see Wallerstein 1995). The liberal consensus, the geoculture of the world-system, was a politics of medium-term increments of reformist change adding up, putatively, to endless (long-term), linear, progress. This vision depicted a golden, extrapolatable, “now” with no allusion to the ideologies of either a future transformation (socialism) or an idyllic past (conservatism). The parallel to Newtonian dynamics is clear. Science itself offered the linear development model, based empirically and epistemologically on independent units. But in the post cold-war world, liberalism has proven unable to deliver on its universalist message of progress. In its neoliberal guise it has failed in its bid to reinstate the geopolitical realities of the world characterized by US hegemony in the post-1945 period, or through structural adjustment to reproduce the conditions of the Kondratieff A-phase economic expansion of the same period. 16 16 Science now provides us with alternative models of physical reality, relationally constituted self-organizing systems and fractal geometry, and of change and transition, complexity theory and chaos theory. These all defy the law of the excluded middle that has been one of the bedrock tenets of legitimate knowledge production, and common sense, for the past five centuries. The Modern World System and Its Structures of Knowledge The recognition of the indeterminacy of meaning in the humanities and the “alternative knowledges” that found a home in the social sciences with the expansion of faculty and student body after 1968 to include those speaking from marginalized subject positions have highlighted the political dimension of knowledge production and undermined the idea of scholarship as a perfectly disinterested activity amenable to objective evaluation. Nonetheless, we have not reached the end of responsibility and social agendas. We are hardly at the “end of history.” To the contrary; we are on the frontier “after history” when time and space can no longer be treated as neutral parameters but must be viewed as socially constructed and interdependent processual categories. The present transition is the last frontier of historical capitalism. The future is decidedly one of transformation and abounds with possibilities. But not all of the possible futures we can envision are equally desirable. Indeed, the alternatives are becoming clear. These may be thought of in terms of the “spirit of Davos” (with reference to the World Economic Forum), a new historical system that like the present world would be “based on privilege, exploitation, and polarization,” or the “spirit of Porto Alegre” (with reference to the World Social Forum), a new historical system “relatively democratic and relatively egalitarian” (Wallerstein 2006: 19). This struggle for the future, then, calls for committed, purposeful action, as no final outcomes are predictable. Lasting for the next 30-50 years perhaps, the transition will be rich in fluctuations, 17 17 17 17 17 that is, social instability—a lack of order already comprises the “new world order.” Unstable systems, in fact, impose fewer constraints, fewer limits. The exercise of free will, for instance in the form of interpretative scholarly work meaningful for these times, is thus less restricted and, capable of massive amplification, at some point will constitute irreversible and determining moral choices for a qualitatively different social world, a new historical system with its own unique set of structures, processes of reproduction, and geoculture. Historical Social Science for Our Times Half a century ago, during the post-1945 period through the mid-1960s say, it was quite clear to academics and laymen alike what the organizing arrangements of the disciplines of knowledge, and the layout of the departments that expressed them institutionally, were. At the upper end of what was indeed a hierarchy of disciplines were the natural sciences; at the bottom of the hierarchy were to be found the humanities; and somewhere in a contested, amorphous, middle space were the social sciences. The departments of the superdisciplines were generally housed in different buildings on university campuses; libraries of the sciences were often separated from those serving the humanities and the social sciences; and the departments themselves published in different sets of proprietary journals. Half a century ago, during the post-1945 period through the mid-1960s say, it was quite clear to academics and laymen alike what the organizing arrangements of the disciplines of knowledge, and the layout of the departments that expressed them institutionally, were. At the upper end of what was indeed a hierarchy of disciplines were the natural sciences; at the bottom of the hierarchy were to be found the humanities; and somewhere in a contested, amorphous, middle space were the social sciences. The departments of the superdisciplines were generally housed in different buildings on university campuses; libraries of the sciences were often separated from those serving the humanities and the social sciences; and the departments themselves published in different sets of proprietary journals. The unquestioned prestige of the sciences was premised on what were genuine accomplishments in explaining real world phenomena and thus at least to a certain extent harnessing those phenomena, as for instance witnessed in the war effort (radar, atom bomb, etc.). There was also, however, an epistemological dimension to this prestige. The sciences purportedly dealt with the natural world in terms of universal truths, in terms of “facts,” which could either be observational (specifics) or theoretical (natural laws). The key term was universal 18 18 in the sense of always, everywhere and untainted by human bias. Furthermore, scientific laws were constructed in such a way as to admit prediction. The humanities, on the other hand, in considering the world of human relations proposed anything but universal knowledge; they were relativistic and explicitly concerned with the unpredictable arena of human values. Historical Social Science for Our Times Finally the social sciences, which were also concerned with the world of human relations, sought to develop a form of knowledge about the social world that was “scientific” in the sense of being unbiased (the “view from nowhere”) and admitting at least a soft form of predictability. The acceptance of this structure, and its hierarchy, as natural and beyond question reached its peak in the immediate post-1945 period, and, like the axial division of labor and the interstate system, it became global in extent. Nonetheless, tensions have remained apparent over the past half century in the resurgences of old epistemological debates such as reductionism versus holism, structure or determinism versus agency or freedom, and order versus disorder— each antinomy manifesting an aspect of the division between facts and values, the sciences and the humanities. The acceptance of this structure, and its hierarchy, as natural and beyond question Today, there is certainly a heightened sense that we live in a “knowledge-based society”. There is, indeed, much discussion of this in the standard literature. Although it is quite true that analysts and entrepreneurs alike have always considered knowledge to be a central aspect of the processes through which capital is accumulated, it is only since the 1960s that the realm of knowledge has been in any way problematized, even in the short run. Fritz Machlup (1962) initiated research in this vein by examining “knowledge industries,” apart from the rest of the service sector of the U.S. economy, and measuring their contribution to the gross national product. He concluded that they would soon overtake the industrial sector. Since then, both the scholarly and popular literature on the “information society” and the “knowledge society” has 19 19 exploded. exploded. Some refer to the phenomenon as living in an age of a “knowledge economy” (e.g., Neef 1998; Cooke 2002; Acs, de Groot and Nijkamp 2002). Others suggest that we are living in an age of “knowledge capitalism.” For his part, Lester Thurow considers the twentieth century as a period of transformation from a natural resource economy to an economy in which the major players are brainpower industries and in which the major sources of comparative advantage are knowledge and skill alone (1996). To cite another particularly well-known observer and analyst, Peter Drucker contends that today we are living through a transformation to a post-capitalist society. Historical Social Science for Our Times He notes that “knowledge is becoming the sole factor of production, sidelining both capital and labor” (1993: 20). I would argue that the transformation is much more fundamental and profound than either of these or many other mainstream analysts suggest; it is the working out of the secular crisis of the processes reproducing the structures of social life of the modern world that emerged from the transition from feudalism to capitalism in Europe. However, it is only visible as a fundamental crisis from the standpoint of the longue durée (see Hopkins and Wallerstein 1996). Nonetheless, the recognition of this crisis is, indeed, the essential starting-point for relevant social analysis today. The crisis was implicit within the sciences over the two decades after 1945 when voices that diverged from the standard positivistic, reductionist and analytic model in the direction of an organic, relational model began to be heard. In one way or another, these new approaches were conceived as appropriate to the study of "systems that are intrinsically complex," as Ross Ashby noted (1991: 249). The issues involved were addressed by, among others, the elaboration of General System Theory (GST) and Cybernetics. 20 20 20 But these have not been easy roads. In matters of application, Anatol Rapoport, an original protagonist of GST, expressed concern about the exploitation of systems approaches in the management of military organizations and in consulting work where the goal was corporate profit-making (1998: 16). Felix Geyer has articulated the larger question: the more realistic—and therefore less parsimonious—a theory, the more complex it becomes, and the more difficult to test the hypotheses and subhypotheses derived from it which are used in collecting and interpreting the data. . . . For the time being, sociology should perhaps . . . force itself to give up the ambition to make accurate medium- and long-term predictions, except in delimited areas of research where complexity is still manageable or can be more or less contained (1995: 28). This statement alludes to both a boundary problem and to the ultimate ambition of the social sciences: prediction. It has become clear that defining a set of interactions for which the external context can be ignored (in the sense of having negligible impact) is extremely problematic. Historical Social Science for Our Times Furthermore, work in contemporary complexity studies shows that prediction itself is possible for only a subset of systems studied even in the natural sciences, and then only under certain, limited circumstances—much less for the world of human interactions. This statement alludes to both a boundary problem and to the ultimate ambition of the social sciences: prediction. It has become clear that defining a set of interactions for which the external context can be ignored (in the sense of having negligible impact) is extremely problematic. context can be ignored (in the sense of having negligible impact) is extremely problematic. Furthermore, work in contemporary complexity studies shows that prediction itself is possible for only a subset of systems studied even in the natural sciences, and then only under certain, limited circumstances—much less for the world of human interactions. Complexity studies emerged directly from research in the sciences and mathematics (see, for instance, Aida et al. 1985; Pagels 1988; Stein 1989; Lee 1992; Waldrop 1992; Byrne 1998). At the very moment of the worldwide triumph of the Newtonian worldview, that is, of a deterministic world of natural laws based on time-reversible dynamics, this new knowledge movement challenging the premises of that worldview began to take root. The rethinking that we are witnessing today represents a synthetic approach as opposed to a reductionist one; indeed, a 21 21 science of complexity holds out the possibility of representing change—that is, “describing our collective reality as a process”—without reverting to reductionism (Casti 1994: 273). It marks a shift away from the Newtonian worldview emphasizing equilibrium and certainty and defining causality as the consistent association of antecedent conditions and subsequent events amenable to experimental replication and hypothesis testing. In 1986, Sir James Lighthill, President of the International Union of Theoretical and Applied Mechanics, went so far as to apologize on behalf of “practitioners of mechanics . . . for having misled the general educated public by spreading ideas about the determinism of systems satisfying Newton’s laws of motion [implying complete predictability] that, after 1960, were to be proved incorrect” (1986: 38). Although all developments were not equally successful, what did turn out to have enormous resonance, in terms of extensive theoretical pertinence and broad areas of application, was the study of chaos. The recognition of the existence of chaotic behavior exhibited by nonlinear systems flew in the face of Laplacian predictability. Historical Social Science for Our Times As these systems evolve over time, they rapidly magnify small perturbations and are thus highly sensitive to small changes in initial conditions. Despite this, there remains evidence of an embedded order underlying the seemingly random evolution of certain dynamical systems. The development of what came to be loosely known as chaos theory on so many fronts opened up the possibility of applying deterministic models, formerly restricted to the “closed universe” of “completely predictable sys- tems,” rather than stochastic models, to certain systems that behave randomly. Such randomness, of course, leaves open a place for chance and therefore creativity and change. But in natural systems, not all theoretically possible states turned out to be realizable. Only some, those that lie on the strange attractor of such systems, will actually appear in nature. Ivar Ekeland has called this an “admirable and subtle mix of chance and necessity” (1998: 13, 12, 15). 22 22 Drawing on an immense body of work by Ilya Prigogine (which won him the Nobel Prize) and carried on with his colleagues in Austin and Brussels where complexity has been understood in terms of system “behavior” rather than of system interactions (Nicolis & Prigogine 1989), Prigogine and Isabelle Stengers (1984) presented chaos not as the opposite but as the source and confederate of order. They considered that a conceptual transformation of science was taking place. This transformation was growing out of the challenge to Newtonian mechanics associated with contemporary research in thermodynamics focusing on nonlinearity (instability, fluctuations, order-out-of-chaos) and the irreversibility of the evolution of far-from-equilibrium, open systems, characterized by self-organizing processes and dissipative structures. In the light of instability and chaos, and the association of the arrow of time with order as well as disorder, Prigogine and Dean Driebe have maintained that the laws of nature now express possibilities in- stead of certainties. There is no longer any contradiction between dynamical and Prigogine, looking for the roots of time, “became convinced that macroscopic irreversibility was the manifestation of the randomness of probabilistic processes on a microscopic scale” (1996: 60). Historical Social Science for Our Times The recognition that probability is more fundamental than trajec- tories implies what Prigogine calls “the end of certainty” (1996), and the end of certainty in scientific prediction connotes (again) an open future of creativity and choice in natural systems and, as a consequence, a vindication of freedom, agency, and creativity in our understanding of social systems, but with particular significance in times of crisis or transition. 23 23 23 In the humanities, it was the turn to culture, or rather the return to culture—and the emergence of cultural studies as a consolidated knowledge movement (see Lee 2003a)—by the independent left in Britain during the 1950s that signaled the crisis of the structures of knowledge. And indeed, this was part of a specific historical conjuncture. The context was formed in the short term by the geopolitical events of 1956 (Hungary, Khrushchev’s “secret” speech, Suez) that figured in the East-West struggle and in the medium-term by the multi-faceted dominance (hegemony) of the United States. On the one hand, the category of culture had enjoyed a continuity of application by the long line of social critics in the literary tradition, but in a way that had eventually depoliticized the analytic perspective it had for so long grounded. On the other hand, the delegitimation of both of the major contemporary approaches to social analysis—in the West, the quantitative, comparative method of the Columbia School and from the East, the orthodox base-superstructure model—opened a space for the deployment of “culture,” repoliticized through efforts of the first New Left, as a primary analytic category. The geopolitics of the mid-1950s came together with the 150-year trajectory of British (social) criticism (e.g., Burke and Paine, Arnold, Pater, Leavis) in the formation of the first New Left and the emergence of cultural studies. Raymond Williams, E. P. Thompson, and Richard Hoggart, each in his own way, contributed to the intellectual and scholarly (as well as political) bases of the movement(s). Notwithstanding the differences they exhibited in practical politics or analytical emphasis, the national perspective and common concerns for education, popular cultural forms and the “lived” experience of class, which they shared, represent a continuity in English cultural criticism that survived in the immediate directions cultural studies took. Historical Social Science for Our Times In Hoggart, Williams, and Thompson, the elite, literary/critical tradition, drained of politics, intersected and combined with the theoretical and practical commitment to both politics 24 24 and history at the popular level on the Left. A renewed and refocused interest in the working class appeared as a common theme in the literature associated with the first New Left and claimed as formative by cultural studies. From “below”, Hoggart recovered an urban working-class experience that was being undermined by commercialism and validated it in the face of elite cultural expressions. From “above”, Williams, in Culture and Society: 1780-1950 (1958), exposed how the forces of reaction had appropriated texts of resistance. Both, however, were limited by the gradual exclusion of the politics of struggle from the tradition of criticism informing their work, a point often made by Thompson. Williams and Thompson shared a dedication to historical analysis inherent to Marxism, but neither Williams nor Hoggart seemed to be writing just history, or sociology, or literary criticism for that matter. Williams and Hoggart approached their subject matter from the humanities, but their work rejected disciplinary boundaries, and the disciplines rejected them. All the same, their work, with that of Thompson, collectively legitimated a return to class politics. The recognition of the inadequacies of received categories of analysis, the emphasis on the relationality of the field, and this decentering and destabilization of the naturalized, taken- for-granted separation of the humanities and the social sciences and the divisions among the social sciences has been fundamental to the cultural studies project with its emphasis on values and interpretation in social analysis. From the social sciences, sciences studies (including "sociology of scientific knowledge," "social studies of science," and "science and technology studies") has offered "exogenous" appraisals of the development of science ranging from the way the social field influenced the directions of the development of science to the contingency of scientific knowledge, its (social) 25 25 25 constructedness, and its local situatedness (e.g., Shapin 1995). New disciplinary/departmental groupings have also been invented, challenging the fact-values divide and illustrating how essentialist categories of difference have functioned to subordinate entire groups. Diverse strands of "feminism and gender studies" have pointed to the constructedness of the categories of "man" and "woman" and have noted how scientific discourse of the female body has functioned to position women in society. Historical Social Science for Our Times Many scholars of "race and ethnicity" in the West have attacked essentialism as well while disputing Western universalism and objectivity. By the same token, many of those studying "non-Western societies" have emphasized alternatives to the Western development model and pointed to the implications of these alternatives for epistemology. The "culture wars" and the "science wars" are striking evidence of the depth of the resistance these new developments have encountered (see Lee 2004b; Santos 2003). They are evidence of the fundamental nature of the debates and conflicts in the modern world over how valid knowledge may be produced, what are the grounds and the scope of its authority, and who may speak in its voice. Most importantly, the developments across the superdisciplines raise the concrete question of what contemporary social action may be considered legitimate. The emphasis in complexity studies—on contingency, context-dependency, multiple, overlapping temporal and spatial frameworks, and deterministic but unpredictable systems displaying an arrow-of-time—suggests, as some scientists are beginning to say, that the natural world as they now see it is beginning to look unstable, complicated, and self-organizing, a world whose present is rooted in its past but whose development is unpredictable and cannot be reversed. In short, it is beginning to more closely resemble the social world, rather than vice versa. The question remains, if complex behavior is not amenable to explanation through hypothesis testing and theory construction because such systems, including now social systems, albeit 26 26 26 deterministic, are inherently unpredictable, how can we proceed? Indeed, the hypothesis testing mentioned earlier may be part of the contemporary epistemological quandary. It is part of a framework oriented towards the development of explanations for particular phenomena and the generalizations on which such explanations rest. This framework includes the corollary that once the theory is known, outcomes of specific interventions can be predicted and, therefore, social science knowledge can be exploited in policy-making. This was the basis for the cross-national comparative research (the analogue of laboratory studies in the natural sciences) on which modernization theory depended. By the 1960s, the empirical failure of this work called seriously into question its theoretical and methodological underpinnings (not incidentally, just as the world-economy entered a period of contraction, U.S. hegemony came to an end, and the politics of knowledge formation became an issue for antisystemic movements). Historical Social Science for Our Times Embodying an analogy between narrative and simulation, social analysts may henceforth feel licensed by the developments in complexity studies to make the shift from fabricating and verifying theories to imagining and evaluating the multiple possible consequences of diverse interpretative accounts of human reality and the actions they entail. Herein lies an alternative for a unified historical social science to the predictive, Newtonian model of social scientific inquiry. It constitutes a mode of constructing authoritative knowledge of the human world, which is of engaging in science, by producing defensible accounts and future scenarios, without chasing the chimera of predictability. Contemporary events in our globally integrated world have shown that methods that specify (often only implicitly) an exemplar and then endeavor to predict the impact of interventions designed to move supposedly autonomous units towards some ideal state perform poorly. This is what both scholars and policy-oriented analysts are experiencing, again today, to their dismay. All 27 27 27 the same, large-scale regularities do persist over time and particularistic "rich description”, or interpretive accounts based on an understanding, verstehen, of local value contexts, or resorting to "human creativity" or "free will" explanations fail as well to capture the interrelatedness of structure and emergence. Furthermore, an assessment of systems approaches uncovers an inherent paradox of social analysis: "the accumulation of knowledge often leads to a utilization of that knowledge both by the social scientists and the objects of their research—which may change the validity of that knowledge" (Geyer 1995: 19). One reading of this is that social knowledge is not universal, but knowledge for specific times, today for our times, fitting nicely with findings in complex systems research and the realization that human interactions constitute such a complex, deterministic but unpredictable, system. Necessity and chance can no longer be viewed as mutually exclusive options in social research. The combination of the conviction that there is a “real” world and that the future, The combination of the conviction that there is a “real” world and that the future, although it is “determined” by the past, is nonetheless unpredictable and the parallel assaults on dualism (e.g., Barrow 1995; Prigogine 1996) challenge the epistemological status of the sciences as unique discoverers, guardians, and purveyors of valid knowledge, that is, truth, by redefining what it means to describe the evolution of natural systems. Historical Social Science for Our Times As Ilya Prigogine has argued, the “sciences are not the reflection of a static rationality to be resisted or submitted to; they are furthering understanding in the same way as are human activities taken as a whole” (1988: 3). He goes so far as to state that “I believe that what we do today depends on our image of the future, rather than the future depending on what we do today” (Prigogine in Snell & Yevtushenko 1992: 28). 28). The fundamental importance of the crisis in knowledge formation is underlined when we must recognize that what has been going on in the sciences had been happening in analogous 28 28 28 movements across the entire range of the structures of knowledge since the 1960s. The equilibrium, consensus model that, it was argued, wrote history and power out of the equation, was contested by intellectuals and activists alike. The theorizations of the challenges to the liberal order (such as the Vietnam War, and the civil rights, feminist, and student movements) were outgrowths of and contributed to challenges to the prevailing structure of knowledge in the long term. Beginning in this period, work from across the disciplines led to conclusions and interpretations incompatible with the premises on which the relational structure of the natural sciences, the social sciences, and the humanities were founded. Developments that indicate a collapse of the frontier separating the humanities from the social sciences have included widespread methodological ecumenism, the rise of structuralism and the concomitant recognition of "values" as an integral part of all knowledge formation, the renewal of an appreciation of the significance of the local and the complex, and the revival of an emphasis on contingency and temporality associated with agency and creativity (see Lee 2003a). Across the disciplines these arguments may be represented as a concern for spatial-temporal wholes constituted of relational structures representing the persisting regularities normally associated with a "scientific" approach on one hand and, on the other hand, the phenomenological time of their reproduction and change (the ineluctable reality of the arrow-of-time) that captures the play of incommensurable differences associated with a "humanistic" approach. Difference, of course, involves values. We are thus presented with a re-fusing of “is” (the realm of facts and the goal of science) and “ought” (the field of values and the challenge of the humanities) in the construction of systematic knowledge of human reality. Historical Social Science for Our Times Values no longer need be, must no longer be, construed simply as a matter of individual ethics or morality in the creation of authoritative knowledge in the social sphere, but must 29 29 29 hereafter be conceived as an integral part of a historical social science. Indeed, authoritative knowledge thus constructed would have no pretensions of universality (validity for all times and places) but rather offer defensible interpretations for particular times and places. Thus, a social science for our times, of necessity singular and transcending disciplinary boundaries, must do two things. First, it must be premised on the indissoluble unity of the regularities of social relations, their structure, and change, their history. Secondly, it must recognize that the latter supposes the integration of values as integral to inquiry, not simply as a matter of the personal inclination of the analyst. Methods, How We Think What We Think One still often hears the claim that all sociology is comparative; indeed, just as Durkheim asserted: "comparative sociology is not a special branch of sociology; it is sociology itself" (1982[1895]: 157). We might also observe that it is now well-accepted that meaning itself is constructed through difference. Furthermore, with the increasing importance given to the study of larger and larger units over the past half century (associated with establishing generalizability and thus the predictability of interventions), more and more social research has consciously and purposefully identified itself as "comparative". Whether one works with a large number of cases in a quantitative setting in order to establish explanations, laws or quasi-laws of cause and effect serving as a basis on which to envisage the probable consequences of some projected action, or few cases and "ideal types" qualitatively to account for historical divergences, the aim is a "balance between competing claims of complexity and generality" (Ragin and Zaret 1983: 731). The common, everyday reality, however, is still an on-going tension between the nomothetic stance of the present-oriented disciplines of sociology, political science, and 30 30 30 economics and the idiographic propensity of history, anthropology, and oriental studies (this last largely superceded by area studies). Indeed, the more recent criticisms of large-scale work in the nomothetic social sciences have revolved around the absence of the historical dimension. In many respects, this represents the recognition, or the return of a recognition, of agency and complexity as co-constitutive of human reality along with the structures that constrain certain possibilities for action and promote others. The mirror image is the criticism of the idiographic social sciences as providing only non-generalizable, particularist descriptions that offer no guide to social action In the English-speaking world during the quarter century following 1945, a particular style of inquiry, theorized as structural-functionalism and operationalized through quantitative techniques and survey data, defined the parameters of authoritative social research, especially in the nomothetic social sciences, sociology, political science, and economics. From the beginning the focus was on "systems analysis", but the methodology was inherently comparative, and most often reductively quantitative. This always implied multiple units of analysis and it was nowhere more apparent than in the macro arena, where attempts were being made to explain differential development on a world scale based on modernization theory. Methods, How We Think What We Think Modernization theory joined policy planners (with their eyes on the East-West struggle) with social scientists (absorbed with explaining inequality). With explicit reference to GST, social structures and institutions were conceptualized as performing functions in systems where a "society" was a self-sufficient social system. Nonetheless, "societies" were ultimately associated with the state; time was transmuted into a function of autonomous society/state units simultaneously positioned at different points on a single reputed temporal hierarchy of development (horizontal history); and intentional 31 31 action modifying social structures was postulated as a primary mechanism of change and "progress." World-systems analysis brought together an intellectual critique of modernization theory, and the way it concealed rather than revealed the working of the world, with the real-world politics of the antisystemic activities of the movements of the 1960s. Modernization theory failed, empirically, on its own terms: fulfilling the political imperative of institutionalizing liberalism and representative democracy, the cultural imperative of implanting meritocracy and entrepreneurialism, and the economic imperative of scratching together enough capital for economic "take-off", simply did not produce the results anticipated. The seeds of failure were in the methods of analysis; the units were not autonomous and comparable, but all formed historically and relationally in the drive for endless accumulation of capital within a single system. For the world-systems perspective the relevant unit of analysis of long-term, large-scale social change is the "historical system". However, it was argued that historical systems were not amorphous, ill-defined "civilizations", but constituted a typology of large-scale, long-lasting structures with clear bounding criteria that included world-empires, world-economies, and mini- systems. The modern world-system or capitalist world-economy is such a historical system. Systemic; it possesses continuities in its relational patterns—in other words, its structures remained qualitatively recognizable over the long term. Historical; it has exhibited irreversible change over the long term—in other words, it came into existence at a specific time and place, underwent a spatio-temporal development that rendered it at all times and places different, and ostensibly will eventually cease to exist. Alone among historical social systems, it expanded to cover the entire globe and incorporated all other previously autonomous systems. It was, then, an 32 32 32 "open system". Its expansion to incorporate new pools of low cost labor represented an intake of "energy" in the form of labor-power. The system could thus overcome the entropy of its accumulation processes, capital corresponding to congealed labor. Methods, How We Think What We Think The salient characteristic of this formalization was that the elements of the modern world-system, including the categories through which social scientists, and activists, sought to understand it, were not pre-existing, timeless entities, but historically constituted by the evolution of the system’s relational structure. Over the past quarter century, this conceptualization has provided fruitful ground for a great deal of work developing theoretical strategies and methodological practices that avoid reification and reductionism. "open system". Its expansion to incorporate new pools of low cost labor represented an intake of "energy" in the form of labor-power. The system could thus overcome the entropy of its accumulation processes, capital corresponding to congealed labor. The salient characteristic of this formalization was that the elements of the modern world-system, including the categories through which social scientists, and activists, sought to understand it, were not pre-existing, timeless entities, but historically constituted by the evolution of the system’s relational structure. Over the past quarter century, this conceptualization has provided fruitful ground for a great deal of work developing theoretical strategies and methodological practices that avoid reification and reductionism. When we consider the practical issues of actual research, promulgating and putting into practice methodological practices appropriate to the world-systems framework, it becomes clear that not only our view of the world must undergo a gestalt shift, the ways in which we arrive at our understanding of that world must as well experience a fundamental transformation. It is from this particular perspective that I want to offer an example (from Lee 2004c) of how the choice of methodological approach constrains our understanding of real-world phenomena. George W. Bush and Usama bin Laden articulate two competing, mutually exclusive visions of the world. They do, however, seem to agree significantly on two things: the nature of the struggle and the final outcome. For Bush, "the war on terror begins with al Qaeda, but it does not end there" and "Americans should not expect one battle, but a lengthy campaign, unlike any other we have ever seen" (Bush 2001: 10, 13). He ended his address on the twentieth of September 2001 by stating that the "course of this conflict is not known, yet its outcome is certain" (23). In a prayer, bin Laden exhorts "Our Lord, make the youths of Islam steadfast and descend patience on them and guide their shots" (in Alexander and Swenan 2001: Appendix 1A: 33 33 22). Methods, How We Think What We Think Both see this as a long war and as an episodic war. Furthermore, they both predict ultimate victory for their respective forces. Victory means different things, however. For Bush, victory comes in terms of the reproduction of the world substantially unaltered; for bin Laden, victory is clearly couched in terms of the transformation of the world as we know it into an Islamic utopia. But how do we, whose profession it is to make sense of the world, much less we as citizens of the world, go about arriving at some understanding of what, on the face of it, seems an impossibly complex situation already cast in terms of diametrically opposed, mutually But how do we, whose profession it is to make sense of the world, much less we as citizens of the world, go about arriving at some understanding of what, on the face of it, seems an impossibly complex situation already cast in terms of diametrically opposed, mutually exclusive world views? The most common response is to look for parallels, to find “cases” that can be assimilated to a general classification and treated comparatively. This is, indeed, what we find in much of the post-9/11 literature. exclusive world views? The most common response is to look for parallels, to find “cases” that can be assimilated to a general classification and treated comparatively. This is, indeed, what we find in much of the post-9/11 literature. For many, the events of 9/11 were most obviously reminiscent of the Japanese bombing of Pearl Harbor, 7 December 1941—the "day that will live in infamy". A complete surprise, it was considered an unprovoked, "sneak", attack on US soil (although tensions between the United States and Japan had been mounting for some time) and determined the entry of the United States on the side of the Allied powers in the Second World War. A second parallel has been suggested in the War of 1812. This was the American front in the wars of Napoleon I. Although what actually caused the outbreak of war were clashes with the British on the western frontier, impressments of sailors and the seizure of U.S. shipping were already causes for strong resentment. A third parallel that has been suggested are the Tripolitan and Algerene Wars. In 1800 the Pasha of Tripoli raised the tribute on Christian Nations and thus on U.S. Methods, How We Think What We Think shipping in the Mediterranean and went so far as to declare war. President Jefferson sent a squadron of warships to North Africa and after a long blockade a peace treaty was signed abolishing the annual tribute 34 34 34 for the United States. The United States ended all tribute to North African states in 1815 and broke the back of Barbary piracy in in that year. Now as cases for comparison with the present situation, each of these prior phenomena presents similarities, and the consequences of designating such parallels are readily apparent. More precisely, the conclusion that can be drawn from each comparison offers a clear prognostic for the future, both in terms of actions and outcomes. In the case of Pearl Harbor, the United States came out of the war the major victor and in a position of such power that no other coalition of states could challenge her militarily, In the case of Pearl Harbor, the United States came out of the war the major victor and in a position of such power that no other coalition of states could challenge her militarily, commercially, or financially. The message is clear: staying the course and bringing overwhelming force to bear, without regard for the cost, will lead to a (new) golden age of Pax Americana and national affluence. Ideologically, this return of the United States to its position of hegemony in the world-system in the immediate post-1945 world not only remains attractive to the political right in the United States, but is, in fact, the stated goal of the Bush administration. Simply asserting the parallel gives the objective a veneer of legitimacy and the actual comparison underwrites the action to be taken and the projected outcome. In the case of the War of 1812, what was at stake was the existence of the United States as a sovereign state and this has been one of the themes of the war on terror. Indeed, the ultimate victory of the United States in the War of 1812 determined a period of nationalistic fervor mixed with isolationism, something that we are already seeing today, where isolationism does not mean pulling back behind national borders, but rather the “go it alone” strategy seeking the maximum of control, including coercive control, over foreign relations. Methods, How We Think What We Think In the case of the wars against the Barbary pirates, it should be remembered that besides the religious or civilizational element, there is a specific mention of such actions in the 35 35 35 Constitution and although the United States at this moment does not assert that there exists a state of war, "the legal state of affairs most closely resembles one we experienced at the founding of the Republic". At the time, pirates and privateers were deemed such a menace that the Constitution gave Congress the "the much narrower power [than declaring war] to 'define and punish Piracies, Felonies committed on the High Seas, and Offenses against the Law of Nations'" (Koh 2001: 158-59). Here the question was whether the United States could project force in a way that would secure the safety of American commerce and its citizens. This is a primary concern of many today, from intellectuals and policy analysts to the protagonists of social movements at the global level. The result of meeting the challenge two hundred years ago suggests the rightness of that decision, and thus of the decision to “meet the challenge” in our own time. Constitution and although the United States at this moment does not assert that there exists a state of war, "the legal state of affairs most closely resembles one we experienced at the founding of the Republic". At the time, pirates and privateers were deemed such a menace that the Constitution gave Congress the "the much narrower power [than declaring war] to 'define and punish Piracies, Felonies committed on the High Seas, and Offenses against the Law of Nations'" (Koh 2001: 158-59). Here the question was whether the United States could project force in a way that would secure the safety of American commerce and its citizens. This is a primary concern of many today, from intellectuals and policy analysts to the protagonists of social movements at the global level. The result of meeting the challenge two hundred years ago suggests the rightness of that decision, and thus of the decision to “meet the challenge” in our own time. As simple comparisons, each of these parallels is defensible and presents a certain face validity; however, comparative sociology demands multiple and autonomous cases, which nonetheless belong to a single category of phenomena that may be characterized by the same set of variables. Methods, How We Think What We Think But how do we proceed when our unit of analysis is singular—a single case? Comparisons may not be the most appropriate mode of inquiry for answering questions about the modern world-system. Systems analysis suggests that analogies, or rather the kind of rigorous analogies that systems scientists call homologies (for our purposes, the investigation of the articulation of diverse instances to the same processes reproducing the long-term structures of the system) may offer the answer. I would, then, like to see how the outcome of our inquiry might be altered by the shift from comparing these cases to treating them as analogies. In doing this, we recognize at the level of method a conception of the processes reproducing the modern world as playing out through social time: the uniqueness and singularity of the structures, the 36 36 fluctuations that account for the long-term dynamic equilibrium of those structures, the trends that manifest the changes in the continuously recognizable system, and finally, the events, Fernand Braudel's "poussière". From this perspective, Pearl Harbor may be seen as a single event, which took place, however, in the context of a hegemonic war. Thus, the analogy must be with the Thirty-Years War of the seventeenth century that ended with the Hegemony of the Dutch and with the Napoleonic Wars that ended with the hegemony of the British. The thirty-years long war of the twentieth century, of course, ended with the hegemony of the United States. That period of hegemony is over. Thus, this analogy is one that suggests that the struggle is for world leadership, a world leadership that the Americans, unlike the Dutch, are unwilling to give up. There is much to suggest that no matter what the expenditure of treasure and lives, it will be to no avail and that the harder the United States fights for a return to the state of the world in the quarter century following the end of the war in 1945, the worse will be the future, both for the United States in the long term and for the world, at least in the short and medium term (see Hopkins and Wallerstein 1996; Wallerstein 2003). The question, of course, is who or what is the challenger for world leadership. This question is the key to understanding the analogy with the War of 1812. Methods, How We Think What We Think By prevailing in this conflict, the United States established its sovereignty within the Interstate System. That organization of mutually recognized (but always partial and fluctuating) territorial autonomy, which structurally, only demands that there be at minimum two state-like entities to avoid the imposition of a world-empire. It assures the reproduction of the world division of labor based on the law of value resulting in exploitation and the polarization of inequalities by regulating flows of capital, goods, and labor across borders and restricting resistance activities to regulating flows of capital, goods, and labor across borders and restricting resistance activities to regulating flows of capital, goods, and labor across borders and restricting resistance activities to 37 37 37 the state rather than the system level. Today that sovereignty is being challenged anew, but not by another state or states as it was two centuries ago, but rather by non-state entities that express a completely different conception of the organization of the legitimate use of force on a world scale; this is a challenge to that very system. The Barbary pirates episode in American history, illustrates what Frederic Lane called "protection rents" (1979: 13) and the way that political and economic processes interact. The two wars were fought for "free trade" and certainly are analogous to the struggles today over the appropriation and accumulation of surplus that the present thirty-year economic contraction has made apparent. All the same, the free trade of two hundred years ago is not the neo-liberalism of today. So far my examples have been one-sided; each of the cases that I have cited is based on comparisons among short-term events with outcomes manifested in the medium-term. This generally includes those who see the present period as paralleling anti-colonial or anti-imperial struggles, with the world experiencing the impact of the “weapons of the weak”. However, in world-systemic terms these are but resistances to the deepening of the structures of domination and exploitation that the expansion of the world-system had extended to the entire globe by the end of the nineteenth century. Those who would sustain that the present period is in some way special in this respect should consider that the expansion of the world-system (and resistance to it) has been one of the processes that not only has defined it but assured its reproduction over the long term by resolving its medium-term crises. Methods, How We Think What We Think Nonetheless, the conditions of neo-imperialism and globalization suggest another long-term element. Usama bin Laden (like his adversaries) might well remember that toward the end of the nineteenth century this expansion began to reach its asymptotic limit with no part of the globe 38 38 38 left un-incorporated in the axial division of labor. Indeed, for bin Laden whose inspiration is to be found in Wahhabism—"a return to the way of 'virtuous ancestors,' a highly regressive, monolithic interpretation of Islam known as Salafiyya, a doctrinal propensity that for centuries encouraged strict adherence to puritanical principles" (Amanat 2001: 36)—the ideal parallel is a return to the Caliphate and the golden age of Islam on the world stage, prior to the full incorporation of the Islamic world. left un-incorporated in the axial division of labor. Indeed, for bin Laden whose inspiration is to be found in Wahhabism—"a return to the way of 'virtuous ancestors,' a highly regressive, But what if we were to ask the same questions that Bush and bin Laden are asking about the present situation, but answer them with an analogy that belongs explicitly to the long-term, structural time of the modern world-system? There are good reasons to believe that, with no room for expansion, the internal contradictions of the processes of the modern world-system may no longer allow for the reproduction of the system as a whole (see Hopkins and Wallerstein 1996). The analogy, then, that I would propose for the present would be the long and episodic Hundred Years' War, the conflict out of which were born the structures of decision making and coercion, production and distribution, and cognition and intentionality that heretofore have been constitutive of our historical system. When Edward III of England made claims on lands on the mainland of Europe and assumed the title of "King of France" in the second quarter of the fourteenth century he did so asserting his feudal rights. At the time England and France were not what we would call “states” today, but realms or royaumes. A century later, the English had achieved little (the government was bankrupt, the Lancastrian dynasty discredited, and civil war was on the horizon). For France, the outcome was very different. They were the big winners, but the system for which they fought, feudalism based on parcelized sovereignty and reciprocal rights and obligations, was 39 39 39 moribund. Methods, How We Think What We Think The cost of winning was the death of the object. The emergence of a new geo-political form laid the groundwork for the Interstate System organized as a relational system (with England and France as its founding parts) that would be formalized by Jean Bodin and institutionalized in the Treaty of Westphalia. So George W. Bush may be right in the sense that he will "win", but winning viewed from a long-term perspective could lead to the transformation of the system that he believes he is trying to protect—much the way France "won" the Hundred Years' War. So, he is probably dead wrong, however, in thinking that he can reestablish U.S. hegemony in the world-system. Usama bin Laden may also be right, but in the sense that even though his struggle certainly will not succeed—indeed, as Gilles Kepel (2003) has argued, militant, fundamentalist Islamism is actually in decline—it may very well lead to, or at least be part of, the transformation of the world as we know it, although probably not in the direction he would desire. The events of 9/11, then, are like the Khobar Towers bombing of 1996, the Kenya and Tanzania embassies bombings in 1998, the attack on the USS Cole in 2000, and the aftermath links it to the events such as the bombings, hijackings, and hostage crises of the 1970s and 1980s and reprisals such as the attack on Muammar el-Qaddafi's compound in 1986. Indeed, Bosnia, Chechnya, Iraq—and 9/11—are specs of Braudelian dust, but they are analogous to the Battle of Crecy and the Jacquerie. Together, they are suggestive of the way analogies among instances of processes may tell a very different story from comparisons of cases. Conclusion In this secular crisis of the structures of knowledge, it is clear that the grand intellectual antinomies that have been the subject of hot debate for so long—holism versus reductionism, structure versus 40 40 agency, determinism versus freedom, order versus disorder, fact versus value—are dependent not just on contradictory epistemological positions, but more surely on a specific ontological view of the world as made up of fundamentally deterministic and predictable systems. We are not living the "new world order" but a transition period of "new world disorder,” a time of massive fluctuations far from equilibrium in the language of complexity studies. Change will not depend only on our normatively motivated action for its initiation. During a period of wide fluctuations in the constitutive processes of a system driven far from equilibrium, including a system of social relations, small fluctuations can have enormous impact even to the extent of effecting total sys- temic transformation—instabilities expanding possibilities, that is, opportunities, by reducing constraints. By the same token, the direction of change will, as complexity studies show, be exquisitely dependent on small fluctuations, for instance, in the form of our value-laden decisions and actions (see Lee 2001; 2003a). “This is the time to make the future—precisely because everything is in flux. This is the time that Immanuel Wallerstein calls transformational timespace, that of Kairos, that of human choice when free will is possible (1991: 147). It is not so much the simple return of agency, but the manifestation of the fundamental relationship between agency and structure—the indivisibility of chance and necessity. As I have argued elsewhere (Lee 2001/02), the definition of valid knowledge claims in terms of "who, what, when, where, why" and the "view from nowhere" is giving way. We may (indeed, I would argue we must), without forsaking our dedication to "science", or even technƝ, turn our attention to producing authoritative knowledge in terms of "for whom, for what, for when, for where" and "from whose point-of-view". The consequences of developments across the disciplines impinge directly on the manner in which scholars make claims for the legitimacy of their interpretations of social reality, and thus amount to overturning the dominant model, of 41 41 knowledge and reality, shaping our understanding of the human world. Conclusion There is no way, however, that we can know if the transformation underway at present, and in which we will all play our part in one way or another, will result in a more substantively rational human world. A knowledge society we have been, and will remain. But the structure of that knowledge is already changing with understandings of the natural world and human reality evolving on non-contradictory bases. Today, in a world at the end of certainly, choice matters: conscious, cognizant choice in variously assembling our exploding stock of information to create knowledge that is both historical and systemic in the form of possible alternative futures along with their inherent value orientations. The issue then truly is choice, choice among those alternatives, the necessity of exercising it, how to exercise it, and on what basis to exercise it to bring about that more substantively rational world. These discussions are especially relevant today when destabilizing structural pressures are forcing change, but unlike the situation a century ago, those of us searching for a way out of the contemporary intellectual, political, and institutional quandaries have been liberated from the Cartesian/Newtonian constraints. The structural sequestration of the spheres of knowledge no longer appears as an unquestioned given. The study of government need not necessarily be isolated from the study of language, or analyses of market operation automatically separated from considerations of culture in departments like Political Science, English, Economics, Sociology or Art History. Indeed, it has become a legitimate proposition to say that they should not be isolated or separated. Defensible, intersubjective interpretations of relationships among constituent parts of concrete wholes on the other hand suggest a realizable mode of scholarly participation in the creation of a world where "social" is no longer forced to serve as the qualifying adjective for a dubious branch of "science." 42 42 The crisis in the field of knowledge, that is, in the structures of cognition and intentionality, is part of the overall exhaustion of the processes reproducing the structures of production and distribution in the economic sphere, and those of coercion and decision-making in the political arena. Since this is a secular, or structural crisis, change does not depend on our normatively motivated action for its initiation. By the same token, the direction of change will, as complexity studies show, be exquisitely dependent on small fluctuations in the form of our value-laden decisions and actions. Conclusion For instance, contemporary economics, in this “far-from-equilibrium” world, despite its allegiance to the principles of formal rationality, could be well placed to contribute to an understanding of the alternatives available today. But this would entail a reexamination of the inherited theoretical approaches and methodological practices—such as the primacy given to the short term in a world of competing “economies,” quantification, and model-building and the premises of ceteris paribus and individualistic decision-making—that currently underpin the discipline. Some alternatives seem evident. Certainly, the idea that we now live in one world should not surprise. But should we not realize also that that world has been an expanding one for the past five centuries, defined by a single world division of labor and only recently has come to encompass the entire globe? Associated with the reality of such a world would be the idea that if such a “historical system” came into existence, it could also very well cease to exist when the processes of its reproduction ran up against asymptotes that no longer allowed its internal contradictions to be overcome. Thus, a historical perspective would permit distinguishing between medium-term conjunctural crises, downturns and eventual upturns, and the exhaustion of long-term trends portending a systemic transformation. All of the above, however, will only 43 43 43 43 be possible if we allow that the study of production and distribution, coercion and decision making, as well as cognition and intentionality, are part of a single historical social science. Works Cited Acs, Z.J., H.L.F. de Groot, and P. Nijkamp, eds. 2002. The Emergence of the Knowledge Economy: A Regional Perspective. Berlin: Springer-Verlag. Aida, Shühei et al. 1985. 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https://openalex.org/W2990278398	https://europepmc.org/articles/pmc6882820?pdf=render	English	null	A Partial Correlation Screening Approach for Controlling the False Positive Rate in Sparse Gaussian Graphical Models	Scientific reports	2,019	cc-by	18,753	www.nature.com/scientificreports www.nature.com/scientificreports Ginette Lafit1,2, Francis Tuerlinckx1, Inez Myin-Germeys2 & Eva Ceulemans1 Ginette Lafit1,2, Francis Tuerlinckx1, Inez Myin-Germeys2 & Eva Ceulemans1 Gaussian Graphical Models (GGMs) are extensively used in many research areas, such as genomics, proteomics, neuroimaging, and psychology, to study the partial correlation structure of a set of variables. This structure is visualized by drawing an undirected network, in which the variables constitute the nodes and the partial correlations the edges. In many applications, it makes sense to impose sparsity (i.e., some of the partial correlations are forced to zero) as sparsity is theoretically meaningful and/or because it improves the predictive accuracy of the fitted model. However, as we will show by means of extensive simulations, state-of-the-art estimation approaches for imposing sparsity on GGMs, such as the Graphical lasso, ℓ1 regularized nodewise regression, and joint sparse regression, fall short because they often yield too many false positives (i.e., partial correlations that are not properly set to zero). In this paper we present a new estimation approach that allows to control the false positive rate better. Our approach consists of two steps: First, we estimate an undirected network using one of the three state-of-the-art estimation approaches. Second, we try to detect the false positives, by flagging the partial correlations that are smaller in absolute value than a given threshold, which is determined through cross-validation; the flagged correlations are set to zero. Applying this new approach to the same simulated data, shows that it indeed performs better. We also illustrate our approach by using it to estimate (1) a gene regulatory network for breast cancer data, (2) a symptom network of patients with a diagnosis within the nonaffective psychotic spectrum and (3) a symptom network of patients with PTSD. In many scientific disciplines, researchers are interested in the linear dependencies and unique relations between larger sets of variables, such as genes1, proteins2, symptoms of a disease3, functional brain connectivity4, etc. There is consensus that computing all pairwise correlations between these variables is misleading, because such correla- tions do not correct for linear relations that might be due to other variables. Therefore, many researchers recur to calculating partial correlation coefficients, which express the remaining linear dependency between two variables, after the effect of the rest of the variables under study is removed. More specifically, Gaussian Graphical Models (GGMs) have become increasingly popular5,6. Scientific Reports \| (2019) 9:17759 \| https://doi.org/10.1038/s41598-019-53795-x Ginette Lafit1,2, Francis Tuerlinckx1, Inez Myin-Germeys2 & Eva Ceulemans1 These models yield an undirected network (i.e., undirected graph) in which the variables are depicted as nodes and the partial correlations among the variables are visualized as the edges among the nodes. The width of an edge reflects the size of the corresponding partial correlation (see Fig. 1).ti hl Often, a sparse GGM is fitted, which implies that many of the partial correlations are forced to zero and thus that the corresponding edges in the network can be dropped. In some applications, the assumption of sparsity is intrinsic to the phenomenon under study. For instance, it has been shown that most genetic networks are sparse7,8. In other applications, the assumption of sparsity is motivated through improved interpretability. Indeed, even if the true model is not sparse the sparsity assumption allows to more accurately estimate the remaining parameters when the amount of information per parameter (n/p) is relatively small9, and prevents overfitting10. i Popular methods to estimate sparse GGMs are the regularized nodewise regression approach of Meinshausen and Bühlmann11, the joint sparse regression (SPACE) approach by Peng, et al.12 and the Graphical lasso (Glasso) proposed by Friedman, Hastie and Tibshirani13. These three approaches optimize different objective functions 1Research Group on Quantitative Psychology and Individual Differences, KU Leuven–University of Leuven, Leuven, 3000, Belgium. 2Center for Contextual Psychiatry, KU Leuven–University of Leuven, Leuven, 3000, Belgium. email: ginette.lafit@kuleuven.be www.nature.com/scientificreports/ Figure 1. The undirected network implied by the toy example. Figure 1. The undirected network implied by the toy example. (see Methods section) but all set some of the estimated parameters, and thus some of the network edges, to zero through 1 penalization. This penalization boils down to summing the absolute values of the estimated parameters and adding this sum to the objective function, after multiplying it by a regularization parameter. This parameter determines the impact of the penalty and has to be tuned by the user. Different tuning approaches have been proposed, based on cross-validation, information criteria, or finite sample derivations. Yet, 1 penalization often does not work well. Indeed, recent studies on the use of 1 penalization in standard regression analysis have shown that it tends to yield too many non-zero regression weights14–16. Translating these results to the estimation of sparse GGMs, we expect regularized nodewise regression, SPACE and the Glasso to often yield false positives, implying that some of the drawn edges should have been dropped. Ginette Lafit1,2, Francis Tuerlinckx1, Inez Myin-Germeys2 & Eva Ceulemans1 We will test this hypothesis in extensive simu- lations, in which we will also evaluate the effect of the tuning approach (i.e., information criteria, k-fold cross validation or finite sample derivations). i p To overcome the problem of incorrectly included edges, we will present a novel approach, that we call Partial Correlation Screening (PCS). Our PCS approach consists of two steps. In the first step, we estimate a sparse partial correlation network using one of the state-of-the art methods mentioned above. In the second step, we try to filter out the false positives that will probably be present in the estimated network. To this end, we screen the resulting partial correlation matrix for values that are smaller in absolute value than a cross-validation based threshold and set these to zero. This novel approach is based on earlier work on thresholding after regularization. Specifically, Saligrama et al.17 and Descloux and Sardy18 proposed the idea of thresholding after applying an 1 regularized procedure in the context of regression analysis. Ha and Sun19 presented a related idea for GGMs that consists of estimating the partial correlation matrix using a ridge penalty and then determining the non-zero entries of the matrix by hypothesis testing. Therefore, we will also evaluate what happens if we replace the 1 penalty by a ridge penalty. We will apply the Partial Correlation Screening approach to the same simulated data to show that it indeed performs better. Finally, we will show how the PCS approach can be used to estimate networks based on real datasets: (1) a gene regulatory network of patients with breast cancer, (2) a symptom network of patients with a diagnosis within the nonaffective psychotic spectrum and (3) a symptom network of patients with Post-Traumatic Stress Disorder (PTSD).hi The rest of the article is organized as follows. In the next section, we first present a toy example to introduce some notation and concepts and to illustrate that state-of-the-art estimation approaches yield networks that dif- fer from the population model. Then, using this toy example we show how our PCS procedure works. Next, we discuss the results of two simulation studies, one based on settings that have been used in other papers on this topic and one based on the estimated network for a real data set. We present applications to real datasets. Next, we discuss our findings and formulate conclusions. Ginette Lafit1,2, Francis Tuerlinckx1, Inez Myin-Germeys2 & Eva Ceulemans1 Finally, the Methods section presents a detailed description of the evaluated tuning approaches for each of the state-of-the-art estimation approaches and of the PCS procedure. Results T The set of edges E contains all node pairs (i, j) that are connected in the network, implying that Xi is conditionally dependent on Xj, given all the remaining variables. Thus, variable pairs that do not belong to the edge set are con- ditionally independent, given all remaining variables. For instance, in this illustration, the network shows an edge between variables X3 and X6. Therefore, these variables are conditionally dependent. However, there is no edge between variables X1 and X2, implying that X1 and X2 are conditionally independent. p y g y p Because the variables are Gaussian distributed, a variable pair (i, j) is conditionally independent if and only if their partial correlation given the rest of the variables is zero5. Let’s denote by Γ the partial correlation matrix. The entries ρij\|V\{i, j} of this matrix are the partial correlations between variables Xi and Xj, conditioned on the rest of variables. For the toy example the matrix Γ equals: Γ =    . . −. . . −. . . . . . . −. . . . −. −. . . . . . . . . −. . . −. −. . −. . −. .    1 00 0 00 0 45 0 00 0 00 0 45 0 00 1 00 0 00 0 00 0 00 0 00 0 45 0 00 1 00 0 00 0 45 0 45 0 00 0 00 0 00 1 00 0 00 0 00 0 00 0 00 0 45 0 00 1 00 0 45 0 45 0 00 0 45 0 00 0 45 1 00 (2) (2) We can now define the neighborhood of each node. The neighborhood of node i consists of all the nodes j that form an edge with node i, implying that the partial correlation of Xi and Xj differs from zero. In the toy example the neighborhood of node 1 is formed by nodes 3 and 6, while the neighborhood of node 2 is empty. Since the true edge set of the toy example is sparse, we can estimate it by means of the Glasso, SPACE, 1 regu- larized nodewise regression (NR) and ridge nodewise regression (Ridge). Unlike Glasso and SPACE which directly estimate the edge structure, NR computes a regression model per node and thus yields two regression weights for each edge. Results T To combine the information in these two weights into one edge, we can consider two vari- ants, NR-AND and NR-OR. The AND rule means that an edge is only included in the model if both regression weights differ from zero, whereas the OR rule is more liberal and selects all edges for which at least one of the regression weights is not set to zero. Ridge estimates the partial correlations by fitting a regression model for each node using an 2 penalty, which shrinks the regression weights towards zero. For each of the estimation methods, a number of approaches have been put forward to tune the regularization parameter, the details of which are provided in the Methods section. For Glasso we will use 10-fold cross valida- tion using two different loss functions: the first approach aims to minimize the negative log-likelihood function (CV1) and the second approach focuses on the sum of the prediction errors of each node (CV2). Moreover, we will apply two selection rules when using cross-validation: selecting the model that yields the lowest value and applying the one-standard-error-rule (1se)20. Additionally, we will consider the Bayesian Information Criterion (BIC) and the Extended Bayesian Information Criterion (EBIC)21. To tune the weight of the 1 penalty term in SPACE and NR, we will apply 10-fold CV, its one-standard-error-rule variant, BIC and the finite sample result (FSR) proposed by Meinshausen and Bühlmann11. Note that in NR the tuning is performed for each separate regression. To optimize the weight of the 2 penalty term in Ridge we will apply 10-fold CV for each separate regression. We note that this considered set of procedures is not intended to be exhaustive. Yet, the set is sufficient to illustrate the problem of efficiently tuning the penalty weight when there is limited information. pfi y g p y g Figure 2 shows the GGMs obtained with the nineteen considered approaches (i.e., nineteen combinations of estimation method (Glasso, SPACE, NR-AND, NR-OR and Ridge) and tuning options (CV, CV-1se, BIC, EBIC, FSR). We observe that Glasso-CV1-1se (panel c), NR-AND-FSR (panel o) and NR-OR-FSR (panel s) yield a network that is more sparse than the true network. Applying Glasso-CV1-1se all edges are set to zero. Whereas with NR-AND-FSR the edges (1, 3) and (3, 6) are set to zero, with NR-OR-FSR only the edge (3, 6) is set to zero. Results T Toy example. The toy data consists of n = 100 observations that are sampled from a p = 6-dimensional mul- tivariate Gaussian distribution. We set the covariance matrix of the distribution Σ to: Toy example. The toy data consists of n = 100 observations that are sampled from a p = 6-dimensional mul- variate Gaussian distribution. We set the covariance matrix of the distribution Σ to: Toy example. The toy data consists of n = 100 observations that are sampled from a p = 6-dimensional mul- tivariate Gaussian distribution. We set the covariance matrix of the distribution Σ to: Σ =    . . −. . . −. . . . . . . −. . . . −. −. . . . . . . . . −. . . −. −. . −. . −. .    1 63 0 00 0 70 0 00 0 63 0 70 0 00 1 00 0 00 0 00 0 00 0 00 0 70 0 00 1 68 0 00 0 70 0 13 0 00 0 00 0 00 1 00 0 00 0 00 0 63 0 00 0 70 0 00 1 63 0 70 0 70 0 00 0 13 0 00 0 70 1 68 (1) Σ =    . . −. . . −. . . . . . . −. . . . −. −. . . . . . . . . −. . . −. −. . −. . −. .    1 63 0 00 0 70 0 00 0 63 0 70 0 00 1 00 0 00 0 00 0 00 0 00 0 70 0 00 1 68 0 00 0 70 0 13 0 00 0 00 0 00 1 00 0 00 0 00 0 63 0 00 0 70 0 00 1 63 0 70 0 70 0 00 0 13 0 00 0 70 1 68 (1) (1) Scientific Reports \| (2019) 9:17759 \| https://doi.org/10.1038/s41598-019-53795-x www.nature.com/scientificreports/ The conditional independence structure of this distribution can be represented by a GGM. The corresponding undirected network is shown in Fig. 1. The six variables X1 to X6 form the set of nodes V = {1, 2, 3, 4, 5, 6}. Results T The other estimation methods yield networks that contain the true set of edges as well as false positives, with the number of false positives varying from ten (Glasso-CV2, panel d; NR-AND-CV-1se, panel m; Ridge-CV, panel t) to one (SPACE-CV-1se, panel i).hi p Our PCS procedure aims to remove these false positive edges. The first step of the procedure is to apply one of the nineteen considered approaches. In the second step, we try to single out the false positives by thresh- olding the entries of the estimated partial correlation matrix. Specifically, only the partial correlations that are larger in absolute value than a given threshold are retained, whereas the others are set to zero and thus removed from the network. The threshold is calibrated by means of 10-fold cross-validation (see Methods section for more information). For the toy example the nineteen computed thresholds range from 0.0001 to 0.283. Figure 3 presents the networks that we obtain by applying these thresholds to the networks in Fig. 2. We observe that PCS-Glasso-CV1 (panel b), PCS-Glasso-CV2 (panel d), PCS-Glasso-CV2-1se (panel e), PCS-Glasso-BIC (panel f), PCS-Glasso-EBIC (panel g), PCS-SPACE-CV (panel h), PCS-SPACE-BIC (panel j), PCS-SPACE-FSR (panel k), PCS-NR-AND-CV (panel l), PCS-NR-AND-CV-1se (panel m), PCS-NR-AND-BIC (panel n), PCS-NR-OR-CV- 1se (panel q), PCS-NR-AND-BIC (panel r) and Ridge-CV (panel t) remove the false positives and yield the true network. For, PCS-SPACE-CV-1se (panel i) none of the false positives are removed. PCS-NR-OR-CV (panel p) discards all but one false positive edge. Obviously, the networks with false negatives (panels c, o and s) cannot be improved by PCS. Simulation study with synthetic data. In this section we perform an extensive simulation study to eval- uate and compare the performance of the different procedures. We will inspect the results obtained with the nineteen combinations used above and study whether they improve when adding PCS. To this end, we replicated the settings used by Liu et al.22, Ravikumar et al.23, Rothman et al.24 and Yuan and Lin25. Scientific Reports \| (2019) 9:17759 \| https://doi.org/10.1038/s41598-019-53795-x www.nature.com/scientificreports/ Design. Each simulated data set is generated by drawing n independent observations from a p-variate Gaussian Figure 2. Estimated undirected networks for the toy example, before applying PCS. igure 2. Estimated undirected networks for the toy example, before applying PCS. Design. Each simulated data set is generated by drawing n independent observations from a p-variate Gaussian distribution with mean zero and partial correlation matrix Γ. Scientific Reports \| (2019) 9:17759 \| https://doi.org/10.1038/s41598-019-53795-x Scientific Reports \| (2019) 9:17759 \| https://doi.org/10.1038/s41598-019-53795-x Results T We considered two possible sample sizes n = {100, 500} and three different values of p = {20, 60, 200}. We inspected four different specifications of the population partial correlation matrix Γ. To illustrate these specifications for p = 60, we visualized them in Fig. 4. 1. Model 1: 2 neighbor Chain Graph, in which ρii\|V\{i} = 1 and ρi, i+1\|V\{i, i+1} = ρi−1, i\|V\{i, i−1} = −0.4, and all other edges are set to 0. 1. Model 1: 2 neighbor Chain Graph, in which ρii\|V\{i} = 1 and ρi, i+1\|V\{i, i+1} = ρi−1, i\|V\{i, i−1} = −0.4, and all other edges are set to 0. 2. Model 2: 3 neighbor Chain Graph, in which ρii\|V\{i} = 1, ρi, i+1\|V\{i, i+1} = ρi−1, i\|V\{i, i−1} = −0.4, ρi, i+2\|V\{i, i+2} = ρi−2, i\|V\{i, i−2} = −0.2, and all other edges are set to 0. edges are set to 0. 2. Model 2: 3 neighbor Chain Graph, in which ρii\|V\{i} = 1, ρi, i+1\|V\{i, i+1} = ρi−1, i\|V\{i, i−1} = −0.4, ρi, i+2\|V\{i, i+2} = ρi−2, i\|V\{i, i−2} = −0.2, and all other edges are set to 0. g 2. Model 2: 3 neighbor Chain Graph, in which ρii\|V\{i} = 1, ρi, i+1\|V\{i, i+1} = ρi−1, i\|V\{i, i−1} = −0.4, ρi, i+2\|V\{i, i+2} = ρi−2, i\|V\{i, i−2} = −0.2, and all other edges are set to 0. Scientific Reports \| (2019) 9:17759 \| https://doi.org/10.1038/s41598-019-53795-x www.nature.com/scientificreports/ 3. Model 3: 2 nearest-neighbor graph. We first specify the inverse of the covariance matrix Σ as follows: we randomly select p points from a unit square and we compute all pairwise distances between the p points. Then, for each node the neighborhood set is found by including the two nodes with the smallest distance. Figure 3. Estimated undirected networks for the toy example, after applying PCS. igure 3. Estimated undirected networks for the toy example, after applying PCS. 3. Model 3: 2 nearest-neighbor graph. We first specify the inverse of the covariance matrix Σ as follows: we randomly select p points from a unit square and we compute all pairwise distances between the p points. Then, for each node the neighborhood set is found by including the two nodes with the smallest distance. Next, the OR-rule is applied to these neighborhood sets to derive the associated undirected network. The off-diagonal elements of the corresponding Σ−1 are randomly chosen from the interval ∪ − −. . Results T We also report the average number of TP and FP values across the 100 replicates. Results. Tables 1 to 3 show the average TPR and FPR scores for the different methods under consideration for the different choices of p. We also report the average number of TP and FP for the different methods in Tables 4 to 6. First, we compare the performance of the methods without conducting PCS. The TPR and FPR scores depend strongly on the model used to generate the data and the values of n and p (i.e., amount of available information). In general, when comparing the performance of the different methods in controlling the amount of false non-zero partial correlations, we observe that for every combination of p and n, SPACE and NR perform better than Glasso. The results for Glasso are affected by the penalty tuning approach: whereas using cross-validation tends to introduce a large number of false positives across all different conditions, applying EBIC yields many false negatives. For NR and SPACE, the results depend on n and p and the data generating model. For n = 100, the best overall though still rather bad performance for Models 1 and 4 is obtained with some of the NR-AND variants and for Models 2 and 3 with some of the SPACE variants. We also note that for Model 2 none of the state-of-the-art methods (excluding Ridge) is able to efficiently estimate the true number of positive edges. Furthermore, in the high-dimensional case (i.e., p > n) all approaches perform badly in controlling the amount of false positive edges. When n = 500, the TPR and FPR values are clearly better than for the low-sample size setting and indicate good overall performance.t y p g g p Turning to the results after applying PCS, we observe in Tables 1 to 6 that PCS-SPACE and PCS-NR estimate networks that contain a smaller number of false positive edges than the state-of-the-art methods without PCS. This improvement is larger for Models 1, 3 and 4 and when n = 100 and n < p, in that PCS is able to control the number of false positive edges without compromising the number of correctly estimated true edges. Furthermore, the performance differences between the different SPACE and NR variants have diminished. PCS-SPACE-BIC has the best overall performance across all the n = 100 conditions. Results T [ 1, 0 5] [0 5, 1]. To ensure that Σ−1 is positive definite, the matrix is transformed as: Σ−1 + (\|λ(Σ−1)min\| + 0.1)Ip where λ(Σ−1)min refers to the smallest eigenvalue and Ip is an identity matrix of dimension p. To compute Γ we normalize Σ−1 and we multiply the off-diagonal elements by (−1). 4 Model 4: Random graph We first specify Σ−1 as follows: each upper triangular element of Σ−1 is set equal www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 4. Heatmaps of the true simulated networks when p = 60. White represents partial correlations equal to zero, and black represents partial correlations different from zero. Figure 4. Heatmaps of the true simulated networks when p = 60. White represents partial correlations equa zero, and black represents partial correlations different from zero. to 0.3 with probability ρ and to zero otherwise. We set the probability ρ = {0.1, 0.01, 0.001} when p = {20, 60, 200}, respectively. Next, we set the lower triangular elements equal to the corresponding upper trian- gular elements. To ensure that Σ−1 is positive definite the matrix is transformed as in model 3. Finally, to compute Γ we normalize Σ−1 and we multiply the off-diagonal elements by (−1). to 0.3 with probability ρ and to zero otherwise. We set the probability ρ = {0.1, 0.01, 0.001} when p = {20, 60, 200}, respectively. Next, we set the lower triangular elements equal to the corresponding upper trian- gular elements. To ensure that Σ−1 is positive definite the matrix is transformed as in model 3. Finally, to compute Γ we normalize Σ−1 and we multiply the off-diagonal elements by (−1). We generated 100 replicates for each cell of the design. An R script to conduct the simulation experiment is provided in the Supplementary Information. Performance measures. Results T To evaluate how well the different methods perform in distinguishing between true non-zero partial correlations and true zero ones, we compute the True Positive Rate (TPR) and False Positive Rate (FPR): = + TPR TP TP FN (3) = + FPR FP TN FP (4) (3) (4) where TP is the number of true positives (true non-zero edges that are estimated as such), TN is the number of true negatives (true zero edges that are recognized as such), FP is the number of false positives (true zero edges that are estimated as non-zero) and FN is the number of false negatives (true non-zero edges that are estimated as zero). The TPR and FPR coefficients take values in the range [0, 1]. For the TPR a value of 0 indicates that the labeling of edges as non-zero is completely wrong, a value of 0.5 indicates that the procedure cannot do better than random prediction and a value of 1 indicates a perfect recovery of the non-zero edges. Similarly, a FPR value of 0 indicates a perfect recovery of the zero edges, a value of 0.5 indicates that the procedure cannot do better than random prediction and a value of 1 indicates that the labeling of edges as zero is completely wrong. We also report the average number of TP and FP values across the 100 replicates. where TP is the number of true positives (true non-zero edges that are estimated as such), TN is the number of true negatives (true zero edges that are recognized as such), FP is the number of false positives (true zero edges that are estimated as non-zero) and FN is the number of false negatives (true non-zero edges that are estimated as zero). The TPR and FPR coefficients take values in the range [0, 1]. For the TPR a value of 0 indicates that the labeling of edges as non-zero is completely wrong, a value of 0.5 indicates that the procedure cannot do better than random prediction and a value of 1 indicates a perfect recovery of the non-zero edges. Similarly, a FPR value of 0 indicates a perfect recovery of the zero edges, a value of 0.5 indicates that the procedure cannot do better than random prediction and a value of 1 indicates that the labeling of edges as zero is completely wrong. Scientific Reports \| (2019) 9:17759 \| https://doi.org/10.1038/s41598-019-53795-x Results T For PCS-Glasso-EBIC the results cannot be improved by PCS, because Glasso-EBIC yields networks with a large number of false negatives. When n = 500, PCS performs almost perfectly in finding the non-zero edges in Models 1, 3 and 4, while for Model 2, the best overall performance is obtained with PCS-NR-OR-BIC when p = 20, 60 and with PCS-NR-OR-FSR when p = 200. Results T Scientific Reports \| (2019) 9:17759 \| https://doi.org/10.1038/s41598-019-53795-x www.nature.com/scientificreports/ n = 100 Model 1 Model 2 Model 3 Model 4 TPR FPR TPR FPR TPR FPR TPR FPR no-PCS PCS no-PCS PCS no-PCS PCS no-PCS PCS no-PCS PCS no-PCS PCS no-PCS PCS no-PCS PCS Glasso-CV 0.999 0.989 0.160 0.008 0.781 0.647 0.240 0.100 0.963 0.901 0.083 0.009 0.989 0.860 0.174 0.014 Glasso-CV-1se 0.992 0.985 0.042 0.019 0.315 0.309 0.019 0.018 0.866 0.849 0.013 0.007 0.853 0.825 0.022 0.013 Glasso-CV2 1.000 0.988 0.316 0.007 0.932 0.653 0.547 0.047 0.993 0.923 0.275 0.003 0.990 0.857 0.200 0.014 Glasso-CV2-1se 0.999 0.988 0.138 0.011 0.794 0.650 0.261 0.095 0.971 0.908 0.107 0.008 0.924 0.858 0.051 0.017 Glasso-BIC 1.000 0.989 0.213 0.008 0.066 0.059 0.014 0.009 0.988 0.927 0.206 0.006 0.659 0.621 0.051 0.011 Glasso-EBIC 0.300 0.299 0.021 0.003 0.000 0.000 0.000 0.000 0.947 0.899 0.054 0.009 0.000 0.000 0.000 0.000 SPACE-CV 0.993 0.991 0.015 0.010 0.667 0.632 0.072 0.054 0.939 0.919 0.016 0.008 0.910 0.876 0.024 0.016 SPACE-CV-1se 0.991 0.989 0.007 0.005 0.550 0.541 0.034 0.032 0.913 0.887 0.005 0.003 0.875 0.833 0.011 0.009 SPACE-BIC 0.994 0.991 0.014 0.007 0.624 0.588 0.044 0.036 0.956 0.907 0.013 0.004 0.938 0.873 0.031 0.015 SPACE-FSR 0.997 0.992 0.026 0.009 0.599 0.571 0.045 0.036 0.964 0.924 0.025 0.007 0.961 0.869 0.044 0.016 NR-AND-CV 0.997 0.979 0.116 0.015 0.819 0.697 0.215 0.047 0.978 0.944 0.084 0.007 0.945 0.838 0.081 0.033 NR-AND-CV-1se 0.942 0.940 0.007 0.004 0.425 0.417 0.022 0.017 0.834 0.817 0.003 0.001 0.623 0.588 0.004 0.003 NR-AND-BIC 0.992 0.987 0.024 0.006 0.522 0.504 0.039 0.022 0.915 0.884 0.012 0.002 0.847 0.793 0.014 0.007 NR-AND-FSR 0.793 0.793 0.000 0.000 0.122 0.113 0.000 0.000 0.730 0.725 0.000 0.000 0.411 0.403 0.000 0.000 NR-OR-CV 1.000 0.967 0.296 0.023 0.906 0.682 0.445 0.041 0.991 0.941 0.267 0.014 0.982 0.749 0.238 0.025 NR-OR-CV-1se 0.988 0.987 0.027 0.013 0.617 0.595 0.078 0.045 0.917 0.887 0.021 0.003 0.838 0.733 0.027 0.008 NR-OR-BIC 0.996 0.979 0.078 0.009 0.709 0.633 0.122 0.033 0.962 0.921 0.061 0.004 0.920 0.782 0.054 0.014 NR-OR-FSR 0.867 0.865 0.001 0.001 0.124 0.116 0.000 0.000 0.767 0.759 0.000 0.000 0.527 0.524 0.001 0.001 Ridge-CV 1.000 0.867 1.000 0.143 1.000 0.572 1.000 0.242 1.000 0.828 1.000 0.046 1.000 0.222 1.000 0.047 n = 500 no-PCS PCS no-PCS PCS no-PCS PCS no-PCS PCS no-PCS PCS no-PCS PCS no-PCS PCS no-PCS PCS Glasso-CV 1.000 1.000 0.076 0.001 0.999 0.981 0.277 0.036 0.988 0.984 0.023 0.014 1.000 1.000 0.071 0.003 Glasso-CV-1se 1.000 1.000 0.034 0.002 0.947 0.942 0.090 0.080 0.943 0.943 0.017 0.012 1.000 1.000 0.016 0.003 Glasso-CV2 1.000 1.000 0.314 0.002 1.000 0.988 0.639 0.011 1.000 1.000 0.264 0.002 1.000 1.000 0.169 0.002 Glasso-CV2-1se 1.000 1.000 0.120 0.002 1.000 0.985 0.393 0.024 1.000 0.999 0.092 0.004 1.000 1.000 0.036 0.004 Glasso-BIC 1.000 1.000 0.233 0.002 0.999 0.983 0.318 0.030 1.000 1.000 0.261 0.001 1.000 1.000 0.113 0.002 Glasso-EBIC 1.000 1.000 0.149 0.002 0.993 0.974 0.170 0.062 1.000 0.999 0.096 0.003 1.000 1.000 0.060 0.002 SPACE-CV 1.000 1.000 0.008 0.002 0.989 0.985 0.041 0.029 1.000 1.000 0.008 0.002 1.000 1.000 0.012 0.001 SPACE-CV-1se 1.000 1.000 0.005 0.001 0.957 0.956 0.018 0.018 1.000 0.999 0.005 0.001 1.000 1.000 0.009 0.001 SPACE-BIC 1.000 1.000 0.007 0.002 0.983 0.978 0.021 0.019 1.000 0.999 0.009 0.001 1.000 1.000 0.026 0.001 SPACE-FSR 1.000 1.000 0.020 0.001 0.993 0.985 0.040 0.029 1.000 0.999 0.023 0.002 1.000 1.000 0.044 0.002 NR-AND-CV 1.000 1.000 0.111 0.003 1.000 0.991 0.273 0.012 1.000 1.000 0.081 0.002 1.000 1.000 0.079 0.001 NR-AND-CV-1se 1.000 1.000 0.001 0.000 0.931 0.930 0.018 0.004 0.998 0.998 0.000 0.000 0.995 0.994 0.001 0.000 NR-AND-BIC 1.000 1.000 0.009 0.001 0.992 0.989 0.055 0.007 0.999 0.999 0.007 0.001 1.000 1.000 0.008 0.001 NR-AND-FSR 1.000 1.000 0.000 0.000 0.612 0.612 0.002 0.002 0.988 0.988 0.000 0.000 1.000 1.000 0.000 0.000 NR-OR-CV 1.000 1.000 0.305 0.002 1.000 0.987 0.510 0.012 1.000 1.000 0.274 0.002 1.000 1.000 0.239 0.001 NR-OR-CV-1se 1.000 1.000 0.003 0.001 0.981 0.979 0.050 0.006 0.999 0.999 0.006 0.000 0.999 0.999 0.011 0.000 NR-OR-BIC 1.000 1.000 0.046 0.001 0.998 0.992 0.148 0.008 1.000 1.000 0.038 0.001 1.000 1.000 0.034 0.001 NR-OR-FSR 1.000 1.000 0.000 0.000 0.662 0.662 0.004 0.002 0.994 0.994 0.000 0.000 1.000 1.000 0.001 0.000 Ridge-CV 1.000 1.000 1.000 0.002 1.000 0.977 1.000 0.021 1.000 0.999 1.000 0.003 1.000 0.996 1.000 0.010 Table 1. Results T Average true positive rate (TPR) and false positive rate (FPR) over 100 replications when p = 20. Note: Standard errors for TPR range from 0.002 to 0.046 and standard errors for FPR range from 0.000 to 0.019. Table 1. Average true positive rate (TPR) and false positive rate (FPR) over 100 replications when p = 20. Note: Standard errors for TPR range from 0.002 to 0.046 and standard errors for FPR range from 0.000 to 0.019. Finally, we study how the sample size and the non-sparsity level influence the height of the estimated threshold in the PCS procedure. For Glasso, SPACE, NR using the AND rule and NR using the OR rule, we estimate a linear mixed effect model with a random intercept in which observations are clustered according to the tuning procedure (i.e., different CV variants, information criteria or finite sample results). The model includes the estimated thresh- olds as the dependent variable and the sample size and the non-sparsity level as predictors. The non-sparsity level is computed as the number of true non-zero partial correlations divided by the total number of edges in the network. For Ridge we estimate the same model using OLS regression. Table 7 shows the obtained regression coefficients for each estimation procedure. We observe that across the different estimation procedures there is a significant negative relation between the sample size and the estimated threshold value. Also, we found a significant negative relation between the non-sparsity level and the threshold value parameter for all methods except Glasso. Simulation study based on real data. In this section we simulate data based on the sparse GGM results obtained by Armour, et al.3 for 20 Post-Traumatic Stress Disorder (PTSD) symptoms of 221 U.S. military veterans. The 20 PTSD symptoms are assumed to form four symptoms clusters: intrusions (B1-B5), avoidance (C1-C2), negative alterations in cognitions and mood (D1-D7), and alterations in arousal and reactivity (E1-E6). Armour, et al.3 applied the Glasso-EBIC approach and used bootstrapping techniques to estimate the parameter accuracy and stability of the partial correlation matrix Γ26. The associated network, shown in Fig. Results T 5, reveals strong positive Scientific Reports \| (2019) 9:17759 \| https://doi.org/10.1038/s41598-019-53795-x www.nature.com/scientificreports/ n = 100 Model 1 Model 2 Model 3 Model 4 TPR FPR TPR FPR TPR FPR TPR FPR no-PCS PCS no-PCS PCS no-PCS PCS no-PCS PCS no-PCS PCS no-PCS PCS no-PCS PCS no-PCS PCS Glasso-CV 1.000 0.981 0.086 0.002 0.655 0.467 0.103 0.018 0.935 0.750 0.079 0.003 1.000 0.915 0.079 0.001 Glasso-CV- 1se 0.998 0.977 0.028 0.003 0.369 0.351 0.010 0.008 0.817 0.714 0.014 0.003 0.982 0.908 0.008 0.001 Glasso-CV2 1.000 0.980 0.161 0.002 0.822 0.496 0.268 0.011 0.957 0.761 0.132 0.003 0.997 0.905 0.055 0.001 Glasso-CV2- 1se 1.000 0.979 0.084 0.002 0.681 0.475 0.123 0.018 0.923 0.740 0.064 0.003 0.978 0.898 0.007 0.001 Glasso-BIC 0.999 0.980 0.051 0.003 0.000 0.000 0.000 0.000 0.722 0.621 0.015 0.003 0.029 0.028 0.000 0.000 Glasso-EBIC 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 SPACE-CV 0.993 0.989 0.004 0.003 0.569 0.531 0.029 0.019 0.834 0.795 0.009 0.005 0.982 0.943 0.004 0.001 SPACE-CV- 1se 0.986 0.984 0.002 0.002 0.474 0.466 0.013 0.012 0.766 0.755 0.003 0.003 0.980 0.941 0.004 0.001 SPACE-BIC 0.997 0.989 0.010 0.003 0.573 0.520 0.025 0.016 0.885 0.809 0.014 0.005 0.997 0.939 0.022 0.001 SPACE-FSR 0.998 0.988 0.009 0.003 0.529 0.507 0.018 0.015 0.889 0.810 0.016 0.005 0.997 0.937 0.022 0.001 NR-AND-CV 0.998 0.972 0.048 0.006 0.687 0.563 0.088 0.013 0.893 0.793 0.047 0.004 0.988 0.882 0.027 0.001 NR-AND- CV-1se 0.940 0.938 0.003 0.002 0.314 0.311 0.007 0.007 0.566 0.537 0.002 0.001 0.733 0.689 0.001 0.000 NR-AND- BIC 0.986 0.974 0.008 0.002 0.348 0.332 0.007 0.004 0.728 0.685 0.005 0.001 0.949 0.902 0.003 0.000 NR-AND- FSR 0.670 0.670 0.000 0.000 0.044 0.038 0.000 0.000 0.308 0.307 0.000 0.000 0.589 0.586 0.000 0.000 NR-OR-CV 1.000 0.943 0.168 0.010 0.822 0.556 0.242 0.014 0.955 0.767 0.159 0.006 0.997 0.836 0.105 0.001 NR-OR-CV- 1se 0.982 0.973 0.017 0.004 0.528 0.494 0.041 0.018 0.767 0.697 0.016 0.002 0.895 0.822 0.012 0.000 NR-OR-BIC 0.996 0.977 0.030 0.003 0.535 0.472 0.032 0.008 0.846 0.769 0.022 0.002 0.977 0.916 0.014 0.001 NR-OR-FSR 0.756 0.756 0.000 0.000 0.044 0.040 0.000 0.000 0.331 0.330 0.000 0.000 0.621 0.618 0.000 0.000 Ridge-CV 1.000 0.457 1.000 0.027 1.000 0.350 1.000 0.010 1.000 0.497 1.000 0.012 1.000 0.242 1.000 0.001 n = = 500 no-PCS PCS no-PCS PCS no-PCS PCS no-PCS PCS no-PCS PCS no-PCS PCS no-PCS PCS no-PCS PCS Glasso-CV 1.000 1.000 0.029 0.000 0.996 0.949 0.136 0.013 0.999 0.992 0.024 0.000 1.000 1.000 0.043 0.000 Glasso-CV- 1se 1.000 1.000 0.015 0.000 0.970 0.932 0.061 0.023 0.989 0.986 0.004 0.001 1.000 1.000 0.001 0.000 Glasso-CV2 1.000 1.000 0.151 0.000 1.000 0.974 0.383 0.004 1.000 0.997 0.206 0.000 1.000 1.000 0.059 0.000 Glasso-CV2- 1se 1.000 1.000 0.076 0.000 1.000 0.964 0.274 0.007 1.000 0.996 0.071 0.000 1.000 1.000 0.004 0.000 Glasso-BIC 1.000 1.000 0.079 0.000 0.985 0.940 0.081 0.021 1.000 0.994 0.060 0.000 1.000 1.000 0.007 0.000 Glasso-EBIC 1.000 1.000 0.038 0.000 0.871 0.865 0.025 0.022 0.998 0.990 0.010 0.001 1.000 1.000 0.003 0.000 SPACE-CV 1.000 1.000 0.001 0.000 0.985 0.979 0.017 0.012 1.000 0.998 0.002 0.000 1.000 1.000 0.004 0.000 SPACE-CV- 1se 1.000 1.000 0.001 0.000 0.947 0.947 0.007 0.007 1.000 0.998 0.002 0.000 1.000 1.000 0.004 0.000 SPACE-BIC 1.000 1.000 0.003 0.000 0.982 0.977 0.011 0.010 1.000 0.999 0.005 0.000 1.000 1.000 0.010 0.000 SPACE-FSR 1.000 1.000 0.006 0.000 0.983 0.978 0.015 0.012 1.000 0.999 0.014 0.000 1.000 1.000 0.023 0.000 NR-AND-CV 1.000 1.000 0.041 0.000 0.999 0.985 0.127 0.002 1.000 0.999 0.046 0.000 1.000 1.000 0.022 0.000 NR-AND- CV-1se 1.000 1.000 0.000 0.000 0.926 0.925 0.008 0.001 0.972 0.972 0.000 0.000 1.000 1.000 0.000 0.000 NR-AND- BIC 1.000 1.000 0.003 0.000 0.975 0.974 0.016 0.001 0.999 0.998 0.003 0.000 1.000 1.000 0.001 0.000 NR-AND- FSR 1.000 1.000 0.000 0.000 0.537 0.537 0.000 0.000 0.965 0.964 0.000 0.000 1.000 1.000 0.000 0.000 NR-OR-CV 1.000 1.000 0.162 0.000 1.000 0.979 0.297 0.003 1.000 1.000 0.157 0.000 1.000 1.000 0.094 0.000 NR-OR-CV- 1se 1.000 1.000 0.001 0.000 0.974 0.970 0.026 0.001 0.995 0.995 0.002 0.000 1.000 1.000 0.005 0.000 NR-OR-BIC 1.000 1.000 0.016 0.000 0.991 0.986 0.053 0.001 1.000 0.999 0.015 0.000 1.000 1.000 0.008 0.000 NR-OR-FSR 1.000 1.000 0.000 0.000 0.563 0.563 0.001 0.000 0.985 0.985 0.000 0.000 1.000 1.000 0.000 0.000 Ridge-CV 1.000 1.000 1.000 0.002 1.000 0.890 1.000 0.024 1.000 0.866 1.000 0.015 1.000 0.761 1.000 0.011 Table 2. Results T Average true positive rate (TPR) and false positive rate (FPR) over 100 replications when p = 60. Note: Standard errors for TPR range from 0.000 to 0.029 and standard errors for FPR range from 0.000 to 0.010. Table 2. Average true positive rate (TPR) and false positive rate (FPR) over 100 replications when p = 60. Note: Standard errors for TPR range from 0.000 to 0.029 and standard errors for FPR range from 0.000 to 0.010. within-cluster connections between nightmares (B2) and flashbacks (B3), blame of self or others (D3) and neg- ative trauma related emotions (D4), detachment (D6) and restricted affect (D7), and hypervigilance (E3) and exaggerated startle response (E4). On top of that, they also find many moderately positive connections within the symptom clusters: for instance, intrusive thoughts (B1) and nightmares (B2), avoidance thoughts (C1) and avoidance remainders (C2), irritability/anger (E1) and self-destructive behaviour (E2), but also between symptom clusters, for instance between loss of interest (D5) and difficulty in concentrating (E5). within-cluster connections between nightmares (B2) and flashbacks (B3), blame of self or others (D3) and neg- ative trauma related emotions (D4), detachment (D6) and restricted affect (D7), and hypervigilance (E3) and exaggerated startle response (E4). On top of that, they also find many moderately positive connections within the symptom clusters: for instance, intrusive thoughts (B1) and nightmares (B2), avoidance thoughts (C1) and avoidance remainders (C2), irritability/anger (E1) and self-destructive behaviour (E2), but also between symptom clusters, for instance between loss of interest (D5) and difficulty in concentrating (E5). Results T Scientific Reports \| (2019) 9:17759 \| https://doi.org/10.1038/s41598-019-53795-x www.nature.com/scientificreports/ n = 100 Model 1 Model 2 Model 3 Model 4 TPR FPR TPR FPR TPR FPR TPR FPR no-PCS PCS no-PCS PCS no-PCS PCS no-PCS PCS no-PCS PCS no-PCS PCS no-PCS PCS no-PCS PCS Glasso-CV 0.999 0.968 0.039 0.001 0.515 0.273 0.033 0.002 0.920 0.723 0.033 0.000 0.990 0.670 0.033 0.000 Glasso-CV-1se 0.999 0.965 0.018 0.001 0.321 0.259 0.005 0.002 0.873 0.720 0.011 0.001 0.956 0.647 0.006 0.000 Glasso-CV2 1.000 0.969 0.065 0.000 0.611 0.283 0.071 0.002 0.932 0.733 0.044 0.000 0.974 0.664 0.011 0.000 Glasso-CV2-1se 0.999 0.967 0.041 0.001 0.517 0.272 0.034 0.002 0.912 0.725 0.028 0.000 0.801 0.646 0.001 0.000 Glasso-BIC 0.996 0.963 0.010 0.001 0.000 0.000 0.000 0.000 0.604 0.520 0.003 0.000 0.000 0.000 0.000 0.000 Glasso-EBIC 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 SPACE-CV 0.987 0.984 0.001 0.001 0.405 0.365 0.006 0.004 0.809 0.785 0.002 0.001 0.930 0.751 0.002 0.000 SPACE-CV-1se 0.978 0.977 0.001 0.001 0.332 0.327 0.003 0.003 0.801 0.780 0.001 0.001 0.930 0.759 0.002 0.000 SPACE-BIC 0.997 0.981 0.006 0.001 0.497 0.365 0.014 0.003 0.908 0.787 0.010 0.001 0.985 0.718 0.016 0.000 SPACE-FSR 0.994 0.983 0.003 0.001 0.443 0.365 0.009 0.003 0.887 0.786 0.007 0.001 0.978 0.733 0.011 0.000 NR-AND-CV 0.995 0.952 0.018 0.002 0.511 0.358 0.029 0.004 0.874 0.749 0.016 0.001 0.944 0.542 0.010 0.000 NR-AND-CV-1se 0.910 0.908 0.001 0.000 0.187 0.170 0.002 0.002 0.615 0.585 0.001 0.000 0.524 0.390 0.001 0.000 NR-AND-BIC 0.973 0.965 0.002 0.001 0.238 0.217 0.002 0.001 0.750 0.706 0.001 0.000 0.839 0.637 0.001 0.000 NR-AND-FSR 0.475 0.475 0.000 0.000 0.010 0.008 0.000 0.000 0.344 0.343 0.000 0.000 0.239 0.237 0.000 0.000 NR-OR-CV 0.999 0.844 0.083 0.009 0.691 0.215 0.107 0.003 0.940 0.622 0.074 0.003 0.979 0.297 0.051 0.000 NR-OR-CV-1se 0.970 0.954 0.008 0.002 0.386 0.338 0.016 0.007 0.787 0.677 0.007 0.000 0.814 0.518 0.006 0.000 NR-OR-BIC 0.990 0.973 0.010 0.002 0.380 0.289 0.009 0.002 0.836 0.762 0.007 0.001 0.913 0.637 0.008 0.000 NR-OR-FSR 0.551 0.551 0.000 0.000 0.010 0.008 0.000 0.000 0.389 0.389 0.000 0.000 0.252 0.249 0.000 0.000 Ridge-CV 1.000 0.961 1.000 0.002 1.000 0.273 1.000 0.002 1.000 0.718 1.000 0.001 1.000 0.629 1.000 0.000 n = 500 no-PCS PCS no-PCS PCS no-PCS PCS no-PCS PCS no-PCS PCS no-PCS PCS no-PCS PCS no-PCS PCS Glasso-CV 1.000 1.000 0.012 0.000 0.986 0.886 0.064 0.004 0.989 0.975 0.006 0.001 1.000 1.000 0.023 0.000 Glasso-CV-1se 1.000 1.000 0.006 0.000 0.962 0.863 0.036 0.005 0.986 0.974 0.004 0.001 1.000 1.000 0.002 0.000 Glasso-CV2 1.000 1.000 0.080 0.000 0.999 0.928 0.218 0.002 0.999 0.985 0.041 0.000 1.000 1.000 0.011 0.000 Glasso-CV2-1se 1.000 1.000 0.042 0.000 0.996 0.908 0.125 0.003 0.999 0.986 0.041 0.000 1.000 1.000 0.001 0.000 Glasso-BIC 1.000 1.000 0.022 0.000 0.906 0.847 0.017 0.007 0.992 0.977 0.008 0.000 1.000 1.000 0.001 0.000 Glasso-EBIC 1.000 1.000 0.008 0.000 0.665 0.664 0.003 0.003 0.987 0.974 0.005 0.001 1.000 1.000 0.000 0.000 SPACE-CV 1.000 1.000 0.000 0.000 0.966 0.957 0.006 0.005 0.999 0.995 0.001 0.000 1.000 1.000 0.002 0.000 SPACE-CV-1se 1.000 1.000 0.000 0.000 0.931 0.931 0.003 0.003 0.999 0.994 0.001 0.000 1.000 1.000 0.002 0.000 SPACE-BIC 1.000 1.000 0.003 0.002 0.973 0.957 0.007 0.004 1.000 0.996 0.004 0.000 1.000 1.000 0.010 0.000 SPACE-FSR 1.000 1.000 0.002 0.001 0.963 0.958 0.006 0.005 1.000 0.996 0.006 0.000 1.000 1.000 0.013 0.000 NR-AND-CV 1.000 1.000 0.013 0.000 0.995 0.977 0.049 0.001 1.000 1.000 0.014 0.000 1.000 1.000 0.008 0.000 NR-AND-CV-1se 1.000 1.000 0.000 0.000 0.873 0.872 0.002 0.000 0.968 0.968 0.000 0.000 0.999 0.999 0.000 0.000 NR-AND-BIC 1.000 1.000 0.001 0.000 0.934 0.932 0.004 0.000 0.998 0.997 0.001 0.000 1.000 1.000 0.001 0.000 NR-AND-FSR 1.000 1.000 0.000 0.000 0.506 0.506 0.000 0.000 0.942 0.942 0.000 0.000 1.000 1.000 0.000 0.000 NR-OR-CV 1.000 1.000 0.073 0.000 0.999 0.962 0.143 0.001 1.000 0.999 0.067 0.000 1.000 1.000 0.037 0.000 NR-OR-CV-1se 1.000 1.000 0.001 0.000 0.944 0.939 0.010 0.000 0.991 0.990 0.001 0.000 1.000 1.000 0.003 0.000 NR-OR-BIC 1.000 1.000 0.005 0.000 0.972 0.968 0.015 0.000 0.999 0.999 0.004 0.000 1.000 1.000 0.004 0.000 NR-OR-FSR 1.000 1.000 0.000 0.000 0.516 0.516 0.000 0.000 0.960 0.960 0.000 0.000 1.000 1.000 0.000 0.000 Ridge-CV 1.000 1.000 1.000 0.000 1.000 0.742 1.000 0.008 1.000 0.965 1.000 0.003 1.000 1.000 1.000 0.000 Table 3. Scientific Reports \| (2019) 9:17759 \| https://doi.org/10.1038/s41598-019-53795-x www.nature.com/scientificreports/ We studied the conditional dependencies of a set of 24 psychopathological symptoms in a sample of 184 patients (189 before patients with missing data were discarded) within the nonaffective psychotic spectrum, that participated in the second wave of the multicenter Genetic Risk and Outcome of Psychosis (GROUP) cohort study43. The symptoms are measured using the Brief Psychiatric Rating Scale (BPRS)44, which captures the following symptoms: Somatic Concern (SmC), Anxiety (Anx), Depression (Dpr), Guilt (Glt), Hostility (Hst), Suspiciousness (Ssp), Unusual Thought (UnT), Grandiosity (Grn), Hallucinations (Hll), Disorientation (Dsr), Conceptual Disorganization (CnD), Excitement (Exc), Elevated mood (ElM), Tension (Tns), Mannerisms (Mnn), Uncooperativeness (Unc), Motor Retardation (MtR), Suicidality (Scd), Self Neglect (SlN), Bizarre Behaviour (BzB), Motor Hyperactivity (MtH), Distractibility (Dst), Emotional Withdrawal (EmW) and Blunted Affect (BlA). Each symptom is rated on a 7-point Likert scale. Because the data is measured on a Likert scale rather than on a continuous one, we apply the nonparanormal transformation proposed by Liu et al.45 that uses the Gaussian copula to transform the data into normal scores. p Table 9 shows the number of edges that result from applying the different methods under consideration. We observe that Ridge-CV, Glasso-CV1, NR-OR-CV and Glasso-BIC estimate the most dense networks and that applying PCS drastically reduces the amount of edges when the original network was not so sparse.f pp y g y g g p Figure 10 shows the network computed by combining the different PCS networks and discarding edges all that occur only once. Cognitive models that study psychosis have postulated that some of the most prominent symptoms are delusional beliefs (grandiosity, suspiciousness, unusual thoughts)46. We indeed observe that there is strong positive relation between Unusual Thoughts (UnT) and Suspiciousness (Ssp) and between Emotional Withdrawal (EmW) and Blunted Affect (BlA). Also, there is a strong positive relation between Unusual Thoughts (UnT) and Grandiosity (Grn), Motor Retardation (MtR) and Elevated mood (ElM), Anxiety (Anx) and Depression (Dpr), Depression (Dpr) and Guilt (Glt), and Tension (Tns) and Distractibility (Dst). Post-traumatic stress disorder symptoms data. Finally, we return to the PTSD data that we studied in Subsection: Simulation Study Based on Real Data. Table 9 shows the number of edges for each of the proce- dures. We observe a similar pattern as in the previous applications. Figure 11 displays the network that results from applying our combination approach to the PCS networks. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Table 9 shows how many edges are obtained with the Glasso, NR, SPACE and Ridge techniques under con- sideration and how much these numbers of edges decrease by applying PCS. It can be concluded that the sparsity level varies considerably depending on the approach used. We observe that when the procedures yield dense networks (i.e. Ridge-CV, Glasso-CV1, Glasso-CV1-1se, Glasso-CV2, Glasso-CV2-1se and NR-OR-CV), applying PCS produces a larger reduction in the number of edges. p g g Given that the results vary considerably across the methods, the next question is how we should deal with this uncertainty when interpreting the networks. We opt to combine the results of the different estimation meth- ods33,34, by computing a network that includes all edges that occur in at least two of the nineteen obtained PCS networks. Note that if we apply this combination approach to the estimated PCS networks for the toy example (see Fig. 3), we would recover the true network. g Figure 8 shows the resulting combined network for the breast cancer data. Figure 9 focuses on the sub-network of the genes that are related with the estrogen receptor gene ERS1 (Panel a) and the gene FOXA1 (Panel b). We can identify some important regularity interactions in the estimated GGM. As a first example, the ESR1 (ESR) gene is partially correlated with SLC39A6 (SLC). This gene functions as a zinc transporter and has been shown to be highly expressed in ESR1-positive tumours and is highly significantly associated with the spread of breast cancer to the lymph nodes32. As a second example, we can inspect the genes that belong to the neighborhood of FOXA1 (FOX). FOXA1 has been found to be predominantly expressed in luminal type A carcinomas35 and may prevent metastatic progression of this type of breast cancer36. We observe an edge between FOXA1 (FOX) and AR (AR) (androgen receptor), which is in line with findings that indicate that AR regulates estrogen receptor expression37. Psychopathological symptoms data. For a long time, modeling approaches to psychopathological data started from the assumption that psychopathological symptoms reflect an underlying mental disorder and thus are caused by this disorder38. This assumption has recently been challenged and an alternative hypothesis has been put forward stating that symptoms are causally active components of a mental disorder39,40. Within this framework, network analysis is then used to study the conditional dependencies between a set of symptoms41,42. www.nature.com/scientificreports/ This combined network recovers the conditional dependencies that Armour et al.3 found to be strongly positive: nightmares (B2) and flashbacks (B3), blame of self or others (D3) and negative trauma related emotions (D4), detachment (D6) and restricted affect (D7), and hypervigilance (E3) and exaggerated startle response (E4). Scientific Reports \| (2019) 9:17759 \| https://doi.org/10.1038/s41598-019-53795-x Results T Average true positive rate (TPR) and false positive rate (FPR) over 100 replications when p = 200. Table 3. Average true positive rate (TPR) and false positive rate (FPR) over 100 replications when p = 200. Note: Standard errors for TPR range from 0.000 to 0.037 and standard errors for FPR range from 0.000 to 0.0 To compare the performance before and after using PCS, we drew n observations from a 20-variate Gaussian distribution with mean zero and partial correlation matrix Γ. We used two sample sizes n = {100, 500} and repli- cated the simulation 100 times. Table 8 shows the average TPR and FPR scores and Figs. 6 and 7 present heatmaps of the frequency with which the entries of the partial correlation matrix are detected as non-zero. We observe that Partial Correlation Screening (PCS) significantly outperforms Glasso, NR and SPACE. When n = 100, PCS-SPACE-BIC has the best performance in terms of the false positive rate, which is in line with the simulation results on synthetic data. For n = 500, all the estimation procedures using PCS show an average TPR higher than 0.999 and an average FPR below 0.020 (see Table 8). Breast cancer data. GGMs have been widely applied to analyze gene expression data, since many authors hypothesize that the complex interactions between genes take the form of sparse pathways or networks27–29. More specifically, given mRNA levels of different patients, researchers have studied the conditional dependencies of genes for a variety of diseases1. We estimate a sparse partial correlation network for gene expression data from a breast cancer study by West et al.30. The dataset contains 7, 129 genes sampled from 49 breast tumor tissues samples: 25 samples from patients diagnosed as estrogen receptor positive and 24 samples from patients diagnosed as estrogen receptor negative. In line with Sheridan et al.31, we focus on a subset of p = 150 genes related to the estrogen receptor gene ERS1. This gene acts as an estrogen-activated transcription factor and has a key role in the proliferation of cancerous cells32. 9 Scientific Reports \| (2019) 9:17759 \| https://doi.org/10.1038/s41598-019-53795-x Discussion In this article, we have demonstrated through an extensive simulation study that the most popular procedures to estimate partial correlation networks, Glasso, SPACE, NR and Ridge, often do not yield the true underlying net- work, no matter which procedure is applied to select the regularization parameter. Results are heavily influenced by sample size and the number of variables (i.e., the lower the sample size and the higher the number of varia- bles, the worse), with high-dimensional problems being especially difficult. We also note that the Glasso results heavily depend on which approach is used to tune the regularization parameter. Specifically, we found that in the high-dimensional setting, using the BIC or EBIC yields many false negatives and thus an overly sparse network. g g g y y g y p Given that the state-of-the-art methods frequently cannot satisfactorily recover the true set of edges, we have presented a novel approach that allows to better control the false positive rate. This procedure boils down to performing an additional second step, after applying one or more state-of-the-art methods of choice. Discussion In this Scientific Reports \| (2019) 9:17759 \| https://doi.org/10.1038/s41598-019-53795-x 10 www.nature.com/scientificreports/ n = 100 Model 1 Model 2 Model 3 Model 4 TP FP TP FP TP FP TP FP no-PCS PCS no-PCS PCS no-PCS PCS no-PCS PCS no-PCS PCS no-PCS PCS no-PCS PCS no-PCS PCS Glasso-CV 18.99 18.80 27.28 1.45 28.91 23.93 36.65 15.30 15.41 14.41 14.38 1.49 11.87 10.32 30.96 2.49 Glasso-CV-1se 18.85 18.71 7.13 3.21 11.64 11.42 2.87 2.70 13.85 13.58 2.22 1.29 10.24 9.90 3.98 2.36 Glasso-CV2 19.00 18.78 53.98 1.24 34.49 24.15 83.68 7.22 15.89 14.77 47.83 0.53 11.88 10.28 35.57 2.53 Glasso-CV2-1se 18.98 18.78 23.59 1.89 29.39 24.04 39.98 14.49 15.53 14.53 18.61 1.38 11.09 10.29 9.06 3.05 Glasso-BIC 19.00 18.79 36.45 1.31 2.44 2.19 2.09 1.33 15.81 14.83 35.93 0.97 7.91 7.45 9.14 1.97 Glasso-EBIC 5.70 5.68 3.62 0.56 0.00 0.00 0.00 0.00 15.15 14.39 9.48 1.60 0.00 0.00 0.00 0.00 SPACE-CV 18.86 18.83 2.62 1.77 24.68 23.39 10.96 8.22 15.02 14.71 2.81 1.34 10.92 10.51 4.20 2.79 SPACE-CV-1se 18.83 18.79 1.18 0.93 20.34 20.00 5.22 4.85 14.61 14.19 0.86 0.56 10.50 10.00 2.01 1.54 SPACE-BIC 18.88 18.82 2.36 1.22 23.07 21.75 6.72 5.49 15.29 14.51 2.21 0.72 11.25 10.48 5.50 2.65 SPACE-FSR 18.94 18.84 4.39 1.49 22.18 21.11 6.93 5.54 15.43 14.78 4.32 1.27 11.53 10.43 7.85 2.93 NR-AND-CV 18.94 18.61 19.87 2.63 30.31 25.80 32.95 7.21 15.65 15.11 14.59 1.26 11.34 10.05 14.41 5.90 NR-AND-CV-1se 17.89 17.86 1.21 0.71 15.73 15.43 3.29 2.54 13.35 13.07 0.54 0.26 7.47 7.05 0.75 0.50 NR-AND-BIC 18.84 18.75 4.09 1.03 19.32 18.66 5.92 3.32 14.64 14.14 2.15 0.27 10.16 9.51 2.44 1.30 NR-AND-FSR 15.06 15.06 0.06 0.04 4.52 4.17 0.02 0.02 11.68 11.60 0.03 0.03 4.93 4.84 0.05 0.05 NR-OR-CV 19.00 18.37 50.63 3.85 33.54 25.22 68.12 6.27 15.86 15.06 46.42 2.42 11.78 8.99 42.36 4.45 NR-OR-CV-1se 18.77 18.75 4.55 2.22 22.83 22.00 11.87 6.90 14.67 14.19 3.61 0.54 10.05 8.80 4.84 1.34 NR-OR-BIC 18.92 18.60 13.38 1.57 26.24 23.43 18.69 5.05 15.39 14.73 10.57 0.66 11.04 9.38 9.70 2.50 NR-OR-FSR 16.48 16.44 0.13 0.09 4.59 4.31 0.03 0.02 12.27 12.14 0.05 0.05 6.32 6.29 0.10 0.10 Ridge-CV 19.00 16.48 171.00 24.42 37.00 21.16 153.00 36.96 16.00 13.24 174.00 8.02 12.00 2.66 178.00 8.40 n = 500 no-PCS PCS no-PCS PCS no-PCS PCS no-PCS PCS no-PCS PCS no-PCS PCS no-PCS PCS no-PCS PCS Glasso-CV 19.00 19.00 12.99 0.16 36.97 36.29 42.31 5.46 15.81 15.74 4.03 2.36 12.00 12.00 12.64 0.47 Glasso-CV-1se 19.00 19.00 5.82 0.34 35.04 34.85 13.74 12.29 15.08 15.08 2.90 2.12 12.00 12.00 2.79 0.56 Glasso-CV2 19.00 19.00 53.73 0.26 37.00 36.55 97.70 1.72 16.00 16.00 45.86 0.39 12.00 12.00 30.01 0.43 Glasso-CV2-1se 19.00 19.00 20.54 0.34 36.99 36.46 60.17 3.69 16.00 15.99 16.05 0.61 12.00 12.00 6.39 0.63 Glasso-BIC 19.00 19.00 39.90 0.35 36.98 36.36 48.67 4.63 16.00 16.00 45.40 0.22 12.00 12.00 20.10 0.41 Glasso-EBIC 19.00 19.00 25.45 0.37 36.74 36.02 26.08 9.54 16.00 15.99 16.65 0.50 12.00 12.00 10.61 0.40 SPACE-CV 19.00 19.00 1.35 0.31 36.60 36.43 6.25 4.38 16.00 16.00 1.44 0.35 12.00 12.00 2.16 0.23 SPACE-CV-1se 19.00 19.00 0.78 0.18 35.41 35.37 2.70 2.69 16.00 15.99 0.84 0.23 12.00 12.00 1.66 0.17 SPACE-BIC 19.00 19.00 1.26 0.26 36.36 36.20 3.24 2.98 16.00 15.99 1.49 0.23 12.00 12.00 4.62 0.17 SPACE-FSR 19.00 19.00 3.42 0.25 36.73 36.46 6.09 4.39 16.00 15.99 3.93 0.41 12.00 12.00 7.83 0.39 NR-AND-CV 19.00 19.00 19.00 0.46 36.99 36.68 41.78 1.76 16.00 16.00 14.18 0.41 12.00 12.00 13.99 0.26 NR-AND-CV-1se 19.00 19.00 0.10 0.05 34.43 34.40 2.69 0.62 15.96 15.96 0.05 0.02 11.94 11.93 0.10 0.01 NR-AND-BIC 19.00 19.00 1.62 0.21 36.70 36.61 8.40 1.12 15.99 15.99 1.27 0.16 12.00 12.00 1.49 0.12 NR-AND-FSR 19.00 19.00 0.02 0.01 22.65 22.65 0.30 0.28 15.81 15.81 0.01 0.01 12.00 12.00 0.05 0.03 NR-OR-CV 19.00 19.00 52.20 0.26 36.99 36.51 78.02 1.89 16.00 16.00 47.73 0.36 12.00 12.00 42.47 0.09 NR-OR-CV-1se 19.00 19.00 0.54 0.16 36.29 36.23 7.68 0.96 15.98 15.98 0.96 0.07 11.99 11.99 1.96 0.06 NR-OR-BIC 19.00 19.00 7.88 0.19 36.94 36.72 22.68 1.18 16.00 16.00 6.59 0.16 12.00 12.00 6.09 0.17 NR-OR-FSR 19.00 19.00 0.07 0.03 24.48 24.48 0.56 0.36 15.90 15.90 0.08 0.05 12.00 12.00 0.09 0.07 Ridge-CV 19.00 19.00 171.00 0.27 37.00 36.15 153.00 3.19 16.00 15.99 174.00 0.57 12.00 11.95 178.00 1.75 Table 4. Discussion Average number of true positive edges (TP) and false positive edges (FP) over 100 replications when p 20 F h d l th b f ti l l ti 19 f M d l 1 37 f M d l 2 16 f Table 4. Average number of true positive edges (TP) and false positive edges (FP) over 100 replications when p = 20. For each model the number of non-zero partial correlations are: 19 for Model 1, 37 for Model 2, 16 for Model 3 and 12 for Model 4. For each model the number of non-zero partial correlations are: 19 for Model 1, 37 for Model 2, 16 for Model 3 and 12 for Model 4. Note: Standard errors for TP range from 0.060 to 0.875 and standard errors for FP range from 0.024 to 2.913. second step, we discard the partial correlation coefficients in the estimated network that are smaller in absolute value than a given threshold, which is obtained through cross-validation. Our novel procedure clearly improved the performance of the estimation methods and tuning approaches considered, especially in the settings where the state-of-the-art methods yielded bad results. Whereas PCS-SPACE-BIC seems to be the best choice for small sample size, which method is applied in the first step hardly matters when sample size increases.h second step, we discard the partial correlation coefficients in the estimated network that are smaller in absolute value than a given threshold, which is obtained through cross-validation. Our novel procedure clearly improved the performance of the estimation methods and tuning approaches considered, especially in the settings where the state-of-the-art methods yielded bad results. Whereas PCS-SPACE-BIC seems to be the best choice for small sample size, which method is applied in the first step hardly matters when sample size increases.h We also applied all approaches to three real data sets. The results again show that our PCS approaches yield more sparse networks than the state-of-the art methods. To deal with the multitude of obtained networks, we proposed to compute a network that combines the different PCS estimates, but discard the edges that occurred in only one network. Although results seemed interpretable, future research should investigate further how to efficiently combine the different estimators or how to optimally select among the nineteen obtained networks. fif In this paper we used standard simulation settings from the literature to demonstrate the problematic behaviour of existing approaches. Scientific Reports \| (2019) 9:17759 \| https://doi.org/10.1038/s41598-019-53795-x Discussion It is important to mention that except for Glasso, none of the state-of-the-art procedures studied in this paper estimates a covariance matrix that is positive definite. Also, it is not guaranteed that this prop- erty still holds after applying the PCS to Glasso. In future research, it would be useful to investigate the behavior of the different approaches under more difficult settings as well as the theoretical properties of the PCS. This would lead to several possible extensions of our method. Discussion One extension targets data in which the assumption of multivariate Scientific Reports \| (2019) 9:17759 \| https://doi.org/10.1038/s41598-019-53795-x www.nature.com/scientificreports/ n = 100 Model 1 Model 2 Model 3 Model 4 TP FP TP FP TP FP TP FP no-PCS PCS no-PCS PCS no-PCS PCS no-PCS PCS no-PCS PCS no-PCS PCS no-PCS PCS no-PCS PCS Glasso-CV 58.99 57.86 146.82 3.62 76.66 54.68 170.65 29.75 44.87 36.01 136.18 5.62 13.00 11.90 139.32 1.40 Glasso-CV-1se 58.90 57.65 47.81 5.26 43.23 41.01 17.20 13.26 39.20 34.25 24.58 5.00 12.77 11.81 14.39 1.05 Glasso-CV2 59.00 57.81 275.50 3.05 96.22 58.05 443.62 18.63 45.95 36.53 227.62 5.43 12.96 11.77 96.47 0.98 Glasso-CV2-1se 58.99 57.77 143.88 3.58 79.65 55.61 203.21 29.94 44.30 35.54 111.01 5.61 12.72 11.67 12.69 1.22 Glasso-BIC 58.97 57.82 87.72 4.57 0.00 0.00 0.00 0.00 34.67 29.82 25.59 4.33 0.38 0.36 0.30 0.01 Glasso-EBIC 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 SPACE-CV 58.57 58.33 7.62 5.16 66.57 62.07 47.32 30.92 40.04 38.15 14.94 8.19 12.77 12.26 7.08 2.01 SPACE-CV-1se 58.16 58.04 3.39 3.11 55.44 54.48 22.12 19.92 36.75 36.25 5.52 4.73 12.74 12.23 6.26 2.16 SPACE-BIC 58.84 58.34 17.20 5.05 67.09 60.84 41.09 26.46 42.46 38.83 24.87 8.61 12.96 12.21 39.09 2.01 SPACE-FSR 58.89 58.28 15.29 4.79 61.88 59.36 30.51 24.70 42.69 38.90 28.35 8.59 12.96 12.18 38.13 2.15 NR-AND-CV 58.90 57.36 82.44 9.60 80.37 65.83 146.24 20.96 42.85 38.08 80.62 7.65 12.85 11.46 46.63 1.66 NR-AND-CV-1se 55.44 55.35 4.94 2.73 36.76 36.34 12.38 10.80 27.19 25.76 3.75 1.41 9.53 8.96 2.63 0.14 NR-AND-BIC 58.16 57.49 12.96 2.98 40.67 38.79 11.08 6.13 34.95 32.89 8.18 2.00 12.34 11.72 5.83 0.87 NR-AND-FSR 39.53 39.51 0.04 0.04 5.09 4.48 0.05 0.03 14.79 14.73 0.02 0.02 7.66 7.62 0.01 0.01 NR-OR-CV 59.00 55.62 287.18 16.60 96.20 65.04 399.67 22.58 45.83 36.83 274.38 10.12 12.96 10.87 184.09 2.44 NR-OR-CV-1se 57.95 57.38 29.18 6.82 61.76 57.75 67.10 30.30 36.81 33.47 27.38 3.39 11.63 10.68 20.23 0.53 NR-OR-BIC 58.76 57.63 51.54 5.84 62.59 55.23 52.46 12.70 40.63 36.92 37.44 4.05 12.70 11.91 25.02 1.53 NR-OR-FSR 44.60 44.59 0.11 0.11 5.12 4.63 0.05 0.04 15.91 15.84 0.05 0.05 8.07 8.04 0.01 0.01 Ridge-CV 59.00 26.94 1711.00 45.40 117.00 41.00 1653.00 17.02 48.00 23.85 1722.00 20.62 13.00 3.14 1757.00 1.60 n = 500 no-PCS PCS no-PCS PCS no-PCS PCS no-PCS PCS no-PCS PCS no-PCS PCS no-PCS PCS no-PCS PCS Glasso-CV 59.00 59.00 2.55 0.13 115.23 114.57 28.81 20.54 47.99 47.91 4.01 0.57 13.00 13.00 6.95 0.13 Glasso-CV-1se 59.00 59.00 2.14 0.19 110.79 110.77 11.24 11.18 47.99 47.92 3.41 0.73 13.00 13.00 6.51 0.10 Glasso-CV2 59.00 59.00 9.88 0.16 115.05 114.48 24.63 20.13 47.99 47.93 23.79 0.35 13.00 13.00 40.12 0.16 Glasso-CV2-1se 59.00 59.00 5.41 0.15 114.92 114.34 18.53 16.74 47.99 47.93 8.93 0.66 13.00 13.00 17.85 0.15 Glasso-BIC 59.00 59.00 48.92 0.24 116.56 111.02 225.04 22.06 47.94 47.63 40.98 0.78 13.00 13.00 76.38 0.08 Glasso-EBIC 59.00 59.00 25.07 0.39 113.45 109.01 101.00 38.68 47.46 47.31 6.31 2.08 13.00 13.00 2.62 0.19 SPACE-CV 59.00 59.00 135.88 0.19 115.28 110.00 133.71 34.43 47.99 47.73 102.61 0.44 13.00 13.00 11.46 0.13 SPACE-CV-1se 59.00 59.00 64.47 0.17 101.85 101.19 41.04 35.93 47.91 47.51 16.72 1.12 13.00 13.00 5.45 0.13 SPACE-BIC 59.00 59.00 258.24 0.12 116.99 113.90 633.67 6.79 48.00 47.84 353.98 0.26 13.00 13.00 104.50 0.09 SPACE-FSR 59.00 59.00 130.22 0.15 116.96 112.82 453.52 11.60 47.99 47.79 122.34 0.34 13.00 13.00 7.68 0.10 NR-AND-CV 59.00 59.00 69.95 0.11 116.91 115.28 209.91 3.73 47.99 47.97 79.27 0.23 13.00 13.00 39.34 0.23 NR-AND-CV-1se 59.00 59.00 0.19 0.11 108.39 108.27 12.52 1.27 46.67 46.65 0.25 0.05 13.00 13.00 0.53 0.01 NR-AND-BIC 59.00 59.00 0.02 0.01 62.85 62.85 0.35 0.35 46.30 46.29 0.02 0.02 13.00 13.00 0.01 0.00 NR-AND-FSR 59.00 59.00 4.30 0.05 114.07 113.92 27.23 1.66 47.93 47.91 4.69 0.12 13.00 13.00 1.98 0.10 NR-OR-CV 59.00 59.00 277.54 0.08 116.98 114.55 491.73 4.79 48.00 47.98 270.86 0.32 13.00 13.00 164.58 0.18 NR-OR-CV-1se 59.00 59.00 2.38 0.31 113.92 113.50 43.28 1.58 47.78 47.75 4.18 0.17 13.00 13.00 9.11 0.01 NR-OR-BIC 59.00 59.00 0.05 0.03 65.82 65.82 0.88 0.79 47.27 47.27 0.07 0.06 13.00 13.00 0.03 0.01 NR-OR-FSR 59.00 59.00 26.60 0.09 115.96 115.33 87.34 2.24 47.99 47.97 25.56 0.15 13.00 13.00 14.55 0.08 Ridge-CV 59.00 59.00 1711.00 3.83 117.00 104.13 1653.00 39.56 48.00 41.55 1722.00 25.85 13.00 9.89 1757.00 19.18 Table 5. Discussion Average number of true positive edges (TP) and false positive edges (FP) over 100 replications when 60 F h d l h b f i l l i 59 f M d l 1 117 f M d l 2 48 f Table 5. Average number of true positive edges (TP) and false positive edges (FP) over 100 replications when p = 60. For each model the number of non-zero partial correlations are: 59 for Model 1, 117 for Model 2, 48 for Model 3 and 13 for Model 4. Note: Standard errors for TP range from 0.000 to 1.385 and standard errors for FP range from 0.000 to 17.702. Table 5. Average number of true positive edges (TP) and false positive edges (FP) over 100 replications when p = 60. For each model the number of non-zero partial correlations are: 59 for Model 1, 117 for Model 2, 48 for Model 3 and 13 for Model 4. Note: Standard errors for TP range from 0.000 to 1.385 and standard errors for FP range from 0.000 to 17.702. normality is violated. Here, our approach can be easily extended to make use of techniques to estimate semipara- metric undirected graphs45,47,48. We also note that in some applications, such as in psychology data or in the high dimensional setting, some variables might be highly linearly correlated. In this setting, the assumption regarding the regularity of the covariance matrix might not hold. A possible solution is to first cluster the strongly correlated variables and then take this cluster structure into account when estimating the GGM using the PCS approach49,50. normality is violated. Here, our approach can be easily extended to make use of techniques to estimate semipara- metric undirected graphs45,47,48. We also note that in some applications, such as in psychology data or in the high dimensional setting, some variables might be highly linearly correlated. In this setting, the assumption regarding the regularity of the covariance matrix might not hold. A possible solution is to first cluster the strongly correlated variables and then take this cluster structure into account when estimating the GGM using the PCS approach49,50. Finally, it is important to note that imposing sparsity might be too stringent in some applications. For instance, in some cases researchers are also interested in detecting partial correlations that are very close to zero. Discussion Moreover, it can also happen that the true network is not so sparse to begin with. In such cases, using approaches based on 1 regularization may affect the validity of the results51. Therefore, we believe that future research should also focus on exploring how the methods proposed in this paper behave when the true underlying network is less sparse or includes some very weak edges. Methods Partial correlation estimation procedures. In this subsection we present the technical details of the state-of-the-arts methods to estimate sparse partial correlation networks and the associated tuning methods for the regularization parameter. Methods Average number of true positive edges (TP) and false positive edges (FP) over 100 replications when p = 200. For each model the number of non-zero partial correlations are: 199 for Model 1, 397 for Model 2, 142 for Model 3 and 23 for Model 4. Note: Standard errors for TP range from 0.000 to 5.256 and standard errors for FP range from 0.000 to 41.561. Table 6. Average number of true positive edges (TP) and false positive edges (FP) over 100 replications when p = 200. For each model the number of non-zero partial correlations are: 199 for Model 1, 397 for Model 2, 142 for Model 3 and 23 for Model 4. Note: Standard errors for TP range from 0.000 to 5.256 and standard errors for FP range from 0.000 to 41.561. Table 6. Average number of true positive edges (TP) and false positive edges (FP) over 100 replications when p = 200. For each model the number of non-zero partial correlations are: 199 for Model 1, 397 for Model 2, 142 for Model 3 and 23 for Model 4. Note: Standard errors for TP range from 0.000 to 5.256 and standard errors for FP range from 0.000 to 41.561. The graphical lasso. Yuan and Lin25 and Rothman et al.24 proposed a penalized maximum likelihood approach to estimate the inverse of the covariance matrix Σ, denoted by Ω = [ωij]. If S denotes the sample covariance matrix, the problem is to minimize the following penalized log-likelihood function: ^  ∑ λ λ ω Ω Ω Ω =     − + \| \|     Ω ≠ S ( ) argmax tr( ) log det( ) , (5) i j ij 1 0 1 (5) where tr(⋅) denotes the trace of a matrix and λ1 > 0 controls the size of the penalty. The penalty term is a proxy of the number of zeros in the precision matrix. The smaller the value of λ1, the more non-zero elements the model includes. Friedman, Hastie and Tibshirani13 proposed an efficient algorithm to implement this method, which is called the Graphical lasso (Glasso). Afterwards, the partial correlation matrix can be computed using the known relation between the entries of the inverse of the covariance matrix and the partial correlation coefficients (see Lemma 1 in Peng et al.12). Methods Scientific Reports \| (2019) 9:17759 \| https://doi.org/10.1038/s41598-019-53795-x Scientific Reports \| (2019) 9:17759 \| https://doi.org/10.1038/s41598-019-53795-x Scientific Reports \| (2019) 9:17759 \| https://doi.org/10.1038/s41598-019-53795-x www.nature.com/scientificreports/ n = 100 Model 1 Model 2 Model 3 Model 4 TP FP TP FP TP FP TP FP no-PCS PCS no-PCS PCS no-PCS PCS no-PCS PCS no-PCS PCS no-PCS PCS no-PCS PCS no-PCS PCS Glasso-CV 198.86 192.63 758.85 11.33 204.29 108.27 637.13 38.70 130.62 102.64 654.20 7.87 22.78 15.40 664.69 1.81 Glasso-CV-1se 198.72 192.02 351.17 13.31 127.57 102.89 91.35 34.79 123.99 102.23 217.95 10.21 21.98 14.87 121.33 1.36 Glasso-CV2 198.94 192.82 1283.12 9.31 242.43 112.48 1385.28 38.17 132.29 104.02 870.41 8.65 22.41 15.27 220.99 1.44 Glasso-CV2-1se 198.85 192.49 804.26 10.45 205.13 108.18 660.23 39.02 129.54 102.98 544.21 8.53 18.43 14.85 13.05 1.54 Glasso-BIC 198.30 191.62 198.60 14.85 0.00 0.00 0.00 0.00 85.78 73.89 58.30 7.97 0.00 0.00 0.00 0.00 Glasso-EBIC 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 SPACE-CV 196.49 195.78 27.26 19.45 160.83 144.91 126.05 73.34 114.93 111.52 29.78 19.71 21.40 17.27 41.19 4.33 SPACE-CV-1se 194.60 194.52 13.02 12.77 131.71 129.85 54.54 51.91 113.74 110.70 24.63 17.89 21.38 17.45 40.76 4.81 SPACE-BIC 198.44 195.26 120.78 14.80 197.37 144.83 265.74 58.57 128.87 111.73 203.32 16.20 22.65 16.52 323.32 3.62 SPACE-FSR 197.85 195.65 59.92 16.99 175.84 145.03 168.31 68.03 125.91 111.68 131.09 16.30 22.49 16.85 222.86 3.64 NR-AND-CV 198.08 189.54 360.86 42.59 202.81 142.22 571.98 80.00 124.14 106.40 306.85 20.49 21.72 12.46 192.20 1.70 NR-AND-CV-1se 181.12 180.78 17.53 8.63 74.24 67.35 37.06 29.53 87.31 83.08 11.94 3.82 12.06 8.98 12.03 0.14 NR-AND-BIC 193.65 192.08 40.27 10.54 94.41 86.01 30.52 17.18 106.49 100.25 24.06 6.20 19.29 14.64 19.94 1.70 NR-AND-FSR 94.51 94.45 0.04 0.04 4.09 3.35 0.00 0.00 48.82 48.77 0.03 0.03 5.50 5.46 0.00 0.00 NR-OR-CV 198.73 167.88 1634.23 176.68 274.34 85.16 2094.31 56.90 133.41 88.36 1459.24 53.96 22.51 6.84 1012.65 1.67 NR-OR-CV-1se 193.09 189.88 167.15 29.94 153.05 134.04 303.67 137.51 111.80 96.10 146.74 8.11 18.72 11.91 112.39 1.12 NR-OR-BIC 196.96 193.57 190.08 33.21 150.86 114.86 178.67 35.82 118.67 108.24 131.56 18.48 20.99 14.65 162.86 2.96 NR-OR-FSR 109.58 109.55 0.14 0.13 4.09 3.37 0.00 0.00 55.29 55.18 0.07 0.07 5.80 5.72 0.00 0.00 Ridge-CV 199.00 191.26 19701.00 30.04 397.00 108.21 19503.00 29.29 142.00 102.00 19758.00 18.46 23.00 14.47 19877.00 1.31 n = 500 no-PCS PCS no-PCS PCS no-PCS PCS no-PCS PCS no-PCS PCS no-PCS PCS no-PCS PCS no-PCS PCS Glasso-CV 199.00 199.00 235.07 0.15 391.50 351.69 1245.58 85.60 140.48 138.47 110.08 10.39 23.00 23.00 453.33 0.05 Glasso-CV-1se 199.00 199.00 116.99 0.25 381.73 342.47 696.92 101.95 140.06 138.34 87.60 12.44 23.00 23.00 39.53 0.06 Glasso-CV2 199.00 199.00 1582.27 0.20 396.71 368.39 4256.19 33.63 141.92 139.90 811.90 2.83 23.00 23.00 223.51 0.05 Glasso-CV2-1se 199.00 199.00 836.46 0.12 395.60 360.63 2440.77 60.64 141.92 139.95 811.90 3.10 23.00 23.00 10.84 0.11 Glasso-BIC 199.00 199.00 435.99 0.21 359.84 336.22 332.29 129.97 140.88 138.74 159.47 8.22 23.00 23.00 10.62 0.09 Glasso-EBIC 199.00 199.00 163.43 0.28 263.95 263.73 52.35 52.17 140.12 138.30 91.93 12.09 23.00 23.00 5.01 0.10 SPACE-CV 199.00 199.00 6.18 0.18 383.35 379.74 120.06 88.04 141.79 141.24 18.01 2.79 23.00 23.00 45.25 0.06 SPACE-CV-1se 199.00 199.00 5.67 0.17 369.73 369.66 59.44 59.05 141.79 141.18 17.56 2.72 23.00 23.00 45.11 0.05 SPACE-BIC 199.00 199.00 58.63 41.28 386.42 379.77 133.83 76.95 141.97 141.42 86.76 1.73 23.00 23.00 201.15 0.12 SPACE-FSR 199.00 199.00 31.80 24.48 382.41 380.19 108.06 93.11 141.98 141.47 110.12 1.32 23.00 23.00 251.95 0.05 NR-AND-CV 199.00 199.00 265.57 0.14 395.08 388.00 952.77 14.26 141.99 141.93 274.47 0.30 23.00 23.00 161.21 0.08 NR-AND-CV-1se 199.00 199.00 0.70 0.25 346.71 346.30 46.21 3.14 137.52 137.47 0.93 0.07 22.97 22.97 6.53 0.00 NR-AND-BIC 199.00 199.00 11.90 0.05 370.60 369.99 81.03 4.87 141.65 141.61 12.19 0.27 23.00 23.00 11.03 0.05 NR-AND-FSR 199.00 199.00 0.01 0.01 200.90 200.90 0.45 0.45 133.82 133.82 0.02 0.02 23.00 23.00 0.01 0.01 NR-OR-CV 199.00 199.00 1441.19 0.05 396.47 381.86 2795.27 23.46 142.00 141.81 1319.48 0.50 23.00 23.00 729.26 0.06 NR-OR-CV-1se 199.00 199.00 12.61 0.38 374.86 372.88 197.90 4.76 140.73 140.65 21.39 0.09 22.99 22.99 60.72 0.00 NR-OR-BIC 199.00 199.00 91.27 0.06 385.83 384.12 299.27 9.28 141.88 141.82 81.60 0.19 23.00 23.00 71.15 0.10 NR-OR-FSR 199.00 199.00 0.07 0.03 205.02 205.02 0.99 0.97 136.27 136.27 0.16 0.04 23.00 23.00 0.01 0.01 Ridge-CV 199.00 199.00 19701.00 0.69 397.00 294.45 19503.00 146.53 142.00 136.96 19758.00 54.15 23.00 23.00 19877.00 0.82 T bl b f d (T ) d f l d ( ) l h Table 6. Methods For the different applications we select the regularization parameter as follows. We generate a grid of 100 equidis- tant possible values for λ1 ranging from 0.001 to \|max(S)\| when p < 100. When p ≥ 100 the sequence limits are p ( )t p p g the entries of the inverse of the covariance matrix and the partial correlation coefficients (see Lemma 1 in Peng et al.12). For the different applications we select the regularization parameter as follows. We generate a grid of 100 equidis- tant possible values for λ1 ranging from 0.001 to \|max(S)\| when p < 100. When p ≥ 100 the sequence limits are (0.05,\|max(S)\|). We propose six approaches to select the optimal value from this grid. The first one is to implement pfi g For the different applications we select the regularization parameter as follows. We generate a grid of 100 equidis- tant possible values for λ1 ranging from 0.001 to \|max(S)\| when p < 100. When p ≥ 100 the sequence limits are (0.05,\|max(S)\|). We propose six approaches to select the optimal value from this grid. The first one is to implement Scientific Reports \| (2019) 9:17759 \| https://doi.org/10.1038/s41598-019-53795-x 13 www.nature.com/scientificreports/ Predictors Estimate SE t p-value Glasso Intercept 0.06495 0.010 6.464 0.001 Non-sparsity 0.00006 0.000 1.130 0.259 n −0.00002 0.000 -13.556 0.000 SPACE Intercept 0.04268 0.006 6.961 0.006 Non-sparsity −0.00202 0.000 −39.938 0.000 n −0.00001 0.000 −4.455 0.000 NR-AND Intercept 0.22450 0.021 10.460 0.002 Non-sparsity −0.00237 0.000 −12.580 0.000 n −0.00023 0.000 −43.530 0.000 NR-OR Intercept 0.21100 0.018 12.035 0.001 Non-sparsity −0.00125 0.000 −7.235 0.000 n −0.00019 0.000 −38.923 0.000 Ridge Intercept 0.21950 0.003 63.860 0.000 Non-sparsity −0.00744 0.000 −24.470 0.000 n −0.00015 0.000 −18.460 0.000 Table 7. Regression coefficients, standard errors (SE), associated Wald’s t-scores and p-values for all predictors in the analysis. Table 7. Regression coefficients, standard errors (SE), associated Wald’s t-scores and p-values for all predictors in the analysis. K-fold cross-validation using the log-likelihood as performance measure (see Section 4.2 in Huang et al.52 and Section 2.3 in Price et al.53). We denote this procedure Glasso-CV1. We split the sample in K subsets. Using all but the k-th subset, we estimate the precision matrix using Glasso and denote this matrix ˆΩ, for different values of λ1. On the basis of the discarded k-th subset we estimate the sample covariance matrix, Sk. Methods Next, for each value of λ1 we compute the following loss function: ∑ λ λ λ Ω Ω = − . = ^ ^ S CV1( ) {tr( ( )) log det( ( ))} (6) k K k 1 1 1 1 (6) We plot CV1(λ1) versus λ1 and we select the tuning parameter that minimizes the loss function CV1(λ1). The second approach uses the one-standard-error-rule20. We denote this procedure Glasso-CV1-1se. Using the loss function in Eq. (6), we first compute the standard deviation of CV11(λ1), …, CV1K(λ1): λ λ λ = … . sd( ) sd(CV1 ( ), , CV1 ( )) (7) K 1 1 1 1 (7) Next, we compute the standard error of CV1(λ1): λ λ = . K se( ) sd( )/ 1 1 λ λ = . K se( ) sd( )/ (8) 1 1 (8) Finally, given the tuning weight that minimizes the cross-validation error in Eq. (6), denoted by ˆλ1, we choose the tuning weight that verifies the following rule: λ λ λ ≤ + ˆ ˆ CV1( ) CV1( ) se( ) (9) 1 1 1 λ λ λ ≤ + ˆ ˆ CV1( ) CV1( ) se( ) 1 1 1 (9) The third approach implements K-fold cross-validation using the prediction errors of each node as perfor- mance measure. We denote this procedure Glasso-CV2. We split the sample in K subsets. Using all but the k-th subset, we estimate the precision matrix using Glasso and denote this matrix ˆΩ, for different values of λ1. Next, for each value of λ1 we compute the following loss function: ˆ ˆ ∑∑ ∑ λ ω ω = −    −    . = = ≠ X X CV2( ) (10) k K i p i k j i ij ii j k 1 1 1 2 (10) We plot CV2(λ1) versus λ1 and we select the tuning parameter that minimizes the loss function CV2(λ1). The fourth procedure selects the tuning weight by applying the one-standard-error-rule on the cross-validation procedure CV2. We denote this procedure Glasso-CV2-1se.hit The fifth and sixth procedures to select the optimal regularization parameter from the 100 considered λ1 values are based on the Bayesian Information Criterion (BIC) or the Extended Bayesian Information Criterion (EBIC). We refer to these procedures as Glasso-BIC and Glasso-EBIC, respectively. Table 7. Regression coefficients, standard errors (SE), associated Wald’s t-scores and p-values for all predict in the analysis. Methods We select the value of λ1 that minimizes the following loss function: L λ λ κ κγ Ω = − + + ˆ n p EBIC( ) 2 ( ( )) log( ) 4 log( ) (11) 1 1 (11) where L ⋅( ) is the value of the log-likelihood function that corresponds to the estimated matrix ˆΩ, κ is the number of edges in the estimated network and γ ∈ [0, 1] is a parameter that controls the penalization of the network. If where L ⋅( ) is the value of the log-likelihood function that corresponds to the estimated matrix ˆΩ, κ is the number of edges in the estimated network and γ ∈ [0, 1] is a parameter that controls the penalization of the network. If Scientific Reports \| (2019) 9:17759 \| https://doi.org/10.1038/s41598-019-53795-x www.nature.com/scientificreports/ p p Figure 5. Heatmap of the true network based on the data on 20 PTSD symptoms. White represents partial correlations equal to zero, and black represents partial correlations different from zero. Figure 5. Heatmap of the true network based on the data on 20 PTSD symptoms. White represents partial correlations equal to zero, and black represents partial correlations different from zero. n = 100 n = 500 TPR FPR TPR FPR no-PCS PCS no-PCS PCS no-PCS PCS no-PCS PCS Glasso-CV 0.999 0.974 0.145 0.013 1.000 1.000 0.070 0.007 Glasso-CV-1se 0.979 0.968 0.052 0.031 1.000 1.000 0.055 0.011 Glasso-CV2 1.000 0.968 0.295 0.007 1.000 1.000 0.274 0.002 Glasso-CV2-1se 0.999 0.972 0.125 0.015 1.000 1.000 0.104 0.008 Glasso-BIC 1.000 0.973 0.229 0.009 1.000 1.000 0.240 0.004 Glasso-EBIC 0.908 0.889 0.088 0.017 1.000 1.000 0.141 0.006 SPACE-CV 0.983 0.983 0.015 0.014 1.000 1.000 0.013 0.009 SPACE-CV-1se 0.978 0.978 0.010 0.009 1.000 1.000 0.009 0.007 SPACE-BIC 0.989 0.988 0.022 0.020 1.000 1.000 0.015 0.010 SPACE-FSR 0.995 0.993 0.032 0.029 1.000 1.000 0.026 0.018 NR-AND-CV 0.995 0.908 0.092 0.017 1.000 1.000 0.077 0.002 NR-AND-CV-1se 0.887 0.879 0.005 0.003 1.000 1.000 0.001 0.001 NR-AND-BIC 0.976 0.931 0.017 0.006 1.000 1.000 0.009 0.001 NR-AND-FSR 0.754 0.751 0.000 0.000 0.999 0.999 0.001 0.000 NR-OR-CV 0.999 0.883 0.270 0.024 1.000 1.000 0.254 0.002 NR-OR-CV-1se 0.965 0.943 0.025 0.011 1.000 1.000 0.007 0.001 NR-OR-BIC 0.991 0.897 0.063 0.007 1.000 0.999 0.038 0.001 NR-OR-FSR 0.855 0.850 0.002 0.002 1.000 1.000 0.002 0.001 Ridge-CV 1.000 0.845 1.000 0.116 1.000 1.000 1.000 0.005 Table 8. Methods Average true positive rate (TPR) and false positive rate (FPR) over 100 simulations based on the PTSD data. Note: Standard errors for TPR range from 0.000 to 0.029 and standard errors for FPR range from 0.000 to 0.028. Table 8. Average true positive rate (TPR) and false positive rate (FPR) over 100 simulations based on the PTSD data. Note: Standard errors for TPR range from 0.000 to 0.029 and standard errors for FPR range from 0.000 to 0.028. γ = 0, the Eq. (11) corresponds to the classical BIC. Positive values of γ lead to stronger penalization. To compute EBIC, we follow the recommendation of Chen and Chen54 and Foygel and Drton21 and set γ to 0.555,56. γ = 0, the Eq. (11) corresponds to the classical BIC. Positive values of γ lead to stronger penalization. To compute EBIC, we follow the recommendation of Chen and Chen54 and Foygel and Drton21 and set γ to 0.555,56. Nodewise regression. Meinshausen and Bühlmann11 proposed to estimate the set of network edges by perform- ing p separate lasso regressions: Nodewise regression. Meinshausen and Bühlmann11 proposed to estimate the set of network edges by perform- ing p separate lasso regressions: ∑ ∑ β λ β λ β =      − + \| \|      β ≠ ≠ ^ X X ( ) argmin 1 2 , (12) i i j i ij j j i ij 2 2 2 ij (12) Scientific Reports \| (2019) 9:17759 \| https://doi.org/10.1038/s41598-019-53795-x www.nature.com/scientificreports/ p p Figure 6. Heatmaps of the frequency with which the edges for the PTSD data based simulations (n = 100) are set to zero by the different methods before applying PCS. White indicates that an edge was excluded from the network in all replications, whereas black reflects that the edge was always retained in the network. Figure 6. Heatmaps of the frequency with which the edges for the PTSD data based simulations (n = 100) are set to zero by the different methods before applying PCS. White indicates that an edge was excluded from the network in all replications, whereas black reflects that the edge was always retained in the network. Figure 6. Heatmaps of the frequency with which the edges for the PTSD data based simulations (n = 100) are set to zero by the different methods before applying PCS. Scientific Reports \| (2019) 9:17759 \| https://doi.org/10.1038/s41598-019-53795-x Methods White indicates that an edge was excluded from the network in all replications, whereas black reflects that the edge was always retained in the network. where βˆi is a vector that contains the p − 1 estimated regression weights of node i and λ2 > 0 is the regularization parameter that controls the number of non-zero elements in the neighborhood of node i. The set of edges can be computed with the AND-rule: where βˆi is a vector that contains the p − 1 estimated regression weights of node i and λ2 > 0 is the regularization parameter that controls the number of non-zero elements in the neighborhood of node i. The set of edges can be computed with the AND-rule: estimate an edge between nodes i and j ⇔ βˆij ≠ 0 and ˆβji ≠ 0 estimate an edge between nodes i and j ⇔ βˆij ≠ 0 and ˆβji ≠ 0 elding the NR-AND procedure. yielding the NR-AND procedure. Scientific Reports \| (2019) 9:17759 \| https://doi.org/10.1038/s41598-019-53795-x www.nature.com/scientificreports/ Alternatively we can use the NR-OR method and compute the edge set with the OR-rule: Figure 7. Heatmaps of the frequency with which the edges for the PTSD data based simulations (n = 100) are set to zero by the different methods after applying PCS. White indicates that an edge was excluded from the network in all replications, whereas black reflects that the edge was always retained in the network. Figure 7. Heatmaps of the frequency with which the edges for the PTSD data based simulations (n = 100) are set to zero by the different methods after applying PCS. White indicates that an edge was excluded from the network in all replications, whereas black reflects that the edge was always retained in the network. Alternatively, we can use the NR-OR method and compute the edge set with the OR-rule: Alternatively, we can use the NR-OR method and compute the edge set with the OR-rule: estimate an edge between nodes i and j ⇔ ˆβij ≠ 0 or ˆβji ≠ 0. estimate an edge between nodes i and j ⇔ ˆβij ≠ 0 or ˆβji ≠ 0. Next, the partial correlation matrix can be computed using the relation between the prediction errors of the best linear predictor of each node and the partial correlation coefficients (see Lemma 1 in Peng et al.12). Methods We select the value of λ2 that minimizes the following loss function: ˆ ∑ ∑ λ β = − = ≠ X X CV( ) , (13) k K i k j i ij j k 2 1 2 (13) where Xi k are the observations in the discarded subset k.h where Xi k are the observations in the discarded subset k.h where Xi k are the observations in the discarded subset k.h re Xi k are the observations in the discarded subset k.h The second approach adapts this cross-validation approach by using the one-standard-error-rule. We denote his procedure NR-CV-1se. The third procedure to select the regularization parameter, NR-BIC, involves computing the Bayesian Information Criterion (BIC) for different values of λ2. For each node, we select the value of λ2 that minimizes the following loss function: n n BIC ( ) RSS( ) log( ) (14) i i i 2 β λ κ = + ˆ (14) where RSS(⋅) is the value of the residual sum of squares for the i-th regression and κi is the number of elements in he estimated neighborhood of node i.h The fourth procedure is NR-FSR and uses a Finite Sample Result. Meinshausen and Bühlmann11 show that under certain assumptions regarding the sparsity and regularity conditions of the covariance matrix and the regression weights, the neighborhood of a node i will contain at most α ∈ (0, 1) false positive edges if the 1 pen- alty parameter is set as: λ α = Φ − α −( ) ( ) 1 n p 2 2 1 2 2 , where Φ−1 is the inverse of the c.d.f. of N(0, 1). We set the bound to the proportion of the false positive edges to α = 0.05. Joint sparse linear regression. Peng et al.12 proposed to estimate the partial correlation matrix by minimizing the following joint sparse regression (SPACE): oint sparse linear regression. Methods Next, the partial correlation matrix can be computed using the relation between the prediction errors of the best linear predictor of each node and the partial correlation coefficients (see Lemma 1 in Peng et al.12). Scientific Reports \| (2019) 9:17759 \| https://doi.org/10.1038/s41598-019-53795-x 17 www.nature.com/scientificreports/ Breast Cancer BPRS PTSD no-PCS PCS no-PCS PCS no-PCS PCS Glasso-CV 2,318 461 172 37 126 67 Glasso-CV-1se 1,799 544 116 77 100 91 Glasso-CV2 1,799 544 124 78 109 68 Glasso-CV2-1se 1,712 640 94 84 95 56 Glasso-BIC 0 0 132 81 114 67 Glasso-EBIC 0 0 0 0 104 100 SPACE-CV 577 576 82 82 70 66 SPACE-CV-1se 433 431 62 62 57 57 SPACE-BIC 437 437 72 72 57 57 SPACE-FSR 562 561 74 74 69 61 NR-AND-CV 608 608 85 85 80 75 NR-AND-CV-1se 287 287 29 29 45 44 NR-AND-BIC 417 417 69 69 58 56 NR-AND-FSR 38 38 26 25 28 28 NR-OR-CV 1,435 820 122 122 113 88 NR-OR-CV-1se 644 644 50 46 56 54 NR-OR-BIC 943 572 87 87 77 77 NR-OR-FSR 105 102 41 39 44 43 Ridge-CV 11,175 631 276 115 190 109 Table 9. Estimated number of edges of the gene regulatory network for the breast cancer data, the symptom network of patients with a diagnosis within the nonaffective psychotic spectrum using the BPRS scale and the symptom network of patients with PTSD. Table 9. Estimated number of edges of the gene regulatory network for the breast cancer data, the symptom network of patients with a diagnosis within the nonaffective psychotic spectrum using the BPRS scale and the symptom network of patients with PTSD. Table 9. Estimated number of edges of the gene regulatory network for the breast cancer data, the symptom network of patients with a diagnosis within the nonaffective psychotic spectrum using the BPRS scale and the symptom network of patients with PTSD. To select the tuning parameter λ2 for each regression separately we generate a grid of 100 possible values using the sequence generated with the function glmnet of the R package glmnet57. We consider four different tuning procedures. First, we can perform K-fold cross-validation. Discarding the k-th subset we estimate the vector of regression weights ˆβi using a lasso regression. Methods Peng et al.12 proposed to estimate the partial correlation matrix by minimizing the ollowing joint sparse regression (SPACE): ∑ ∑ ∑ λ ρ ω ω λ ρ Γ =         −    + \| \|      ρ ω = ≠ ≤< ≤ ^ X X ( ) argmin 1 2 , (15) i p i j i ij V i j jj ii j i j p ij 3 , 1 { , } 2 3 1 ij ii  (15) where ωii is the residual variance of the optimal prediction of Xi given all remaining variables, which is equivalent to the the i-th diagonal element of the matrix Ω and λ3 > 0 is the regularization parameter that controls the num- ber of non-zero elements in the partial correlation matrix Γ. Scientific Reports \| (2019) 9:17759 \| https://doi.org/10.1038/s41598-019-53795-x www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 8. Estimated gene regulatory network for the breast cancer data. Figure 8. Estimated gene regulatory network for the breast cancer data. Given a grid of 100 equidistant values for λ3 ranging from Φ − − . ( ) n 1 p 1 0 9 2 2 to Φ − − − ( ) n 1 e p 1 1 4 2 2 , there are four different procedures to calibrate the tuning parameter λ3. We first propose to perform K-fold cross-validation, yielding SPACE-CV. We first split the sample into K subsets and select the parameter value that minimizes the following loss function:  ∑∑ ∑ λ ρ ω ω = − . = = ≠ ^ ^ ^ X X CV( ) (16) k K i p i k j i ij V i j jj ii j k 3 1 1 { , } 2 (16) The second procedure again adapts this cross-validation approach by using the one-standard-error-rule. We denote this procedure SPACE-CV-1se. Th h d d l h l l h B I f The second procedure again adapts this cross-validation approach by using the one-standard-error-rule. We denote this procedure SPACE-CV-1se.h The third procedure to select the regularization parameter involves computing the Bayesian Information Criterion (BIC) for the 100 values of λ3. Methods First, we compute for each node the residual sum of squares: ∑ ρ ω ρ ω ω = − ≠ ˆ ˆ ˆ ˆ ˆ X X RSS ( , ) , i ij V i j ii i j i ij V i j jj ii j { , } { , } 2   Next, we select the value of λ3 by minimizing: Next, we select the value of λ3 by minimizing:  ( ) n n BIC( ) RSS ( , ) log( ) (17) i p i ij V i j ii i 3 1 { , } ^ ^ ∑ λ ρ ω κ = + = (17) where κi is the number of elements in the estimated neighborhood of node i. where κi is the number of elements in the estimated neighborhood of node i. where κi is the number of elements in the estimated neighborhood of node i. where κi is the number of elements in the estimated neighborhood of node i. Scientific Reports \| (2019) 9:17759 \| https://doi.org/10.1038/s41598-019-53795-x 19 www.nature.com/scientificreports/ Figure 9. Estimated sub-network of genes in the neighborhood of ESR1 and FOXA1 for the breast cancer data. Figure 9. Estimated sub-network of genes in the neighborhood of ESR1 and FOXA1 for the breast cancer data. Figure 10. Estimated symptoms network of patients with a diagnosis within the nonaffective psychotic spectrum using the BPRS data. Figure 10. Estimated symptoms network of patients with a diagnosis within the nonaffective psychotic spectrum using the BPRS data. Scientific Reports \| (2019) 9:17759 \| https://doi.org/10.1038/s41598-019-53795-x 20 www.nature.com/scientificreports/ Figure 11. Estimated symptoms network for the PTSD data. Figure 11. Estimated symptoms network for the PTSD data. The fourth procedure SPACE-FSR is based on the Finite Sample Result by Peng et al.12. These authors show that under certain assumptions regarding the sparsity and regularity conditions of the covariance matrix and the regression weights, the neighborhood of a node i will contain at most α ∈ (0, 1) false positive edges if the penalty parameter is set as: ( ) n ( ) 1 p 3 1 2 2 λ α = Φ − α − , where Φ−1 is the inverse of the c.d.f. of N(0, 1). We again set the bound to the proportion of the false positive edges to α = 0.05. Partial correlation estimation using ridge regression. Methods Ha and Sun19 proposed to estimate a penalized partial cor- relations matrix using a ridge penalty. We apply a simpler version of their method by performing p separate ridge regressions: ∑ ∑ δ λ δ λ δ =      − +      δ ≠ ≠ ^ X X ( ) argmin 1 2 , (18) i i j i ij j j i ij 4 2 4 2 ij (18) where δˆi is a vector that contains the p − 1 estimated regression weights for node i and λ4 > 0 is the regularization parameter that controls the amount of shrinkage of the regression weights toward zero in the neighborhood of node i. The partial correlation matrix is computed using the relation between the prediction errors of the best linear predictor of each node and the partial correlation coefficients (see Lemma 1 in Peng et al.12). where δˆi is a vector that contains the p − 1 estimated regression weights for node i and λ4 > 0 is the regularization parameter that controls the amount of shrinkage of the regression weights toward zero in the neighborhood of node i. The partial correlation matrix is computed using the relation between the prediction errors of the best linear predictor of each node and the partial correlation coefficients (see Lemma 1 in Peng et al.12). p pfi g To select the tuning parameter λ4 for each regression separately we generate a grid of 100 possible values using the sequence generated with the function glmnet of the R package glmnet57. We select the regularization param- eter by performing K-fold cross-validation. Discarding the k-th subset we estimate the vector of regression weights ˆδi using ridge regression. We select the value of λ4 that minimizes the following loss function: ˆ ∑ ∑ λ δ = − = ≠ X X CV( ) , (19) k K i k j i ij j k 4 1 2 (19) where Xi k are the observations in the discarded subset k. We denote this procedure Ridge-CV. Partial correlation screening procedure. In this subsection we present the technical details of the Partial Correlation Screening (PCS) algorithm. The procedure estimates the set of edges in two steps. In the first step, we Partial correlation screening procedure. In this subsection we present the technical details of the Partial Correlation Screening (PCS) algorithm. Methods The procedure estimates the set of edges in two steps. In the first step, we Scientific Reports \| (2019) 9:17759 \| https://doi.org/10.1038/s41598-019-53795-x www.nature.com/scientificreports/ determine a sparse partial correlation network, denoted by ˆ ˆρ Γ = \| [ ] ij V i j \{ , } , using one of the methods that we dis- cussed in the previous subsection. determine a sparse partial correlation network, denoted by ˆ ˆρ Γ = \| [ ] ij V i j \{ , } , using one of the methods that we dis- cussed in the previous subsection. In the second step of the algorithm, we detect unimportant pairs of variables by thresholding the partial cor- relations estimated in the first step. For i ∈ V and a threshold parameter τ ∈ (0, 1), we estimate the neighborhood of node i as follows A ρ τ = ∈ \| \| > . τ \| ˆ ˆ j V i { \{ }: } (20) i ij V i j , \{ , } A ρ τ = ∈ \| \| > . τ \| ˆ ˆ j V i { \{ }: } i ij V i j , \{ , } (20) m outputs the estimated set of edges for a given threshold τ: The algorithm outputs the estimated set of edges for a given threshold τ: ˆ ˆρ τ = ∈ \| \| > . τ \| E i j V {( , ) : } ij V i j \{ , } ˆ ˆρ τ = ∈ \| \| > . τ \| E i j V {( , ) : } (21) ij V i j \{ , } (21) Finally, the prediction error of the regression of each node i conditioned on the variables that belong to the estimated neighborhood set A τˆi, is given by A ∑ ε θ = − τ τ ∈ τ ˆ ˆ ˆ X X , i i j ij j , , i, where ˆθ τ i, is the vector of estimated regression coefficients of node i ∈ V given the variables in the estimated neigh- borhood set A τˆi, . Choice of the tuning parameter. To select the threshold parameter τ, we perform K-fold cross validation. 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Res. 10, 2295–2328 (2009). 6. Garety, P. A., Kuipers, E., Fowler, D., Freeman, D. & Bebbington, P. A cognitive model of the positive symptoms of psychosis. Psychol Medicine 31, 189–195 (2001).h 46. Garety, P. Competing interestsh p g The authors declare no competing interests. Scientific Reports \| (2019) 9:17759 \| https://doi.org/10.1038/s41598-019-53795-x 23 www.nature.com/scientificreports/ Additional information Supplementary information is available for this paper at https://doi.org/10.1038/s41598-019-53795-x. Correspondence and requests for materials should be addressed to G.L. 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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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) 9:17759 \| https://doi.org/10.1038/s41598-019-53795-x 24
https://openalex.org/W3022383804	https://www.nature.com/articles/s41467-020-15986-3.pdf	English	null	Comprehensive aptamer-based screening identifies a spectrum of urinary biomarkers of lupus nephritis across ethnicities	Nature communications	2,020	cc-by	16,464	1 Department Biomedical Engineering, University of Houston, Houston, TX, USA. 2 Rheumatology and Rehabilitation Department, Faculty of Medicine, Minia University, Minya, Egypt. 3 Center for Clinical Research and Evidence-Based Medicine, University of Texas Health Science Center at Houston, Houston, TX, USA. 4 Department of Rheumatology, Albert Einstein College of Medicine, Bronx, NY, USA. 5 Department of Molecular Biology, Rockefeller University, New York, NY, USA. 6 Department of Rheumatology, New York University, New York, NY, USA. 7 Azrieli Faculty of Medicine, Bar-Ilan University, Zefat, Israel. 8 Research Institute, Galilee Medical Center, Nahariya, Israel. 9 Department of Medicine, Tuen Mun Hospital, New Territories, Hong Kong, China. 10 Division of Rheumatology, Johns Hopkins University School of Medicine, Baltimore, MD, USA. 11 University Hospital Kidney & Liver Clinic, University of Texas Southwestern Medical Center, Dallas, TX, USA. ✉email: cmohan@central.uh.edu NATURE COMMUNICATIONS \| (2020) 11:2197 \| https://doi.org/10.1038/s41467-020-15986-3 \| www.nature.com/naturecommunications ARTICLE ARTICLE Results Aptamer-based screening of LN urine. Urine samples from 23 human subjects (7 active LN, 8 inactive SLE, 8 healthy controls, all female, age range 23–42 years) were initially screened for the levels of 1129 distinct human proteins using a pre-fabricated aptamer-based-targeted proteomic assay, that is commercially available10. In this assay, streptavidin-coated beads labeled with 1129 unique aptamers are added to each urine sample to allow them to bind to their designated protein targets17. After incu- bation, the beads are removed from the sample, the proteins attached to the aptamers are biotinylated and all aptamer–protein complexes are cleaved from the initial streptavidin beads and re- coupled to a new bead, with the biotinylated protein attaching to the bead. The aptamers are then removed from the beads and quantitated using a DNA microarray10. The assay readouts (measured as relative ﬂuorescence units or RFU) were normalized to urinary creatinine levels. In this assay, 326 proteins were sig- niﬁcantly elevated in SLE urine compared to healthy control urine, while 284 proteins were signiﬁcantly elevated in active LN urine compared to inactive SLE urine, with 198 proteins over- lapping between these two comparisons, as displayed in the heatmap in Fig. 1a. SLE and LN are both heavily inﬂuenced by genetics3, and African-Americans are three times more likely to develop SLE than Caucasians4. Likewise, disease manifestations are variably expressed among patients, with African-Americans being more likely to develop ESRD5, although inﬂuence from environmental triggers or socioeconomic factors cannot be ruled out5,6. Although patient demographics are widely known to affect SLE disease manifestations and outcomes, there are virtually no stu- dies investigating this phenomenon in the context of disease biomarkers; most SLE biomarkers studies focus on one demo- graphic group or all ethnic groups combined, which yield results that may not be equally predictive in all demographic groups of SLE patients. The proteins that were signiﬁcantly elevated in the urine of patients with active LN clustered into 20 pathways with at least 10 upregulated proteins each, as determined by Ingenuity Pathway Analysis. One of these pathways was enriched for several molecules involved in inﬂammation, including IL1R1, IL1RAP, IL1RAPL2, IL15RA, IL17F, IL18R1, E-selectin, TLR2, CD86, and several signaling molecules, including MAKAPK2, MAPKAPK3, and MAPKAPK5, all of which were signiﬁcantly elevated in the urine of patients with active LN, as indicated by the pink-colored nodes in Fig. 1b. Results Another network of urine proteins elevated in LN urine included various complement proteins (C3, C5, CFB, CFI), several chemokines (CCL2 (MCP-1), CCL11 (Eotaxin), CCL13, CCL16, CCL17 (TARC), CCL23 (MIP-3), CCL28, CXCL1 (KC; Gro1), CXCL4 (PF-4), CXCL5, CXCL6, CXCL11 (iTAC)), and other molecules implicated in inﬂammation, including IL6R, MMP8, and SERPINA4 (Kallistatin; alpha-1- anti-trypsin), as displayed in Fig. 1c. A third upregulated network interconnected several members of the TNF/TNF-receptor superfamily, including TNFSF12 (TWEAK), TNFRSF12A (TWEAK-R, Fn14), TNFSF4 (OX40L), TNFSF14 (LIGHT), TNFSF6 (FasLG; CD95L), TNFRSF6B, TNFRSF13c (BAFF-R), TNFRFSF9 (41BBL; CD137), TNFRSF21 (CD358; DR6), and TNFRSF25 (DR3), as shown in Supplementary Fig. 1a. Other upregulated pathways include proteins important for extracellular matrix turnover and/or ﬁbrosis (Collagen, COL8A, TNNI2, PDGFB, PDGFAb, PDGFBB, CTGF), metalloprotease family members (TIMP1, TIMP3, ADAM9, ADAMTS1, ADAMTS4), NOTCH family members (NOTCH2, NPTCH3, DLL1), and cadherins (CDH2, CDH5, CDH15), as displayed in Supplemen- tary Fig. 1b and 1c. Traditional biomarker discovery study design is typically based on prior understanding of established pathophysiological path- ways underlying LN, with a focus on selected molecules (such as speciﬁc growth factors, cytokines, and chemokines) directly related to those pathways, which stamps a bias on the types of biomarkers identiﬁed. In contrast, large-scale proteomic approa- ches have transformed the discovery of urinary biomarkers from a highly skewed search to a comprehensive unbiased screen. A couple of studies in LN have utilized non-targeted proteomic approaches, including isobaric tag for relative and absolute quantitation (iTRAQ) mass spectrometry7, and electrospray ionization quadrupole time-of-ﬂight tandem mass spectrometry (ESI-Q-TOF MS/MS)8. In contrast to the above mass spectrometry-based approa- ches, which typically uncover high abundance proteins, afﬁnity- based approaches using various ligands (such as antibodies) have the potential to uncover lower abundance proteins, due to the use of speciﬁc, high-afﬁnity ligands. A few screening studies in LN have utilized afﬁnity-based techniques such as antibody- based or aptamer-based arrays, with only one study using antibody-based arrays in the context of SLE9. The aptamer- based screen used in the present study has the power of simultaneous interrogation of over 1100 unique proteins, with a dynamic ranger larger than that of a traditional enzyme-linked immunosorbent assay (ELISA). It is based on specially designed aptamers, which are synthetic, single-stranded DNA-based molecular recognition elements, to selectively recognize and quantify a wide spectrum of proteins in body ﬂuids or cell lysates10. Comprehensive aptamer-based screening identiﬁes a spectrum of urinary biomarkers of lupus nephritis across ethnicities Samantha Stanley1, Kamala Vanarsa1, Samar Soliman 1,2, Deena Habazi1, Claudia Pedroza 3, Gabriel Gidley 1, Ting Zhang1, Shree Mohan1, Evan Der 4, Hemant Suryawanshi5, Thomas Tuschl5, Jill Buyon6, Chaim Putterman 4,7,8, Chi Chiu Mok9, Michelle Petri 10, Ramesh Saxena 11 & 1✉ Samantha Stanley1, Kamala Vanarsa1, Samar Soliman 1,2, Deena Habazi1, Claudia Pedroza 3, Gabriel Gidley 1, Ting Zhang1, Shree Mohan1, Evan Der 4, Hemant Suryawanshi5, Thomas Tuschl5, Jill Buyon6, Chaim Putterman 4,7,8, Chi Chiu Mok9, Michelle Petri 10, Ramesh Saxena 11 & Chandra Mohan 1✉ Chandra Mohan 1✉ Emerging urinary biomarkers continue to show promise in evaluating lupus nephritis (LN). Here, we screen urine from active LN patients for 1129 proteins using an aptamer-based platform, followed by ELISA validation in two independent cohorts comprised of 127 inactive lupus, 107 active LN, 67 active non-renal lupus patients and 74 healthy controls, of three different ethnicities. Urine proteins that best distinguish active LN from inactive disease are ALCAM, PF-4, properdin, and VCAM-1 among African-Americans, sE-selectin, VCAM-1, BFL- 1 and Hemopexin among Caucasians, and ALCAM, VCAM-1, TFPI and PF-4 among Asians. Most of these correlate signiﬁcantly with disease activity indices in the respective ethnic groups, and surpass conventional metrics in identifying active LN, with better sensitivity, and negative/positive predictive values. Several elevated urinary molecules are also expressed within the kidneys in LN, based on single-cell RNAseq analysis. Longitudinal studies are warranted to assess the utility of these biomarkers in tracking lupus nephritis. 1 TURE COMMUNICATIONS \| (2020) 11:2197 \| https://doi.org/10.1038/ ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15986-3 L L upus nephritis (LN), one of the most severe complications of systemic lupus erythematosus (SLE), is a condition where the kidneys become inﬂamed and eventually lose function. It is estimated that ~60% of all SLE patients will develop LN1, and in 10–15% of those patients the disease will progress to end-stage renal disease (ESRD)1. The current gold standard for diagnosis of renal involvement is a renal biopsy; while biopsies are highly informative, they cannot be serially repeated and come with attendant concerns, including the invasive nature of the proce- dure, and the possibility that the sample taken may not be representative of the entire kidney. It has been documented that early detection and prompt treatment can have a signiﬁcant impact on morbidity and mortality in LN2, but current diagnostic techniques are not optimal for early detection. Comprehensive aptamer-based screening identiﬁes a spectrum of urinary biomarkers of lupus nephritis across ethnicities Hence, an easily measurable biomarker for LN with high predictive value is highly desirable, and this has sparked signiﬁcant research interest in this direction. group-speciﬁc differences in their biomarker potential. More- over, several of the urinary biomarkers elevated in LN urine are expressed within the kidneys in LN, either within resident renal cells or inﬁltrating immune cells. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15986-3 algorithm, implicated urine calpastatin, CTAP-III, NAP-2, Histone H1.2, PF-4, VEGF sR3, C6, and TFPI as some of the most discriminatory molecules with the largest impact on distinguishing the study groups (Fig. 1e). Of these top 50 proteins, 21 proteins were not pursued further for the following reasons, as detailed in Supplementary Table 2: average intensity levels of protein in all subject groups were <1000 RFU (IL-12Rb2, p27Kip1, PFD5, RUXF, TLR2, TWEAKR, VEGF sR3), previous studies had already documented the elevations of these markers (IgM, MIF), or there was a strong correlation (correlation coefﬁcient r > 0.95) with another chosen protein (CAMK1, CTAP-III, Cytochrome P450, GOT1, HGFA, Histone H1.2, LCMT1, PAFAHβ1, PGP9.5, PSME1, SP-D, TXD-12). ELISA kits were purchased for the remaining 29 proteins. Of these, the purchased ELISA kits did not work for urine AIF1 and 40s ribosomal protein SA (Supplementary Fig. 2). algorithm, implicated urine calpastatin, CTAP-III, NAP-2, Histone H1.2, PF-4, VEGF sR3, C6, and TFPI as some of the most discriminatory molecules with the largest impact on distinguishing the study groups (Fig. 1e). Of these top 50 proteins, 21 proteins were not pursued further for the following reasons, as detailed in Supplementary Table 2: average intensity levels of protein in all subject groups were <1000 RFU (IL-12Rb2, p27Kip1, PFD5, RUXF, TLR2, TWEAKR, VEGF sR3), previous studies had already documented the elevations of these markers (IgM, MIF), or there was a strong correlation (correlation coefﬁcient r > 0.95) with another chosen protein (CAMK1, CTAP-III, Cytochrome P450, GOT1, HGFA, Histone H1.2, LCMT1, PAFAHβ1, PGP9.5, PSME1, SP-D, TXD-12). ELISA kits were purchased for the remaining 29 proteins. Of these, the purchased ELISA kits did not work for urine AIF1 and 40s ribosomal protein SA (Supplementary Fig. 2). Validation of proteomic hits in a primary independent cohort. With the remaining 27 biomarker candidates (for which func- tional ELISA kits were identiﬁed), pilot ELISA testing was carried out using a limited cohort of 36 subjects, comprises 12 active LN, 12 inactive SLE, and 12 healthy controls. With 15 of these 27 tested proteins, the urine levels in active LN were not signiﬁcantly higher than that in inactive SLE (as listed in Supplementary Table 2). In contrast, 12 urinary proteins continued to show signiﬁcantly higher levels in active LN in this pilot ELISA test (as indicated in bold in Supplementary Table 2), and were hence pursued further. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15986-3 d The 50 urine proteins (ranked in order of fold-change) that were signiﬁcantly elevated in active LN compared to inactive SLE are displayed. Further details regarding these proteins are in included in Supplementary Data (red dots represent active LN; blue dots represent inactive SLE; black dots represent healthy controls). e Random forest classiﬁcation analysis identiﬁcation of the 20 most discriminatory urine proteins with the largest impact on distinguishing the study groups, ordered by their GINI coefﬁcient. Source data are provided as a Source Data ﬁle. Fig. 1 Aptamer-based screening of lupus nephritis urine samples for 1129 proteins. a Heatmap representation of aptamer-based screening results of the 1129 proteins analyzed in 23 human urine samples (7 active LN, 8 inactive SLE, 8 healthy controls). 284 urinary proteins were found to be elevated (p < 0.05, fold-change ≥2, Mann–Whitney U-test) when comparing active LN to inactive SLE (top), while 326 urinary proteins were elevated (p < 0.05, fold- change ≥2; Mann–Whitney U-test) when comparing all SLE subjects to healthy controls (bottom). Each map shows the relative concentrations of these proteins after normalizing against urinary creatinine. Each column represents a patient sample, while rows correspond to a creatinine-normalized protein level measured using the screening assay. Proteins that are above the mean value (for each biomarker) are yellow, while those below are blue, with proteins comparable to the mean value are black. b, c The proteins that were signiﬁcantly elevated in the urine of patients with active LN clustered into 20 pathways with at least 10 upregulated proteins each, as determined by Ingenuity Pathway Analysis, of which 2 are displayed. Additional pathways are included in Supplementary Data. Molecules elevated in LN urine are shaded pink. Documented and putative interactions between the displayed molecules are indicated by solid and interrupted arrows, respectively, based on literature review. d The 50 urine proteins (ranked in order of fold-change) that were signiﬁcantly elevated in active LN compared to inactive SLE are displayed. Further details regarding these proteins are in included in Supplementary Data (red dots represent active LN; blue dots represent inactive SLE; black dots represent healthy controls). e Random forest classiﬁcation analysis identiﬁcation of the 20 most discriminatory urine proteins with the largest impact on distinguishing the study groups, ordered by their GINI coefﬁcient. Source data are provided as a Source Data ﬁle. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15986-3 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15986-3 Actives vs inactive SLE vs Healthy 40S ribosomal protein SA Activin A AIF1 ALCAM BAFF Receptor BFL1 C4 C6 Calpastatin CAMK1 CTAP-III Cytochrome P450 3A4 FcgRIIBC Fibronectin GOT1 Hemopexin HGFA Histone H1.2 HPG- HSP 60 IgM IL-12 RB2 IL-16 LCMT1 LY86 MCP-1 MIF NAP-2 p27Kip1 PAFAH beta subunit Peroxiredoxin-6 PF-4 PFD5 PGK1 PGP9.5 Properdin PSME1 RUXF sE-Selectin SOD SP-D Tenascin TFPI TIMP-1 TLR2 TWEAKR TXD12 VCAM-1 VE-Cadherin VEGF sR3 0 2 4 6 8 10 12 Log 10(RFU/[mg/dL]) Healthy Inactive Active Healthy SLE GINI coefficient (Relative lmportance of biomarker) HC1 HC2 HC3 HC4 HC5 HC6 HC7 HC8 ILN1 ILN2 ILN3 ILN4 ILN5 ILN6 ILN7 ILN8 ALN1 ALN2 ALN3 ALN4 ALN5 ALN6 ALN7 HC1 HC2 HC3 HC4 HC5 HC6 HC7 HC8 ILN1 ILN2 ILN3 ILN4 ILN5 ILN6 ILN7 ILN8 ALN1 ALN2 ALN3 ALN4 ALN5 ALN6 ALN7 Calpastatin CTAPIII NAP-2 Histone H1.2 PE-4 VEGF sR3 C6 TFPI Fibronectin Activin A Hemopexin IL-12 RB2 AIF1 TLR2 SP-D TXD12 VCAM-1 40S ribosomal protein SA LY86 Properdin 3 2 1 0 –1 –2 –3 3 2 1 0 –1 –2 –3 a b c d e 0.00 0.02 0.04 0.06 0.08 0.10 Fig. 1 Aptamer-based screening of lupus nephritis urine samples for 1129 proteins. a Heatmap representation of aptamer-based screening results of the 1129 proteins analyzed in 23 human urine samples (7 active LN, 8 inactive SLE, 8 healthy controls). 284 urinary proteins were found to be elevated (p < 0.05, fold-change ≥2, Mann–Whitney U-test) when comparing active LN to inactive SLE (top), while 326 urinary proteins were elevated (p < 0.05, fold- change ≥2; Mann–Whitney U-test) when comparing all SLE subjects to healthy controls (bottom). Each map shows the relative concentrations of these proteins after normalizing against urinary creatinine. Each column represents a patient sample, while rows correspond to a creatinine-normalized protein level measured using the screening assay. Proteins that are above the mean value (for each biomarker) are yellow, while those below are blue, with proteins comparable to the mean value are black. b, c The proteins that were signiﬁcantly elevated in the urine of patients with active LN clustered into 20 pathways with at least 10 upregulated proteins each, as determined by Ingenuity Pathway Analysis, of which 2 are displayed. Additional pathways are included in Supplementary Data. Molecules elevated in LN urine are shaded pink. Results This platform has been successfully applied in bio- marker screens of several diseases, including Alzheimer’s dis- ease11, pulmonary tuberculosis12, and others13–16, but not in autoimmune diseases. In the current study, this aptamer-based screen is applied to identify potential urinary biomarkers in LN. Identiﬁed biomarker candidates are further validated in inde- pendent cross-sectional cohorts to validate the screening hits. Interestingly, the validated molecules exhibit striking ethnic- Next, to validate the observed elevations in the primary screen, we limited further analysis to the 50 proteins (ranked in order of fold-change) that were signiﬁcantly elevated in active LN compared to inactive SLE, as displayed in Fig. 1d, and detailed in Supplementary Table 1. As can be seen in this table, most of these proteins were signiﬁcantly elevated in the urine of active LN patients (compared to inactive disease), even after multiple testing correction (q < 0.05). Random forest analysis, a machine learning NATURE COMMUNICATIONS \| (2020) 11:2197 \| https://doi.org/10.1038/s41467-020-15986-3 \| www.nature.com/naturecommunications 2 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15986-3 Proteins that are above the mean value (for each biomarker) are yellow, while those below are blue, with proteins comparable to the mean value are black. b, c The proteins that were signiﬁcantly elevated in the urine of patients with active LN clustered into 20 pathways with at least 10 upregulated proteins each, as determined by Ingenuity Pathway Analysis, of which 2 are displayed. Additional pathways are included in Supplementary Data. Molecules elevated in LN urine are shaded pink. Documented and putative interactions between the displayed molecules are indicated by solid and interrupted arrows, respectively, based on literature review. d The 50 urine proteins (ranked in order of fold-change) that were signiﬁcantly elevated in active LN compared to inactive SLE are displayed. Further details regarding these proteins are in included in Supplementary Data (red dots represent active LN; blue dots represent inactive SLE; black dots represent healthy controls). e Random forest classiﬁcation analysis identiﬁcation of the 20 most discriminatory urine proteins with the largest impact on distinguishing the study groups, ordered by their GINI coefﬁcient. Source data are provided as a Source Data ﬁle. Fig. 1 Aptamer-based screening of lupus nephritis urine samples for 1129 proteins. a Heatmap representation of aptamer-based screening results of the 1129 proteins analyzed in 23 human urine samples (7 active LN, 8 inactive SLE, 8 healthy controls). 284 urinary proteins were found to be elevated (p < 0.05, fold-change ≥2, Mann–Whitney U-test) when comparing active LN to inactive SLE (top), while 326 urinary proteins were elevated (p < 0.05, fold- change ≥2; Mann–Whitney U-test) when comparing all SLE subjects to healthy controls (bottom). Each map shows the relative concentrations of these proteins after normalizing against urinary creatinine. Each column represents a patient sample, while rows correspond to a creatinine-normalized protein level measured using the screening assay. Proteins that are above the mean value (for each biomarker) are yellow, while those below are blue, with proteins comparable to the mean value are black. b, c The proteins that were signiﬁcantly elevated in the urine of patients with active LN clustered into 20 pathways with at least 10 upregulated proteins each, as determined by Ingenuity Pathway Analysis, of which 2 are displayed. Additional pathways are included in Supplementary Data. Molecules elevated in LN urine are shaded pink. Documented and putative interactions between the displayed molecules are indicated by solid and interrupted arrows, respectively, based on literature review. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15986-3 Documented and putative interactions between the displayed molecules are indicated by solid and interrupted arrows, respectively, based on literature review. d The 50 urine proteins (ranked in order of fold-change) that were signiﬁcantly elevated in active LN compared to inactive SLE are displayed. Further details regarding these proteins are in included in Supplementary Data b c b Actives vs inactive SLE vs Healthy Healthy Inactive Active Healthy SLE HC1 HC2 HC3 HC4 HC5 HC6 HC7 HC8 ILN1 ILN2 ILN3 ILN4 ILN5 ILN6 ILN7 ILN8 ALN1 ALN2 ALN3 ALN4 ALN5 ALN6 ALN7 HC1 HC2 HC3 HC4 HC5 HC6 HC7 HC8 ILN1 ILN2 ILN3 ILN4 ILN5 ILN6 ILN7 ILN8 ALN1 ALN2 ALN3 ALN4 ALN5 ALN6 ALN7 3 2 1 0 –1 –2 –3 3 2 1 0 –1 –2 –3 a b c 40S ribosomal protein SA Activin A AIF1 ALCAM BAFF Receptor BFL1 C4 C6 Calpastatin CAMK1 CTAP-III Cytochrome P450 3A4 FcgRIIBC Fibronectin GOT1 Hemopexin HGFA Histone H1.2 HPG- HSP 60 IgM IL-12 RB2 IL-16 LCMT1 LY86 MCP-1 MIF NAP-2 p27Kip1 PAFAH beta subunit Peroxiredoxin-6 PF-4 PFD5 PGK1 PGP9.5 Properdin PSME1 RUXF sE-Selectin SOD SP-D Tenascin TFPI TIMP-1 TLR2 TWEAKR TXD12 VCAM-1 VE-Cadherin VEGF sR3 0 2 4 6 8 10 12 L (RFU/[ /dL]) d d GINI coefficient (Relative lmportance of biomarker) Calpastatin CTAPIII NAP-2 Histone H1.2 PE-4 VEGF sR3 C6 TFPI Fibronectin Activin A Hemopexin IL-12 RB2 AIF1 TLR2 SP-D TXD12 VCAM-1 40S ribosomal protein SA LY86 Properdin e 0.00 0.02 0.04 0.06 0.08 0.10 e ( p ) Fig. 1 Aptamer-based screening of lupus nephritis urine samples for 1129 proteins. a Heatmap representation of apta ( p ) Fig. 1 Aptamer-based screening of lupus nephritis urine samples for 1129 proteins. a Heatmap representation of aptamer-based screening results of the 1129 proteins analyzed in 23 human urine samples (7 active LN, 8 inactive SLE, 8 healthy controls). 284 urinary proteins were found to be elevated (p < 0.05, fold-change ≥2, Mann–Whitney U-test) when comparing active LN to inactive SLE (top), while 326 urinary proteins were elevated (p < 0.05, fold- change ≥2; Mann–Whitney U-test) when comparing all SLE subjects to healthy controls (bottom). Each map shows the relative concentrations of these proteins after normalizing against urinary creatinine. Each column represents a patient sample, while rows correspond to a creatinine-normalized protein level measured using the screening assay. ARTICLE Listed are the Spearman correlation coefﬁcients between the two platforms, for each protein, and the associated signiﬁcance (p < 0.05; *p < 0.001). N/A Not done. Table 1 Correlation between aptamer screen and ELISA assay results. Next, we performed Lasso regression analysis to identify multi- marker panels that may better predict active LN status in the primary validation cohort. Besides the 12 urine proteins, we included race, age, and prednisone usage as additional variables. The combination with the best predictive model encompassed 8 of the 12 urine proteins, together with race, but excluded age and prednisone usage. This octuplex panel exhibited outstanding ability to discriminate active LN from inactive SLE with a ROC AUC value of 0.98, as shown in Fig. 3a, and also identiﬁed race as a signiﬁcant confounding variable. The diagnostic utility of biomarkers vary with ethnicity. As the above analyses identiﬁed race as a signiﬁcant confounding factor, we examined the performance of these markers within each ethnic group. Among African-American patients, the best bio- markers that distinguished active LN from inactive disease, with statistical signiﬁcance, were urine PF-4 (AUC = 0.88), VCAM-1 (AUC = 0.87), properdin (AUC = 0.85), ALCAM (AUC = 0.84), and FcgRIIBC (AUC = 0.82), followed by MCP-1, hemopexin, Twenty-four urine samples (8 active LN, 8 inactive SLE, 8 healthy controls) were assayed on both platforms (aptamer-based screening and ELISA assays). Listed are the Spearman correlation coefﬁcients between the two platforms, for each protein, and the associated signiﬁcance (p < 0.05; *p < 0.001). N/A Not done. ARTICLE ALCAM Peroxiredoxin-6 * ** * ns * ns ns ns ns ns African-American pg/mg African-American pg/mg Caucasian pg/mg Caucasian pg/mg 50,000 40,000 30,000 20,000 10,000 0 40,000 30,000 20,000 10,000 Healthy control Inactive SLE Active LN Healthy control Inactive SLE Active LN 10,000 8000 4000 2000 2000 1500 1000 500 0 6000 0 0 ALCAM BFL-1 Calpastatin FcgRIIBC Hemopexin MCP-1 Peroxiredoxin-6 PF-4 Properdin sE-Selectin TFPI VCAM1 ** * ** * ns * ns ns ns ns ns * ns ns * ns ** ns ** * ns * ** ns * ns ns * * ns * ns ns * *** * ns ns ns * * ns ns ** ** ns * ns * * ns * * ns ** ns ns ns ns ns ns * ns ns * African-American pg/mg African-American pg/mg Caucasian pg/mg Caucasian pg/mg 50,000 100,000 200,000 300,000 50,000 100,000 150,000 200,000 250,000 0 6000 12,000 18,000 24,000 0 5000 10,000 15,000 8000 4000 3000 2000 1000 0 8000 6000 4000 2000 0 6000 4000 2000 0 2500 2000 1500 1500 1000 500 0 1500 1000 500 0 600 400 200 0 600 800 400 200 0 1000 500 0 0 100,000 200,000 300,000 400,000 0 5 × 105 1 × 106 2 × 106 2 × 106 3 × 106 0 5.0 × 106 1.0 × 107 1.5 × 107 2.0 × 107 0 2.0 × 106 4.0 × 106 6.0 × 106 8.0 × 106 1.0 × 107 0 0 300 200 100 0 600 400 200 8000 6000 4000 2000 0 600 400 200 0 0 40,000 30,000 20,000 10,000 0 40,000 30,000 20,000 10,000 Healthy control Inactive SLE Active LN Healthy control Inactive SLE Active LN Healthy control Inactive SLE Active LN Healthy control Inactive SLE Active LN Healthy control Inactive SLE Active LN Healthy control Inactive SLE Active LN Healthy control Inactive SLE Active LN Healthy control Inactive SLE Active LN Healthy control Inactive SLE Active LN Healthy control Inactive SLE Active LN Healthy control Inactive SLE Active LN Healthy control Inactive SLE Active LN 10,000 8000 4000 2000 2000 1500 1000 500 0 6000 0 0 Fig. 2 ELISA validation of elevated urinary proteins in African-American and Caucasian lupus nephritis patients. Plotted are ELISA-validation results for 12 urine proteins in African-American and Caucasian subjects. ARTICLE ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15986-3 ELISA-validation assay exceeded 0.8, with these values exceeding 0.9 for ALCAM, peroxiredoxin-6, PF-4, properdin, and VCAM-1 (Table 1). the 12 urine proteins interrogated outperformed traditional laboratory measures including C3/C4 and anti-dsDNA in discriminating active LN from inactive SLE, with improved AUC values and statistical signiﬁcance, as detailed in Table 3. Among these, urine VCAM-1, PF-4, and properdin performed the best in distinguishing active from inactive disease, with AUC values ≥79%. These three urine proteins exhibited fold-increase values ranging from 2.4 to 8.9, comparing patients with active LN to those with inactive disease. They also exhibited superior sensitivity, NPV and PPV values compared to the traditional yardsticks, and several of the other proteins interrogated. As detailed in Table 4, urine ALCAM, BFL-1, calpastatin, hemo- pexin, MCP-1, PF-4, properdin, sE-selectin, and VCAM-1 maintained signiﬁcant association with active renal disease status, after adjusting for race, age, prednisone usage, and multiple testing correction, as determined by multivariable logistic regression analysis. The 12 shortlisted urine proteins from the 1129 initially screened were ELISA tested in a cross-sectional cohort of 95 subjects with African-American and Caucasian ethnicity (comprised of 47 inactive SLE patients, 27 active LN patients, and 21 healthy controls), and creatinine-normalized (Fig. 2). The demographic attributes, clinical features, and medication history of these subjects are presented in Table 2, parsed by ethnic group. Each urinary protein was analyzed to determine how well it distinguished SLE patients from healthy controls, and active LN from inactive SLE, using receiver operating curve analysis. 10 of Table 1 Correlation between aptamer screen and ELISA assay results. Molecule Pearson correlation ALCAM 0.926*** BFL-1 N/A Calpastatin 0.728* FcgRIIB/C N/A Hemopexin 0.752* MCP-1 0.771* Peroxiredoxin-6 0.969* PF-4 0.977* Properdin 0.927*** sE-Selectin N/A TFPI 0.805* VCAM-1 0.981*** Twenty-four urine samples (8 active LN, 8 inactive SLE, 8 healthy controls) were assayed on both platforms (aptamer-based screening and ELISA assays). Listed are the Spearman correlation coefﬁcients between the two platforms, for each protein, and the associated signiﬁcance (p < 0.05; p < 0.001). N/A Not done. Table 1 Correlation between aptamer screen and ELISA assay results. Molecule Pearson correlation ALCAM 0.926* BFL-1 N/A Calpastatin 0.728* FcgRIIB/C N/A Hemopexin 0.752* MCP-1 0.771* Peroxiredoxin-6 0.969* PF-4 0.977* Properdin 0.927*** sE-Selectin N/A TFPI 0.805* VCAM-1 0.981*** Twenty-four urine samples (8 active LN, 8 inactive SLE, 8 healthy controls) were assayed on both platforms (aptamer-based screening and ELISA assays). ARTICLE Each protein was tested using the following sample group: 27 active SLE samples (14 African-American, 13 Caucasian), 47 inactive SLE samples (19 African-American, 28 Caucasian), and 21 healthy controls (14 African-American, 7 Caucasian). The plots show the mean concentration in urine for each disease group after normalizing against urinary creatinine. p < 0.05, p < 0.01, and *p < 0.001, as determined using Mann–Whitney U-test. Further details regarding these proteins are included in Supplementary Data. Source data are provided as a Source Data ﬁle. BFL-1 ns ns * ns ** 300 200 100 0 600 400 200 0 Healthy control Inactive SLE Active LN FcgRIIBC Hemopexin MCP-1 * * ns ns ns ** ns * ns * ns ns ns ns 8000 4000 3000 2000 1000 0 8000 6000 4000 2000 0 6000 4000 2000 0 2500 2000 1500 1000 500 0 0 5.0 × 106 1.0 × 107 1.5 × 107 2.0 × 107 0 2.0 × 106 4.0 × 106 6.0 × 106 8.0 × 106 1.0 × 107 Healthy control Inactive SLE Active LN Healthy control Inactive SLE Active LN Healthy control Inactive SLE Active LN Calpastatin Properdin ** ns * ns ns * 0 6000 12,000 18,000 24,000 0 5000 10,000 15,000 Healthy control Inactive SLE Active LN VCAM1 ns ns * ns ns * 100,000 200,000 300,000 400,000 0 5 × 105 1 × 106 2 × 106 2 × 106 3 × 106 0 Healthy control Inactive SLE Active LN PF-4 ** ns ** * ns * 10 20 30 5 10 15 20 25 8000 6000 4000 2000 0 600 400 200 0 Healthy control Inactive SLE Active LN Properdin PF-4 Properdin sE-Selectin TFPI ** ** * * * ns * ns ns * * * ns ns * ns * * ns 100,000 200,000 300,000 50,000 100,000 150,000 200,000 250,000 1500 1000 500 0 1500 1000 500 0 600 400 200 0 600 800 400 200 0 0 1 2 3 4 0 Healthy control Inactive SLE Active LN Healthy control Inactive SLE Active LN Healthy control Inactive SLE Active LN Inactive SLE Active LN Fig. 2 ELISA validation of elevated urinary proteins in African-American and Caucasian lupus nephritis patients. Plotted are ELISA-validation results for 12 urine proteins in African-American and Caucasian subjects. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15986-3 These proteins were also tested in a subset of urine samples included in the aptamer-based screening assay in order to assess the correlation between the two platforms—ELISA readouts and the aptamer-based screening results; these data are outlined in Table 1. For most proteins, the correlation of the biomarker results between the aptamer screening result and the NATURE COMMUNICATIONS \| (2020) 11:2197 \| https://doi.org/10.1038/s41467-020-15986-3 \| www.nature.com/naturecommunications 3 ARTICLE (%) 14 (100%) 19 (100%) 14(100%) 13 (100%) 28(100%) 7(100%) SLEDAI, median (IQR) 11(10–14) 2 (0–4) N/A 12(10–12) 0 (0–0) N/A rSLEDAI, median (IQR) 8 (8–12) 0 (0–0) N/A 8 (8–8) 0 (0–0) N/A PGA, median (IQR) 2.1 (1.9–2.5) 1 (0.5–1.2) N/A 1.5 (1.5–1.8) 0.5 (0–0.6) N/A Protein:Cr ratio (mg/mg) 2.7 ± 1.5 0.4 ± 0.3 N/A 1.3 ± 1.0 0.2 ± 0.1 N/A eGFR (ml/min/1.73 m2) 121 ± 40 100 ± 45 N/A 83 ± 24 77 ± 26 N/A Serum creatinine (mg/dl) 0.8 ± 0.3 1 ± 0.5 N/A 0.9 ± 0.2 0.9 ± 0.3 N/A Anti-dsDNA+ve/total tested 7/14 6/17 N/A 7/13 1/28 N/A Hypocomplementemia/total 5/13 5/19 N/A 7/13 4/28 N/A Concurrent medication use, n (%) Prednisone 10 (71%) 15 (79%) N/A 10 (77%) 9 (32%) N/A Immunosuppressants 12 (86%) 12 (63%) N/A 7 (54%) 16 (57%) N/A Plaquenil 14 (100%) 18 (95%) N/A 8 (62%) 23 (82%) N/A NSAID 3 (21%) 2 (11%) N/A 3 (23%) 1 (4%) N/A Anti-hypertensives 10 (71%) 12 (63%) N/A 12 (92%) 23 (82%) N/A Diuretic 4 (29%) 5 (26%) N/A 3 (23%) 4 (14%) N/A ACE inhibitor or ARB 8 (57%) 11 (58%) N/A 8 (62%) 18 (64%) N/A Statin 3 (21%) 3 (16%) N/A 3 (23%) 16 (57%) N/A Anti-dsDNA+ve refers to number of subjects who were positive for anti-dsDNA antibodies. Source data are provided as a Source Data ﬁle. Table 2 Patient cohort used for the cross-sectional validation studies. Table 3 Urine Cr-normalized biomarker levels in a primary validation cohort. ARTICLE Each protein was tested using the following sample group: 27 active SLE samples (14 African-American, 13 Caucasian), 47 inactive SLE samples (19 African-American, 28 Caucasian), and 21 healthy controls (14 African-American, 7 Caucasian). The plots show the mean concentration in urine for each disease group after normalizing against urinary creatinine. p < 0.05, p < 0.01, and *p < 0.001, as determined using Mann–Whitney U-test. Further details regarding these proteins are included in Supplementary Data. Source data are provided as a Source Data ﬁle. Fig. 2 ELISA validation of elevated urinary proteins in African-American and Caucasian lupus nephritis patie Fig. 2 ELISA validation of elevated urinary proteins in African-American and Caucasian lupus nephritis patients. Plotted are ELISA-validation results for 12 urine proteins in African-American and Caucasian subjects. Each protein was tested using the following sample group: 27 active SLE samples (14 African-American, 13 Caucasian), 47 inactive SLE samples (19 African-American, 28 Caucasian), and 21 healthy controls (14 African-American, 7 Caucasian). The plots show the mean concentration in urine for each disease group after normalizing against urinary creatinine. p < 0.05, p < 0.01, and p < 0.001, as determined using Mann–Whitney U-test. Further details regarding these proteins are included in Supplementary Data. Source data are provided as a Source Data ﬁle. NATURE COMMUNICATIONS \| (2020) 11:2197 \| https://doi.org/10.1038/s41467-020-15986-3 \| www.nature.com/naturecommunications 4 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15986-3 Table 2 Patient cohort used for the cross-sectional validation studies. African-American cohort Caucasian cohort Active LN Inactive SLE Healthy control Active LN Inactive SLE Healthy control N = 14 N = 19 N = 14 N = 13 N = 28 N = 7 Age (years) 31.2 ± 8.6 33.1 ± 10.3 32.2 ± 5.1 44.8 ± 10.8 48.9 ± 10.7 50.1 ± 5.7 Female, no. ARTICLE Urine protein Fold-changea Active/inactive SLE/healthy Comparison of active LN vs inactive SLE Cut-off ROC AUC Sensitivity Speciﬁcity NPV PPV ALCAM 3.0 40.7 4875 0.74* 0.52 0.92 0.78 0.77 BFL-1 3.3* 4.3 11 0.74 0.70 0.81 0.68 0.83 Calpastatin 63 51.0* 325 0.74**** 0.52 0.98 0.93 0.78 FcγRIIBC 3.2* 2.7 367 0.69 0.59 0.79 0.62 0.77 Hemopexin 1.5* 2.8 327459 0.76** 0.96 0.57 0.57 0.96 MCP-1 2.9 4.4 203 0.71* 0.78 0.62 0.54 0.83 Peroxiredoxin-6 7.2 2.8 1 0.58 0.30 0.87 0.57 0.68 PF-4 8.9** 84.7 39 0.81** 0.78 0.83 0.72 0.87 Properdin 6.2 6.2 2062 0.79** 0.74 0.89 0.80 0.86 sE-Selectin 2.6 22.2* 3 0.73** 0.82 0.66 0.58 0.86 TFPI 1.9 6.3** 146 0.65* 0.63 0.75 0.59 0.78 VCAM-1 2.5** 0.2 10324 0.85 0.82 0.79 0.69 0.88 C3 0.9 N/A 146 0.39 0.19 0.92 0.56 0.66 C4 1 N/A 30 0.49 0.33 0.81 0.50 0.68 Anti-DNA 3.7 N/A 40 0.66 0.52 0.81 0.61 0.75 The primary validation cohort of 95 subjects comprise 47 inactive SLE patients, 27 active LN patients, and 21 healthy controls. Source data are provided as a Source Data ﬁle. ap-values by Mann–Whitney U-test (p < 0.05; p < 0.01; p < 0.001; *p < 0.0001). Table 3 Urine Cr-normalized biomarker levels in a primary validation cohort. calpastatin, PF-4, and properdin (Supplementary Table 3 and Fig. 2). In particular, urine sE-selectin outperformed anti-DNA in terms of sensitivity, NPV and PPV, and matched anti-DNA in terms of speciﬁcity, when comparing Caucasian patients with active LN to those with inactive SLE (Supplementary Table 3). Also striking was the ﬁnding that the absolute urine levels of ALCAM and FcgRIIBC were signiﬁcantly higher among African- American LN patients compared to Caucasian LN patients (p < 0.05, Mann–Whitney U-test; Fig. 2; Supplementary Table 3). calpastatin, PF-4, and properdin (Supplementary Table 3 and Fig. 2). In particular, urine sE-selectin outperformed anti-DNA in terms of sensitivity, NPV and PPV, and matched anti-DNA in terms of speciﬁcity, when comparing Caucasian patients with active LN to those with inactive SLE (Supplementary Table 3). Also striking was the ﬁnding that the absolute urine levels of ALCAM and FcgRIIBC were signiﬁcantly higher among African- American LN patients compared to Caucasian LN patients (p < 0.05, Mann–Whitney U-test; Fig. 2; Supplementary Table 3). calpastatin and TFPI, all exceeding the performance of C3/C4 and anti-DNA (Supplementary Table 3 and Fig. 2). ARTICLE Although the most discriminatory biomarkers tracked with proteinuria, they are unlikely to be simply the consequence of proteinuria as several other proteins that shared similar molecular weights with these biomarkers were not elevated in the urine (Supplementary Fig. 3). Although the correlation between urine biomarkers and renal pathology LN class was feeble, it should be pointed out that the time interval between the biopsy and urine procurement ranged from 1 month to 20 years; hence, the urine biomarker levels may not be reﬂective of historical renal pathology data. Table 4 Association of urine biomarkers with active LN status after adjusting for confounding factors. Active LN vs inactive SLE Protein Race as co-factor Race + age co-factors Race + prednisone co-factors p-value q-value p-value q-value p-value q-value ALCAM * * * * ns BFL-1 * * Calpastatin * * * * * FcgRIIBC ns ns ns ns ns ns Hemopexin MCP-1 ** * ** * ** * Peroxiredoxin-6 ns ns ns ns ns ns PF-4 * * * * * * Properdin * * * * * * sE-Selectin TFPI ns ns ns ns ns ns VCAM-1 *p < 0.001, p < 0.01, p < 0.05, ns = p > 0.05. The same nomenclature is used for signiﬁcance after multiple testing correction (q-values). Table 4 Association of urine biomarkers with active LN status after adjusting for confounding factors. Hence, our secondary validation cohort was composed of 80 inactive SLE patients, 80 active LN patients, 67 active non-renal lupus patients, and 53 healthy controls. The demographic attributes, clinical features, and medication history of these subjects are presented in Supplementary Table 5. In this second validation cohort, four of the eight urine proteins interrogated, including ALCAM (AUC = 0.93), VCAM-1 (AUC = 0.92), TFPI (AUC = 0.88), and PF-4 (AUC = 0.83), outperformed C3/C4 and anti-dsDNA in discriminating active LN from inactive SLE, with improved AUC values and statistical signiﬁcance, as detailed in Table 5 and Fig. 3b. The sensitivities and speciﬁcities of these biomarkers in the Asian cohort were also re-calculated using the same cut-off values used for the primary validation cohort, and this is presented in Supplementary Table 6. ARTICLE Even though the remaining four urine proteins, calpastatin, hemopexin, peroxir- edoxin-6, and properdin, did not outperform anti-dsDNA in distinguishing active LN from inactive SLE patients, they were all signiﬁcantly elevated in active LN when compared with inactive SLE patients, thus offering independent validation of the urinary biomarkers uncovered in this study, across multiple ethnic groups. More importantly, as plotted in Fig. 3b, several of the interrogated urine proteins were signiﬁcantly elevated in active LN compared to active non-renal lupus in this cohort, alluding to their renal speciﬁcity. In particular, urine ALCAM (AUC = 0.84), calpastatin (AUC = 0.68), properdin (AUC = 0.75), TFPI (AUC = 0.78), and VCAM-1 (AUC = 0.73) exhibited the best ability to distinguish active renal involvement from active non-renal lupus. Interestingly, in patients with active LN, the urine levels of ALCAM, TFPI, and VCAM-1 were signiﬁcantly higher in Asian patients when compared to either African-American or Cauca- sian patients (p < 0.05, Mann–Whitney U-test), alluding to potential genetic contributions underpinning these differences. p gy We next subjected the 12 assayed urine proteins, ethnicity, and various clinical metrics to an unsupervised Bayesian network analysis, which uses probability distributions to represent the inter-dependencies of all changing variables in a model and how they relate to each other. As one would predict, the three clinical indices monitored, SLEDAI, rSLEDAI, and disease status (active LN vs inactive lupus) were strongly linked to each other, with strong positive correlation (Fig. 4b). Likewise, proteinuria, pyuria, and hematuria were strongly linked to rSLEDAI. The fact that these “ground truth” relationships were correctly identiﬁed by the unsupervised Bayesian network algorithm offers internal valida- tion of this probabilistic association approach. This independent analysis identiﬁed urine ALCAM as having the strongest impact on “disease status”, and sE-selectin as having the strongest impact on SLEDAI (Fig. 4b), with race being an important confounding factor, as we have already established above. Indeed, these two urine proteins exhibited the largest impact on all other nodes in this network, based on their “node force”, which is proportional to the size of each node. Expression of urinary biomarkers within the kidneys in LN. The proteins noted to be elevated in the urine of LN patients could have originated from two potential sources—from the circulating blood, or from within the kidneys. ARTICLE These same molecules also surpassed conventional laboratory yardsticks, in terms of assay sensitivity, PPV and NPV. Among the ﬁve urine proteins with the highest discriminatory potential (highest AUC values) in African-American patients, urine VCAM-1, and PF-4 exhibited the highest sensitivity (0.93) and PPV (0.93–0.94), whereas urine ALCAM and properdin exhibited the highest speciﬁcity (0.90–0.95) and NPV (0.86–0.92) (Supplementary Table 3). calpastatin and TFPI, all exceeding the performance of C3/C4 and anti-DNA (Supplementary Table 3 and Fig. 2). These same molecules also surpassed conventional laboratory yardsticks, in terms of assay sensitivity, PPV and NPV. Among the ﬁve urine proteins with the highest discriminatory potential (highest AUC values) in African-American patients, urine VCAM-1, and PF-4 exhibited the highest sensitivity (0.93) and PPV (0.93–0.94), whereas urine ALCAM and properdin exhibited the highest speciﬁcity (0.90–0.95) and NPV (0.86–0.92) (Supplementary Table 3). Given the observed variation in urine biomarkers across ethnicities, we next assayed these biomarkers in a second validation cohort of Asian patients. Power calculations based on ELISA results from the primary validation cohort indicate that we would need an average sample size of 52 per group (Supplementary Table 4). Different biomarkers emerged as being useful among Cauca- sian patients, where the most discriminatory urine proteins were sE-selectin (AUC = 0.87), VCAM-1 (AUC = 0.84), BFL-1 (AUC = 0.81), and hemopexin (AUC = 0.80), followed by 5 5 TURE COMMUNICATIONS \| (2020) 11:2197 \| https://doi.org/10.1038/s41467-020-15986-3 \| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15986-3 patients. Thus, within the African-American cohort, ALCAM, FcgRIIBC, hemopexin, peroxiredoxin-6, properdin, and VCAM-1 were positively correlated with PGA, SLEDAI, rSLEDAI scores, and proteinuria (p < 0.05, Mann–Whitney U-test; R > 0.4), with PF-4 showing a similar trend (as dot-plotted in Fig. 4a). Inter- estingly, urine ALCAM and VCAM-1 also positively correlated with ESR. These proteins exhibited poor correlation with C3/C4 and anti-DNA (Fig. 4a). Conversely, within the Caucasian cohort, urine BFL-1, sE-Selectin, and VCAM- 1 displayed a positive correlation with PGA, SLEDAI, rSLEDAI, and proteinuria (p < 0.05, Mann–Whitney U-test; R > 0.4), with a similar trend being noted for Hemopexin (Fig. 4a; Supplementary Table 3). Within the Asian cohort, urine ALCAM, peroxiredoxin-6, TFPI, and VCAM-1 all signiﬁcantly correlated with PGA, SLEDAI, and rSLEDAI (p < 0.05, Mann–Whitney U-test; R > 0.4) (Fig. 4a). Several urine proteins displayed a negative correlation with C3/ C4 and a positive correlation with anti-DNA, as summarized in Fig. 4a. NATURE COMMUNICATIONS \| (2020) 11:2197 \| https://doi.org/10.1038/s41467-020-15986-3 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15986-3 h di t d ll l t ti MCP 1 d i d i t t d t h i h b tili d ithi th t t f ALCAM Calpastatin Hemopexin * * * ns ns ns * Peroxiredoxin-6 ns ** **** * * ** ns PF-4 Properdin TFPI VCAM1 ns ns ns ns * ** * ** * ** pg/mg pg/mg Active LN vs Inactive SLE AUC = 0.98 Specificity 0.0 1.0 0.8 0.6 0.4 0.2 0.0 0.2 0.4 0.6 0.8 1.0 Parameter RaceW 1.37 0 0 0 0.30 0.62 0 0.09 0.02 0 0.54 0.50 0.56 0 0.25 Pred = 10 Age ALCAM BFL1 Calpastatin FcgRIIBC Hemopexin MCP-1 Peroxiredoxin-6 PF4 Properdin sE-Selectin TFPI VCAM1 Coefficient Sensitivity 400,000 300,000 200,000 100,000 10,000 150,000 0 1000 2000 3000 4000 5000 0 2000 4000 6000 0 5 × 106 5 × 107 1.5 × 107 2 × 107 100,000 50,000 0 8000 6000 4000 2000 0 Healthy control Inactive SLE Active NR Active LN Healthy control Inactive SLE Active NR Active LN Healthy control Inactive SLE Active NR Active LN Healthy control Inactive SLE Active NR Active LN Healthy control Inactive SLE Active NR Active LN Healthy control Inactive SLE Active NR Active LN Healthy control Inactive SLE Active NR Active LN Healthy control Inactive SLE Active NR Active LN 0 800,000 600,000 400,000 200,000 0 4000 5000 3000 2000 1000 0 a b Fig. 3 Testing the utility of multi-marker panels, and validity of markers in other ethnicities. a Lasso Regression Analysis to identify multi-marker panels. We performed Lasso regression analysis to identify multi-marker panels that may better predict active LN status in the primary validation cohort, as detailed in Methods. Besides the 12 urine proteins, we included race, age, and prednisone usage as additional variables. The combination with the best predictive model encompassed 8 of the 12 urine proteins, together with race, but excluded age and prednisone usage. This octuplex panel exhibited outstanding ability to discriminate active LN from inactive SLE with a ROC AUC value of 0.98. b Plotted are ELISA-validation results in a second cohort, comprised of Asian subjects. ARTICLE To assess whether the 12 proteins interrogated in this study might also be expressed with the kidneys in LN, we turned to another OMICs platform. Single-cell RNAseq analysis of renal biopsy tissue from LN has recently detailed the expression of all genes within LN kidneys, with imputed cell-of-origin information. Two publically available single-cell RNAseq datasets which contained 1624 kidney single- cell RNAseq proﬁles from 21 LN patients and 3 healthy controls and 363 cells from 10 patients, respectively, were combined using canonical correlation analysis, and interrogated for expression of the 12 urine biomarkers described above. Correlation with clinical and laboratory metrics. Most of the urine proteins that exhibited signiﬁcant potential to discriminate active LN from inactive disease were also associated with disease activity indices such as SLEDAI, renal-SLEDAI (rSLEDAI), and physician global assessment (PGA). Nine of the validated urine proteins were signiﬁcantly associated with rSLEDAI and PGA, after adjusting for race, age, prednisone usage, and multiple testing correction, as determined by multivariable linear regres- sion analysis (Supplementary Table 7). This was further con- ﬁrmed by a correlation analysis within each ethnic group of Strong intra-renal expression was noted for MCP-1 (CCL2), calpastatin, peroxiredoxin-6, ALCAM, TFPI, and VCAM-1, within LN kidneys (Fig. 5a). Intra-renal endothelial cells in LN expressed sE-selectin, TFPI, VCAM-1, all of which one would 6 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15986-3 Strong renal tubular expression of ALCAM, calpastatin, MCP- 1, TFPI, VCAM-1, and peroxiredoxin-6 were observed. ALCAM, MCP-1, calpastatin, peroxiredoxin-6, and TFPI were expressed within mesangial cells as well, while inﬁltrating leukocytes expressed ALCAM, BFL-1, calpastatin, FcGR2B, properdin, and peroxiredoxin-6, as portrayed in Fig. 5a. Despite the limited number of healthy control samples (N = 3), renal expression of VCAM-1 was signiﬁcantly higher in LN, with similar trends being noted for MCP-1, calpastatin, and FcGR2B (Fig. 5b). have predicted, as well as calpastatin, MCP-1, and peroxiredoxin- 6. Strong renal tubular expression of ALCAM, calpastatin, MCP- 1, TFPI, VCAM-1, and peroxiredoxin-6 were observed. ALCAM, MCP-1, calpastatin, peroxiredoxin-6, and TFPI were expressed within mesangial cells as well, while inﬁltrating leukocytes expressed ALCAM, BFL-1, calpastatin, FcGR2B, properdin, and peroxiredoxin-6, as portrayed in Fig. 5a. Despite the limited number of healthy control samples (N = 3), renal expression of VCAM-1 was signiﬁcantly higher in LN, with similar trends being noted for MCP-1, calpastatin, and FcGR2B (Fig. 5b). untargeted techniques have been utilized within the context of SLE7,8,18,19. Although these platforms do not rely on antigen or ligand binding, the data generated by these untargeted platforms may be noisy and may not reliably detect or measure low- abundance proteins. Targeted assays, like the aptamer-based screening platform, have the potential to accurately detect and quantify low-abundance proteins through protein-ligand inter- actions. To date, the human proteome project has successfully identiﬁed over 30,000 human proteins20, and the targeted pro- teomic platforms available at the time of this study offer coverage of <2000 proteins10,21. The aptamer-based screening platform utilized within this study is, to date, one of the largest proteomic screening platforms available, and its use of aptamer ligands has led to the successful identiﬁcation of biomarkers in several diseases10–16. Importantly, the hits that were identiﬁed using this initial screening platform were successfully validated by ELISA, with good correlation ratios being noted between these two platforms (Table 1). Reassuringly, NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15986-3 a Lasso Regressi he utility of multi-marker panels, and validity of markers in other ethnicities. a Lasso Regression Analysis to identify m Fig. 3 Testing the utility of multi-marker panels, and validity of markers in other ethnicities. a Lasso Regression Analysis to identify multi-marker panels. We performed Lasso regression analysis to identify multi-marker panels that may better predict active LN status in the primary validation cohort, as detailed in Methods. Besides the 12 urine proteins, we included race, age, and prednisone usage as additional variables. The combination with the best predictive model encompassed 8 of the 12 urine proteins, together with race, but excluded age and prednisone usage. This octuplex panel exhibited outstanding ability to discriminate active LN from inactive SLE with a ROC AUC value of 0.98. b Plotted are ELISA-validation results in a second cohort, comprised of Asian subjects. Each protein was tested using the following sample group: 80 active SLE samples, 80 inactive SLE samples, 67 active non- renal lupus samples, and 53 healthy controls. The plots show the mean concentration in urine for each disease group after normalizing against urinary creatinine. p < 0.05, p < 0.01, and *p < 0.001, as determined using Mann–Whitney U-test. Source data are provided as a Source Data ﬁle. g g y p , y g y y p We performed Lasso regression analysis to identify multi-marker panels that may better predict active LN status in the primary validation cohort, as detailed in Methods. Besides the 12 urine proteins, we included race, age, and prednisone usage as additional variables. The combination with the best predictive model encompassed 8 of the 12 urine proteins, together with race, but excluded age and prednisone usage. This octuplex panel exhibited outstanding ability to discriminate active LN from inactive SLE with a ROC AUC value of 0.98. b Plotted are ELISA-validation results in a second cohort, comprised of Asian subjects. Each protein was tested using the following sample group: 80 active SLE samples, 80 inactive SLE samples, 67 active non- renal lupus samples, and 53 healthy controls. The plots show the mean concentration in urine for each disease group after normalizing against urinary creatinine. p < 0.05, p < 0.01, and p < 0.001, as determined using Mann–Whitney U-test. Source data are provided as a Source Data ﬁle. have predicted, as well as calpastatin, MCP-1, and peroxiredoxin- 6. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15986-3 Each protein was tested using the following sample group: 80 active SLE samples, 80 inactive SLE samples, 67 active non- renal lupus samples, and 53 healthy controls. The plots show the mean concentration in urine for each disease group after normalizing against urinary creatinine. p < 0.05, p < 0.01, and p < 0.001, as determined using Mann–Whitney U-test. Source data are provided as a Source Data ﬁle. NATURE COMMUNICATIONS \| (2020) 11:2197 \| https://doi.org/10.1038/s41467-020-15986-3 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15986-3 Active LN vs Inactive SLE AUC = 0.98 Specificity 0.0 1.0 0.8 0.6 0.4 0.2 0.0 0.2 0.4 0.6 0.8 1.0 Parameter RaceW 1.37 0 0 0 0.30 0.62 0 0.09 0.02 0 0.54 0.50 0.56 0 0.25 Pred = 10 Age ALCAM BFL1 Calpastatin FcgRIIBC Hemopexin MCP-1 Peroxiredoxin-6 PF4 Properdin sE-Selectin TFPI VCAM1 Coefficient Sensitivity a Active LN vs Inactive SLE AUC = 0.98 Specificity 0.0 1.0 0.8 0.6 0.4 0.2 0.0 0.2 0.4 0.6 0.8 1.0 Sensitivity ALCAM * * * PF-4 pg/mg 400,000 300,000 200,000 100,000 Healthy control Inactive SLE Active NR Active LN 0 b ALCAM Calpastatin * * ns ns ns * PF pg/mg 400,000 300,000 200,000 100,000 0 1000 2000 3000 4000 5000 0 5 × 106 5 × 107 1.5 × 107 2 × 107 Healthy control Inactive SLE Active NR Active LN Healthy control Inactive SLE Active NR Active LN H c 0 b b Calpastatin ns ns ns ** * 0 1000 2000 3000 4000 5000 0 5 × 106 5 × 107 1.5 × 107 2 × 107 Healthy control Inactive SLE Active NR Active LN Hemopexin * ** ns Healthy control Inactive SLE Active NR Active LN Hemopexin Peroxiredoxin-6 ns **** * * ** ns VCAM1 Healthy control Inactive SLE Active NR Active LN Healthy control Inactive SLE Active NR Active LN 4000 5000 3000 2000 1000 0 TFPI VCAM1 ns * ** * Healthy control Inactive SLE Active NR Active LN Healthy control Inactive SLE Active NR Active LN 800,000 600,000 400,000 200,000 0 PF-4 Properdin TFPI ns ns ns ns * ** * ** pg/mg 10,000 150,000 0 2000 4000 6000 100,000 50,000 0 8000 6000 4000 2000 0 Healthy control Inactive SLE Active NR Active LN Healthy control Inactive SLE Active NR Active LN Healthy control Inactive SLE Active NR Active LN VCAM1 ** * ** Healthy control Inactive SLE Active NR Active LN Active NR Fig. 3 Testing the utility of multi-marker panels, and validity of markers in other ethnicities. a Lasso Regression Analysis to identify multi-marker panels. Fig. 3 Testing the utility of multi-marker panels, and validity of markers in other ethnicities. Discussion l As elaborated further below, most of the newly identiﬁed biomarker candidates have important biological functions relating to inﬂammation or auto- immunity (Supplementary Table 8). Of importance, this screen- ing platform has been effective in identifying several urine biomarkers of LN that correlate with traditional yardsticks of SLE/LN, but outperform those metrics in terms of sensitivity, NPV, and PPV. Besides representing one of the largest targeted proteomic screen conducted in LN, this study is also unique in highlighting the importance of tailoring the biomarkers to patient ethnicity. p Properdin (CFP) is a plasma glycoprotein of the complement system that is important in the stabilization of alternative pathway convertases. Properdin was reported to be elevated in the kidney tissue of LN (but not SLE patients without nephritis) but decreased in both serum of SLE patients and in patients with poststreptococcal/membranoproliferative glomerulonephritis33–35. Properdin has also been implicated in other inﬂammatory dis- eases, including arthritis36, and IgA nephropathy37. In this study, urine properdin exhibits similar diagnostic characteristics as PF- 4, in that it has the ability to distinguish active LN in both ethnic groups of patients, but with the best performance being noted among African-American LN patients, in whom urine properdin exhibited the highest speciﬁcity and NPV values in identifying active LN patients, compared to other competing markers and conventional yardsticks. Urine properdin also emerged as one of the few proteins that distinguished active LN from active non- renal lupus (Fig. 3), alluding to its renal speciﬁcity. Among African-American patients, the most discriminatory biomarkers that distinguished active LN from inactive disease were urine ALCAM, PF-4, properdin, and VCAM-1. Activated leukocyte cell adhesion molecule (ALCAM/CD166), a trans- membrane glycoprotein expressed primarily on activated T cells, is involved in multiple immune and inﬂammatory responses22–24. ALCAM is also a known recruiter for white blood cells in chronic kidney disease25, and is upregulated within the glomeruli in animal models of LN26. Serum levels of ALCAM correlate with SLE disease activity27. In this study, ALCAM surpassed conven- tional metrics in identifying active LN, with better sensitivity, speciﬁcity, PPV and NPV, among African-American patients and Asian patients (but not among Caucasian patients), correlating signiﬁcantly with PGA, SLEDAI, rSLEDAI, and proteinuria. When all SLE patients were combined, urine ALCAM levels had the strongest bearing on disease activity status, in an unsu- pervised Bayesian network analysis. Discussion l Large-scale proteomic screening technologies have revolutionized the study design and workﬂow for biomarker research. Samples that could previously only be used to measure <100 proteins can now be screened for several thousands of proteins simultaneously using multiple proteomic platforms. Mass spectrometry-based 7 7 NATURE COMMUNICATIONS \| (2020) 11:2197 \| https://doi.org/10.1038/s41467-020-15986-3 \| www.nature.com/naturecommunicatio ARTICLE ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15986-3 Table 5 Urine Cr-normalized biomarkers in the second (Chinese) validation cohort. Urine protein Fold-changea Active/inactive AR/ANR SLE/healthy Comparison of active LN vs inactive SLEa Cut-off ROC AUC Sens. Spec. NPV PPV ALCAM 14 4.3 10.8 15K 0.93 0.9 0.91 0.9 0.91 Calpastatin 7.1 3.1 3.3 0 0.74 0.66 0.78 0.7 0.75 Hemopexin 2.3 1.1 2.6 630K 0.74 0.85 0.56 0.79 0.66 Peroxiredoxin-6 6.6** 1.8* 3.4** 0 0.75 0.56 0.91 0.68 0.87 PF-4 24 1.8 11** 0 0.83 0.74 0.88 0.77 0.86 Properdin 13 10.8 26 0 0.74 0.61 0.84 0.68 0.79 TFPI 6.2 2.4 4.9 200 0.88 0.8 0.89 0.82 0.89 VCAM-1 8.9 2.0 21 30,200 0.92 0.9 0.88 0.9 0.88 C3 0.5 0.8* N/A 13 0.12 1 0 0 0.5 C4 0.5** 0.8 N/A 3 0.19 1 0 0 0.5 Anti-DNA 2.4** 1.3 N/A 215 0.82**** 0.63 0.99 0.98 0.72 The cohort comprises 80 inactive SLE patients, 67 active non-renal (ANR), 80 active LN patients, and 53 healthy controls. Source data are provided as a Source Data ﬁle. ap-values by Mann–Whitney U-test (p < 0.05; p < 0.01; p < 0.001; **p < 0.0001). Table 5 Urine Cr-normalized biomarkers in the second (Chinese) validation cohort. disease in all African-American, Caucasian, and Asian patients. Importantly, healthy subjects exhibited minimal levels of urinary PF-4, that are comparable to those seen in inactive LN patients, while active LN patients consistently exhibited marked increases in PF-4 levels (fold-change >5). It was a better disease marker among African-American SLE patients, where its sensitivity, NPV and PPV were among the highest, well surpassing that of anti- DNA and complement. this platform re-identiﬁed several urine protein biomarkers that had previously been implicated as biomarkers for LN, including adiponectin, NGAL, and TWEAK; although all of these proteins were elevated in the urine of active LN patients in the aptamer- based screen, they did not rank within the top 50, based on fold- change or p-values. This observation offers independent valida- tion of this proteomic screening platform. Discussion l Urine ALCAM also emerged as one of the few proteins that distinguished active LN from active non-renal lupus (Fig. 3). Indeed, intra-renal expression within LN kidneys was noted within inﬁltrating leukocytes, renal tubular cells and mesangial cells, based on RNAseq analysis. p g g p y VCAM-1 (vascular cell adhesion molecule, CD106) is a cell adhesion molecule expressed on endothelial cells. VCAM-1 has been implicated as a biomarker in atherosclerosis38–40, and extensively studied in the context of SLE and LN41–43. In this study, VCAM-1 was one of top two urine biomarkers that had the potential to distinguish active LN from inactive LN within all ethnic cohorts, with respectable sensitivity, NPV and PPV values, compared to the traditional yardsticks. Urine VCAM-1 correlated signiﬁcantly with PGA, SLEDAI, rSLEDAI, and proteinuria in all ethnic groups. Urine VCAM-1 also emerged as one of the few proteins that distinguished active LN from active non-renal lupus (Fig. 3), alluding to its renal speciﬁcity. Indeed, it is strongly expressed within LN kidneys, based on renal single-cell RNAseq analysis, particularly on endothelial cells, renal tubular cells, and inﬁltrating leukocytes (Fig. 5). g y Platelet factor 4 (PF-4, or CXCL4) is CXC chemokine that is chemotactic for monocytes and neutrophils28,29. PF-4 has been implicated as a possible urinary biomarker for LN30. PF-4 auto- antibodies have been found in SLE patients31, and studies suggest that these antibodies may correlate to disease activity32. Of the 12 urinary proteins examined in this study, urinary PF-4 was one of ﬁve proteins that was able to distinguish active LN from inactive Among Caucasian patients, the most discriminatory urine proteins were sE-selectin, VCAM-1, BFL-1, and Hemopexin. E- Selectin mediates immune cell adhesion, allowing neutrophils to adhere to vascular endothelial cells. E-selectin has been impli- cated in several cancers44–46, and as a potential biomarker for SLE NATURE COMMUNICATIONS \| (2020) 11:2197 \| https://doi.org/10.1038/s41467-020-15986-3 \| www.nature.com/naturecommunications 8 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15986-3 African-American Cohort Caucasian Cohort Chinese Cohort ALCAM BFL1 Calpastatin FcgRIIBC Hemopexin MCP–1 Peroxiredoxin 6 PF4 Properdin sE–Selectin TFPI VCAM1 ALCAM BFL1 Calpastatin FcgRIIBC Hemopexin MCP–1 Peroxiredoxin 6 PF4 Properdin sE–Selectin TFPI VCAM1 ALCAM Calpastatin Hemopexin –1.0 –0.5 0 +0.5 +1.0 Peroxiredoxin 6 PF4 Properdin TFPI VCAM1 eGFR PGA (0–3) SLEDAI rSLEADI ESR UrPrCrRatio C3 C4 DNA Biopsy Class eGFR PGA (0–3) SLEDAI rSLEADI ESR UrPrCrRatio C3 C4 DNA PGA (0–3) SLEDAI rSLEADI C3 C4 DNA Biopsy Class 10° 10–1 10–2 10–3 ≤10–4 a b Fig. 4 Correlation and association of urine biomarkers of lupus nephritis with clinical and laboratory indices. a Creatinine-normalized urine levels of the 12 proteins (listed vertically) were Pearson correlated with various clinical and laboratory yardsticks, as listed on the x axis, in both the African-American (14 active LN, 19 inactive SLE, 14 healthy controls), Caucasian (13 active LN, 28 inactive SLE, 7 healthy controls), and Asian (80 active LN, 80 inactive SLE, 67 active non-renal lupus, 53 healthy control) patient cohorts. It should be noted that the renal biopsy data included is not from concurrent biopsies, but from previous biopsies, executed 1-mo to 20 years before urine procurement. Positive and negative correlations are denoted by orange and blue circles respectively, while statistical signiﬁcance is denoted using gray-scale boxes. b The levels of the 12 urine proteins in the combined cohort (27 active LN, 47 inactive SLE, and 21 healthy controls) and their respective clinical features were subjected to Bayesian network analysis using BayesiaLab. The network shown was constructed in an unsupervised manner, using the EQ algorithm and a structural coefﬁcient of 0.4. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15986-3 The circular nodes that make up the Bayesian Network represent the variables of interest, including urine biomarkers (purple-colored), clinical indices (green-colored), other features (colored ) d di t t ( ti LN i ti SLE di l d b ) Th i f h d d t th “ d f ” hi h i l t d t it African-American Cohort ALCAM BFL1 Calpastatin FcgRIIBC Hemopexin MCP–1 Peroxiredoxin 6 PF4 Properdin sE–Selectin TFPI VCAM1 eGFR PGA (0–3) SLEDAI rSLEADI ESR UrPrCrRatio C3 C4 DNA Biopsy Class a Caucasian Cohort Chinese Cohort ALCAM BFL1 Calpastatin FcgRIIBC Hemopexin MCP–1 Peroxiredoxin 6 PF4 Properdin sE–Selectin TFPI VCAM1 ALCAM Calpastatin Hemopexin –1.0 –0.5 0 +0.5 +1.0 Peroxiredoxin 6 PF4 Properdin TFPI VCAM1 eGFR PGA (0–3) SLEDAI rSLEADI ESR UrPrCrRatio C3 C4 DNA PGA (0–3) SLEDAI rSLEADI C3 C4 DNA Biopsy Class 10° 10–1 10–2 10–3 ≤10–4 a b b Fig. 4 Correlation and association of urine biomarkers of lupus nephritis with clinical and laboratory indices. a Creatinine-normalized urine levels of the 12 proteins (listed vertically) were Pearson correlated with various clinical and laboratory yardsticks, as listed on the x axis, in both the African-American (14 active LN, 19 inactive SLE, 14 healthy controls), Caucasian (13 active LN, 28 inactive SLE, 7 healthy controls), and Asian (80 active LN, 80 inactive SLE, 67 active non-renal lupus, 53 healthy control) patient cohorts. It should be noted that the renal biopsy data included is not from concurrent biopsies, but from previous biopsies, executed 1-mo to 20 years before urine procurement. Positive and negative correlations are denoted by orange and blue circles respectively, while statistical signiﬁcance is denoted using gray-scale boxes. b The levels of the 12 urine proteins in the combined cohort (27 active LN, 47 inactive SLE, and 21 healthy controls) and their respective clinical features were subjected to Bayesian network analysis using BayesiaLab. The network shown was constructed in an unsupervised manner, using the EQ algorithm and a structural coefﬁcient of 0.4. The circular nodes that make up the Bayesian Network represent the variables of interest, including urine biomarkers (purple-colored), clinical indices (green-colored), other features (colored gray) and disease status (active LN vs inactive SLE vs no disease; colored brown). The size of each node denotes the “node force”, which is related to its impact on other nodes in the network, based on conditional probabilities. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15986-3 Healthy LN 0 20 40 60 80 100 Healthy LN 0 50 100 150 Healthy LN 0 50 100 150 Healthy LN 0 20 40 60 80 CCL2 MCP-1 VCAM1 CAST Calpastatin FCGR2B FcgRIIIBC p = 0.13 p = 0.02 p = 0.09 p = 0.1 b c −20 −10 0 10 20 30 −30 −20 −10 0 10 20 30 tSNE_1 tSNE_2 Tubules Fibroblasts Leukocytes Endothelial Mesangial PRDX6 PF4 −30 −20 −10 0 10 20 30 tSNE_1 TFPI −30 −20 −10 0 10 20 30 tSNE_1 VCAM1 HPX CFP −30 −20 −10 0 10 20 30 tSNE_1 SELE −30 −20 −10 0 10 20 30 tSNE_1 FCGR2B −20 −10 0 10 20 30 tSNE_2 CCL2 CAST −20 −10 0 10 20 30 −30 −20 −10 0 10 20 30 tSNE_1 tSNE_2 ALCAM −30 −20 −10 0 10 20 30 tSNE_1 BCL2A1 a Fig. 5 Renal single-cell RNAseq expression of potential urine biomarkers in LN and healthy controls. a Feature plots of potential urine biomarkers where each dot is a cell and the color intensity (gray = low, to red = high) indicates expression of the indicated gene within each cell, as deduced by RNAseq data analysis of 21 LN renal tissue samples. CCL2 = MCP-1; CAST = calpastatin; HPX = hemopexin; PRDX6 = peroxiredoxin-6; BCL2A1 = BFL-1; SELE = sE- selectin. b tSNE plot of 1624 cells obtained from the renal biopsy of 21 LN patients and 3 healthy controls. Clusters are derived from PCA and graph-based clustering analysis and color-coded based on identiﬁed cell type. c Student’s t-test comparison of expression of CCL2 (MCP-1), VCAM-1, CAST (calpastatin), and FCG2RB between LN patients (N = 21) and healthy controls (N = 3) within the single-cell RNAseq dataset. The ends of the box represent the upper and lower quartiles, so the box spans the interquartile range. The median is marked by a horizontal line inside the box. The whiskers are the two lines outside the box that indicate 1.5 times the interquartile range from the upper or lower quartile. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15986-3 PRDX6 PF4 −30 −20 −10 0 10 20 30 tSNE_1 TFPI −30 −20 −10 0 10 20 30 tSNE_1 VCAM1 HPX CFP −30 −20 −10 0 10 20 30 tSNE_1 SELE −30 −20 −10 0 10 20 30 tSNE_1 FCGR2B −20 −10 0 10 20 30 tSNE_2 CCL2 CAST −20 −10 0 10 20 30 −30 −20 −10 0 10 20 30 tSNE_1 tSNE_2 ALCAM −30 −20 −10 0 10 20 30 tSNE_1 BCL2A1 a a Healthy LN 0 20 40 60 80 100 Healthy LN 0 50 100 150 Healthy LN 0 50 100 150 Healthy LN 0 20 40 60 80 CCL2 MCP-1 VCAM1 CAST Calpastatin FCGR2B FcgRIIIBC p = 0.13 p = 0.02 p = 0.09 p = 0.1 b c −20 −10 0 10 20 30 −30 −20 −10 0 10 20 30 tSNE_1 tSNE_2 Tubules Fibroblasts Leukocytes Endothelial Mesangial tSNE_1 tSNE_1 tSNE_1 tSNE_1 tSNE_1 tSNE_1 Fig. 5 Renal single-cell RNAseq expression of potential urine biomarkers in LN and healthy controls. a Feature plots of potential urine biomarkers where each dot is a cell and the color intensity (gray = low, to red = high) indicates expression of the indicated gene within each cell, as deduced by RNAseq data analysis of 21 LN renal tissue samples. CCL2 = MCP-1; CAST = calpastatin; HPX = hemopexin; PRDX6 = peroxiredoxin-6; BCL2A1 = BFL-1; SELE = sE- selectin. b tSNE plot of 1624 cells obtained from the renal biopsy of 21 LN patients and 3 healthy controls. Clusters are derived from PCA and graph-based clustering analysis and color-coded based on identiﬁed cell type. c Student’s t-test comparison of expression of CCL2 (MCP-1), VCAM-1, CAST (calpastatin), and FCG2RB between LN patients (N = 21) and healthy controls (N = 3) within the single-cell RNAseq dataset. The ends of the box represent the upper and lower quartiles, so the box spans the interquartile range. The median is marked by a horizontal line inside the box. The whiskers are the two lines outside the box that indicate 1.5 times the interquartile range from the upper or lower quartile. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15986-3 Healthy LN 0 20 40 60 80 100 Healthy LN 0 50 100 150 Healthy LN 0 50 100 150 Healthy LN 0 20 40 60 80 CCL2 MCP-1 VCAM1 CAST Calpastatin FCGR2B FcgRIIIBC p = 0.13 p = 0.02 p = 0.09 p = 0.1 c 0 b b −20 −10 0 10 20 30 −30 −20 −10 0 10 20 30 tSNE_1 tSNE_2 Tubules Fibroblasts Leukocytes Endothelial Mesangial c Fig. 5 Renal single-cell RNAseq expression of potential urine biomarkers in LN and healthy controls. a Feature plots of potential urine biomarkers where each dot is a cell and the color intensity (gray = low, to red = high) indicates expression of the indicated gene within each cell, as deduced by RNAseq data analysis of 21 LN renal tissue samples. CCL2 = MCP-1; CAST = calpastatin; HPX = hemopexin; PRDX6 = peroxiredoxin-6; BCL2A1 = BFL-1; SELE = sE- selectin. b tSNE plot of 1624 cells obtained from the renal biopsy of 21 LN patients and 3 healthy controls. Clusters are derived from PCA and graph-based clustering analysis and color-coded based on identiﬁed cell type. c Student’s t-test comparison of expression of CCL2 (MCP-1), VCAM-1, CAST (calpastatin), and FCG2RB between LN patients (N = 21) and healthy controls (N = 3) within the single-cell RNAseq dataset. The ends of the box represent the upper and lower quartiles, so the box spans the interquartile range. The median is marked by a horizontal line inside the box. The whiskers are the two lines outside the box that indicate 1.5 times the interquartile range from the upper or lower quartile. Renal RNAseq analysis indicated strong expression of MCP-1, calpastatin, peroxiredoxin-6, ALCAM, TFPI, and VCAM-1 within the inﬂamed kidneys, suggesting that intra-renal cells may be the dominant source of these urinary biomarkers in LN, though this needs to be validated by immunohistochemistry. Given this RNAseq expression data, it is not surprising that these same urine proteins, urine calpastatin, ALCAM, TFPI, and VCAM-1, are also the best at discriminating active LN from active non-renal lupus (Fig. 3). On the other hand, the diseased kidneys in LN may not be the dominant source of BFL-1, hemopexin, properdin or PF-4 in the urine; although this pre- diction is consistent with the known biology of these molecules, this hypothesis warrants further testing of paired serum and urine samples from the same subjects. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15986-3 The links (arcs) that interconnect the nodes represent informational or causal dependencies among the variables, including the correlation coefﬁcients between neighboring nodes (as indicated), with the thickness of the link being proportional to the correlation coefﬁcient. analysis. Its diagnostic performance in other ethnic groups clearly warrants further analysis in expanded patient cohorts. and LN47–49. In this study, while sE-Selectin emerged as the most discriminatory urinary marker within Caucasian LN patients, it was not promising within the African-American cohort. Among Caucasian patients, urine sE-selectin exhibited improved sensi- tivity, NPV and PPV values, compared to the traditional yard- sticks, and correlated signiﬁcantly with PGA, SLEDAI, rSLEDAI, and proteinuria. Urine sE-selectin also exhibited the strongest bearing on SLEDAI and race, in an unsupervised Bayesian net- work analysis. Strong intra-renal expression within LN kidneys was also noted within endothelial cells, based on RNAseq BFL-1 is an anti-apoptosis protein within the BCL2 protein family. BFL-1 is a direct transcription target from the NF-kB pathway and is known to be upregulated by several inﬂammatory signals, including GM-CSF, IL-1, and CD40. It is believed to play a role in leukocyte activation and survival50–52, and has been shown to be overexpressed in B cells of SLE patients53. This study implicates that BFL-1 may be a reliable biomarker for LN within Caucasian patients (p < 0.0001, Mann–Whitney U-test; AUC = 9 NATURE COMMUNICATIONS \| (2020) 11:2197 \| https://doi.org/10.1038/s41467-020-15986-3 \| www.nature.com/naturecommunications 9 ARTICLE Methods P ti t Random forest classiﬁcation analysis, a machine learning algorithm for dimensionality reduction, was executed using 1000 bootstrap sampling iterations, in order to identify the relative importance of each biomarker candidate in disease classiﬁcation, as mea- sured by the GINI index, using the sklearn.ensembl R package. For the top 20 urine potential biomarkers identiﬁed by random forest classiﬁcation, the fold-change in SLE vs healthy controls, and the fold-change in active LN vs inactive SLE were plotted as a radial plot, using Excel. Bayesian network analysis was performed using the BayesiaLab software (Bayesia, version 7.0.1). The dataset for unsupervised learning included data pertaining to 95 subjects (comprises 47 inactive SLE patients, 27 active LN patients, and 21 healthy controls), including the following parameters: the urine levels of 12 protein biomarkers, race, disease status (active renal vs inactive SLE) and disease features or measures (proteinuria, pyuria, hematuria, SLEDAI, rSLEDAI, PGA, and eGFR). Continuous data were discretized into 3 bins using the R2-GenOpt algorithm and the Maximum Weight Spanning Tree algorithm was used for unsupervised learning of the network. Under these conditions, all parameters were connected in the generated network model. Aptamer-based screen. Urine samples for the initial aptamer-based screen were obtained from the Renal Clinic of UT Southwestern Medical Center; these samples consisted of 7 patients with active renal disease and SLE (rSLEDAI > 0), 8 patients with SLE but no active renal disease (rSLEDAI = 0, SLEDAI ≤6), and 8 healthy controls. Urine samples were morning midstream collections, collected, aliquoted, and stored frozen till the time of assay. All samples were clariﬁed by centrifugation before use. These samples were screened using an aptamer-based screening plat- form pioneered by Somalogic10. This assay uses aptamer–protein interactions to detect proteins within a sample. In the assay, aptamer-coated streptavidin beads are ﬁrst added to the sample to allow the aptamers to bind to the proteins. Next, the bound proteins are biotinylated, and the aptamer–protein complexes are cleaved from the streptavidin beads. These aptamer–protein complexes are then conjugated to a second streptavidin bead, and aptamers are separated from the proteins. The aptamers are then collected from the sample and quantitated by hybridization to a DNA microarray. The ﬁnal output is the relative ﬂuorescence unit (RFU) for each protein; these RFU values were then normalized to urinary creatinine and statis- tically analyzed to determine which proteins were increased in patients with active LN or SLE. Methods P ti t Patients, sample collection and sample preparation. Patients were recruited from the University of Texas Southwestern Medical Center’s Renal Clinic, Dallas, TX, Johns Hopkins University School of Medicine, Baltimore, MD or Tuen Mun Hospital, Hong Kong, China. All patients gave informed consent, and this study was approved by the institutional review board of Johns Hopkins University School of Medicine, UT Southwestern Medical Center, Tuen Mun Hospital, and the University of Houston. Patients who had a known history of lupus nephritis were included, as well as patients with SLE but no history of lupus nephritis. Patients with renal failure and pediatric patients were excluded from this study. Clean-catch midstream urine samples were collected in sterile containers and either placed on ice or refrigerated within 1 h of sample collection. The samples were then aliquoted and stored at −80 °C. At each sample collection visit, the patients were assessed by the attending physician, and the following data were obtained: SLEDAI (Systemic Lupus Erythematosus Disease Activity Index), rSLEDAI (renal SLEDAI), PGA, weight, blood pressure, complete blood count, platelets, erythrocyte sedimentation rate, creatinine, cholesterol, C3, C4, anti- dsDNA, anti-cardiolipin, urinalysis, and urine protein/Cr ratio. For all patients, the hybrid SLEDAI was used, where proteinuria was scored if >0.5 g/24 h. The rSLEDAI summates the renal components of the SLEDAI, including hematuria (>5 red blood cells/high-power ﬁeld), pyuria (>5 white blood cells/high-power ﬁeld), proteinuria (>0.5 g/24 h), and urinary casts. Active LN was deﬁned as biopsy- proven LN with rSLEDAI > 0. None of the active LN patients in this study had isolated hematuria or pyuria. Heatmap, cluster analysis. Data from the aptamer-based screening assay were used to generate heatmaps that cluster proteins with similar expression patterns together. There were two heatmaps generated for this analysis; one heatmap focuses on proteins that were signiﬁcantly elevated in active LN when compared to inactive LN, and the second focuses on proteins that were signiﬁcantly elevated in SLE when compared to healthy controls. For both heatmaps, proteins were clas- siﬁed as signiﬁcantly elevated if they had a p-value of <0.05 and a fold-change >2. The data from each group was imported into MATLAB for clustering analysis and heatmap generation. For clustering, proteins were clustered in an unsupervised manner based on Euclidean distance with a maximum cluster size of 20. Random forest classiﬁcation and Bayesian network analysis. ARTICLE For these 4 patients, the average time interval between the renal biopsy and urine procurement was RNAseq analysis of renal tissue from LN. Publically available single-cell RNAseq data from patients with biopsy-proven LN and healthy controls was obtained from Immport using accession numbers SDY997 EXP15077 and from the NCBI Short- Read Archive (SRA) under the accession number PRJNA37999263. For both datasets, post quality control expression matrices contained both skin and kidney cells and were subsetted to only include kidney cells for downstream analysis, yielding 1624 cells from 21 patients and 3 healthy controls1 and 363 cells from 10 patients64. Datasets were combined using canonical correlation analysis using the Seurat package for R as previously described65. Graph-based clustering and tSNE was performed on the kidney single-cell proﬁles using the Seurat package for R66. Principal component analysis yielding 12 principal components was used to derive the clusters. Cluster identity was assigned by comparing differentially expressed genes between the clusters to canonical markers. Feature plots were also created using the Seurat package for R67. Gene expression comparisons between LN and healthy control tubular cells were performed by ﬁrst creating a per-patient tubular cell proﬁle and then using the Student’s t-test. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15986-3 0.808), but baseline levels of BFL-1 are much higher in African- American patients, making it unreliable as a biomarker among these patients (p > 0.05, Mann–Whitney U-test). Hemopexin, expressed by hepatocytes, and induced by several inﬂammatory factors exhibits an anti-inﬂammatory effect and can induce proteinuria54–56. Hemopexin has been implicated as a urinary biomarker for pediatric LN57, and is elevated in serum of SLE patients; however, it does not correlate with disease activity/ severity58. The data presented in this study indicate that urinary hemopexin may differentiate active LN from inactive LN regardless of patient demographics (p < 0.05, Mann–Whitney U- test; AUC > 0.7), but its potential use among Caucasian patients may be limited due to high background levels even among healthy individuals. Several aspects of this study could be improved upon. Sample sizes could certainly be expanded to uncover markers that are less discriminatory. Given that this study pursued the validation of only 27 urine proteins, a large number of additional proteins (Supplementary Table 1) await systematic validation in future studies. Since these studies were not performed with urine Among Asian patients, the most discriminatory urine proteins were ALCAM, VCAM-1, TFPI, and PF-4. TFPI (tissue factor pathway inhibitor), mainly produced by the endothelial cells and megakaryocytes, is the primary inhibitor of the initiation of blood coagulation59. It has been reported that urinary TFPI is elevated in active LN, correlating with rSLEDAI60,61. NATURE COMMUNICATIONS \| (2020) 11:2197 \| https://doi.org/10.1038/s41467-020-15986-3 \| www.nature.com/naturecommunications 10 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-15986-3 samples obtained at the time of renal biopsy, we were not able to ascertain the relationship between urine biomarkers and renal pathology features, with one exception. We have recently exam- ined the performance of one of the urine proteins examined here, VCAM-1, in patients from whom concurrent renal biopsies were available62. Importantly, urine VCAM-1 surpass C3/C4, anti- DNA, and proteinuria in predicting concurrent renal pathology changes, including endocapillary proliferation, glomerular leu- kocyte inﬁltration, ﬁbrinoid necrosis, cellular crescents, and interstitial inﬂammation, all of which are manifestations of renal pathology activity62. Clearly, similar types of analyses need to be executed to assess the biomarker potential of the other proteins described in this manuscript, in predicting concurrent renal pathology. In addition, a longitudinal study is warranted to investigate how these molecules relate to disease pathology and progression over time. Frequently timed collections would be needed in order to identify urine proteins that may herald impending renal ﬂares. Finally, these ﬁndings call for mechanistic studies that investigate the pathogenic roles of ALCAM, PF-4, properdin, VCAM-1, and sE-selectin in mediating LN. (urine protein/creatinine <0.5 or 24-hour urine protein <0.5). For these 4 patients, the average time interval between the renal biopsy and urine procurement was 16 months. The second validation cohort included 80 inactive SLE patients, 80 active LN patients, 67 active non-renal lupus patients and 53 healthy controls with Asian ethnicity. Potential biomarkers were validated using commercially available ELISA assays, following manufacturer instructions, in a blinded fashion, where the operator was unaware of the disease group, before data analysis. The vendors and dilutions used are summarized in Supplementary Data. The absolute levels of urine proteins were determined using standard curves run on each ELISA plate, and normalized by urine creatinine. The degree of correlation between the aptamer screening platform and the ELISA-validation platform are presented in Supple- mentary Data. Brieﬂy, diluted urine samples were added in pre-coated 96-well microplates. After sample incubation, detection antibodies were added, followed by streptavidin-HRP, and substrate. A microplate reader (ELX808 from BioTek Instruments, Winooski, VT) was used to read the optical density at 450 nm. The optimal urine concentration was determined based on a standard curve derived for each molecule, and this is detailed in Supplementary Data. Inter-assay and inter- day variability in these assays were negligible, as summarized in Supplementary Fig. 4. (urine protein/creatinine <0.5 or 24-hour urine protein <0.5). NATURE COMMUNICATIONS \| (2020) 11:2197 \| https://doi.org/10.1038/s41467-020-15986-3 \| www.nature.com/naturecommunications References 31. Alpert, D. et al. Anti-heparin platelet factor 4 antibodies in systemic lupus erythaematosus are associated with IgM antiphospholipid antibodies and the antiphospholipid syndrome. Ann. Rheum. Dis. 67, 395–401 (2008). 1. Cameron, J. S. Lupus nephritis. J. Am. Soc. Nephrol. 10, 413–424 (1999). 2. Borchers, A. T. et al. 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All measurements were standardized before running LASSO. Ten-fold cross validation was carried out to select the tuning parameter lambda. The largest value of lambda such that error is within 1 standard error of the minimum was selected to ﬁt LASSO. Then active status was predicted based on the estimated LASSO coefﬁcients. ROC curve was plotted with area under the curve to demonstrate the discriminative power of the group of variables selected by LASSO. Race was purposely kept in the model throughout cross validation and LASSO ﬁtting process. We examined two models with LASSO: Model 1 includes all biomarkers and race, and Model 2 includes all biomarkers and race, age and prednisone administration. All the calculations were done in R 3.4.1. q-values were computed using the qvalue package. ROC curves were plotted and areas under the ROC curve were calculated using the pROC package. Cross validation was conducted and LASSO coefﬁcients were estimated using the glmnet package. 15. 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Lolyeva, M. et al. Novel role for ALCAM in lymphatic network formation and function. FASEB J. 27, 978–990 (2013). 23. Levesque, M. C., Heinly, C. S., Whichard, L. P. & Patel, D. D. Cytokine- regulated expression of activated leukocyte cell adhesion molecule (CD166) on monocyte-lineage cells and in rheumatoid arthritis synovium. Arthritis Rheum. 41, 2221–2229 (1998). Reporting summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article. 24. Wagner, M. et al. ALCAM and CD6—multiple sclerosis risk factors. J. Neuroimmunol. 276, 98–103 (2014). Code availability y 28. Stoeckle, M. Y. & Barker, K. A. Two burgeoning families of platelet factor 4- related proteins: mediators of the inﬂammatory response. New Biol. 2, 313–323 (1990). Various R packages were used, and no unique code was written for this work. Received: 15 May 2019; Accepted: 2 April 2020; Received: 15 May 2019; Accepted: 2 April 2020; 29. Deuel, T. F., Senior, R. 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Biomarker group comparisons were analyzed using the Mann–Whitney U-test as several datasets were not normally distributed. Statistical p-values and q-values (p-values adjusted for false discovery rate, for multiple testing correction) were computed for each biomarker. The Spearman method was used for the correlation analysis, and the Kruskal–Wallis test was used for multiple comparisons. Cross-sectional validation study using ELISA. For the primary cross-sectional study, 95 subjects with African-American and Caucasian ethnicities were included, comprised of 47 inactive SLE patients, 27 active LN patients and 21 healthy con- trols. These subjects were drawn from two medical centers: Johns Hopkins Uni- versity School of Medicine, Baltimore, MD and UT Southwestern Medical Center, Dallas, TX. Out of the 27 active LN patients, 23 had proteinuria. 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Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. y p y 59. Wood, J. P., Ellery, P. E., Maroney, S. A. & Mast, A. E. Biology of tissue factor pathway inhibitor. Blood 123, 2934–2943 (2014). y 60. 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Single cell RNA sequencing to dissect the molecular heterogeneity in lupus nephritis. Competing interests 51. D’Souza, B., Rowe, M. & Walls, D. The bﬂ-1 gene is transcriptionally upregulated by the Epstein-Barr virus LMP1, and its expression promotes the survival of a Burkitt’s lymphoma cell line. J. Virol. 74, 6652–6658 (2000). The authors declare no competing interests. © The Author(s) 2020 Reprints and permission information is available at http://www.nature.com/reprints JCI Insight 2, e93009 (2017). © The Author(s) 2020 13 NATURE COMMUNICATIONS \| (2020) 11:2197 \| https://doi.org/10.1038/s41467-020-15986-3 \| www.nature.com/naturecommunications
https://openalex.org/W3089527954	https://ria.ua.pt/bitstream/10773/31601/1/RodriguesAgraRequejoDelage-INFORMS2021-PostPrint.pdf	English	null	Lagrangian Duality for Robust Problems with Decomposable Functions: The Case of a Robust Inventory Problem	INFORMS journal on computing	2,020	cc-by	19,941	Cite as: F. Rodrigues, A. Agra, C. Requejo, E. Delage. Lagrangian duality for robust problems with de- composable functions: the case of a robust inventory problem. INFORMS Journal on Computing, 33 (2), Pages 419-835, 2021. [doi: 10.1287/ijoc.2020.0978] Abstract We consider a class of min-max robust problems in which the functions that need to be robustiﬁed can be decomposed as the sum of arbitrary functions. This class of problems includes many practical problems such as the lot-sizing problem under demand uncertainty. By considering a Lagrangian relaxation of the uncertainty set we derive a tractable ap- proximation, called the dual Lagrangian approach, that we relate with both the classical dualization approximation approach and an exact approach. Moreover we show that the dual Lagrangian approach coincides with the aﬃne decision rule approximation approach. The dual Lagrangian approach is applied to a lot-sizing problem, where demands are assumed to be uncertain and to belong to the uncertainty set with a budget constraint for each time period. Using the insights provided by the interpretation of the Lagrangian multipliers as penal- ties in the proposed approach, two heuristic strategies, a new guided iterated local search heuristic and a subgradient optimization method, are designed to solve more complex lot- sizing problems where additional practical aspects, such as setup costs, are considered. Computational results show the eﬃciency of the proposed heuristics which provide a good compromise between the quality of the robust solutions and the running time required in their computation. Keywords:Lagrangian relaxation; robust optimization; lot-sizing; demand uncertainty; aﬃne approximation; budgeted uncertainty polytope Lagrangian duality for robust problems with decomposable functions: the case of a robust inventory problem Filipe Rodrigues, Agostinho Agra, Cristina Requejo Department of Mathematics, University of Aveiro, Portugal {fmgrodrigues, aagra, crequejo}@ua.pt Erick Delage Department of Decision Sciences, HEC Montr´eal, Montreal, Qc, H3T 2A7, Canada Group for Research in Decision Analysis (GERAD), Montreal, Qc, H3T 1J4, Canada erick.delage@hec.ca Erick Delage 1 Introduction Dealing with uncertainty is very important when solving practical problems where some decisions need to be taken before the real data is revealed. This is the case of inventory man- agement problems where some decisions, such as the quantities to be produced or ordered, need to be taken without knowing the exact demands. A recent and popular approach to deal with 1 such uncertain optimization problems is Robust Optimization (RO). RO was ﬁrst introduced by Soyster (1973) who proposed a model for linear optimization where constraints had to be satisﬁed for all possible data values. Ben-Tal and Nemirovski (1999), El-Ghaoui and Lebret (1997), Bertsimas and Sim (2003, 2004) propose computationally tractable approaches to handle uncertainty and avoid excessive conservatism. For a recent paper on a less conservative variant of RO see Roos and den Hertog (2017). For general reviews on RO see Ben-Tal et al. (2009) and Bertsimas et al. (2011). Although current research on RO is being very useful and diﬀerent approaches have been proposed, there is a large gap in the research devoted to applying those approaches to complex mixed-integer problems. This is the case of many practical production planning problems with demand uncertainty, which motivated our work. While deterministic production planning prob- lems have been extensively studied both from a practical and a theoretical viewpoint (Pochet and Wolsey 2006), robust applications are still scarce. Two seminal works on robust inventory problems consists of i) the study of robust basestock levels, by Bienstock and ¨Ozbay (2008), where a decomposition approach to solve the true min-max problem to optimality is proposed (henceforward denoted by BO approach), and ii) the dualization approach introduced by Bertsi- mas and Thiele (2006) (henceforward denoted by BT approach) to inventory problems adapted from the general approach proposed by Bertsimas and Sim (2004). The two approaches have been applied to more complex problems. The decomposition approach for the min-max prob- lem using the budget polytope is investigated by Agra et al. (2016b) for a larger class of robust optimization problems where the ﬁrst-stage decisions can be represented by a permutation. The general decomposition procedure, regarded as row-column generation, is described for general robust optimization problems by Zeng and Zhao (2013). The BO approach is also used to solve more complex inventory problems, for example, the robust maritime inventory problem (Agra et al. 1 Introduction 2018a), and a production and inventory problem with the option of remanufacturing (At- tila et al. 2017). The dualization approach is also very popular since it often leads to tractable models. Wei et al. (2011) extended the results presented by Bertsimas and Thiele (2006) to a production and inventory problem with remanufacturing, where uncertainty is considered on returns and demands. For the application of the dualization approach to a robust inventory routing problem see Solyali et al. (2012). Solving the true min-max problem to optimality, using for instance a decomposition algo- rithm, can be impractical for many inventory problems, while the dualization approach may produce solutions which are too conservative. In order to circumvent both the diﬃculty of solving the min-max problem and the conservativeness of the dualization approach, other ap- proaches, such as the use of aﬃne decision rules (Ben-Tal et al. 2004, Chen and Zhang 2009) have been proposed. The aﬃne decision rules often lead to less conservative solutions than the ones obtained with the dualization approach, and in some cases they can lead to optimal solutions, see Bertsimas et al. (2011), Bertsimas and Goyal (2012), Kuhn et al. (2011), Iancu et al. (2013). Furthermore, for special uncertainty sets the use of aﬃne decision rules leads to computationally tractable aﬃnely adjustable robust counterpart (AARC) models. In particular, when the uncertainty set is a polyhedron the resulting AARC model is linear, see Ben-Tal et al. (2004). Georghiou et al. (2019) propose an approach that combines the aﬃne decision rules with the extreme point reformulation used in the exact decomposition methods. Tractable AARC models can also be obtained for lot-sizing problems when the demands are uncertain and belong to the uncertainty set with a budget constraint for each time period. However, when additional aspects are included in the lot-sizing problems, such tractable models can become computationally hard to solve even for small size instances. The results in this 2 paper express how diﬃcult can be to solve the AARC model when setup costs are considered in the basic lot-sizing problem. Such results justify the need for developing simpler tractable models as well as the use of approximation heuristic schemes. For a survey on adjustable robust optimization see Yanikoglu et al. (2018). 1 Introduction For a deeper discussion of other conservative approximations for the min-max problem obtained through relaxations of the uncertainty set we refer to Ardestani-Jaafari and Delage (2016) and Gorissen and den Hertog (2013). For many practical production planning and inventory management problems some data, such as demands, are not known in advance, and several decisions need to be taken before the data is revealed. Frequently, such decisions are taken before the start of the planning horizon and are not adjustable to the data when it is revealed. That is the case of decisions such as the amount of each item to produce in each time period, when complex aspects such as setups, sequence dependent changeovers, etc. are present (Pochet and Wolsey 2006). Adjusting the production to the known demands can imply new setups, creating diﬀerent sequences of products that may not be implementable. Another example in inventory management occurs in maritime transportation, where the distribution must be planned in advance and can hardly be adjusted to the demands given the long transportation times. Motivated by such applications we focus on robust problems in which the functions to be robustiﬁed can be decomposed as the sum of arbitrary functions. This class of problems was also investigated by Delage et al. (2018). The authors proposed a new robust formulation for generic uncertainty sets where it is assumed that the functions to be robustiﬁed are decomposed into the sum of functions, each one involving a diﬀerent nonadjustable variable, which is not the case we consider in this paper. C t ib ti Contributions In this paper, for a class of RO problems with decomposable functions, we propose a refor- mulation of the inner maximization subproblem occurring in a min-max model, known as ad- versarial problem. This reformulation starts by creating copies of both the uncertain variables and the uncertainty set in a way that the uncertainty set becomes constraint-wise independent. Further, a set of additional constraints is imposed enforcing that all the copies take identical values. By relaxing those constraints in the usual Lagrangian way, we obtain a mixed integer linear model, called Lagrangian dual model, that permits to directly relate the min-max ap- proach with the dualization approach (obtained when such constraints are ignored). With the obtained model, it is possible to derive eﬃcient heuristic approximation schemes that use the information from the Lagrangian multipliers to obtain solutions with lower true cost. Our main contributions are the following: Our main contributions are the following: 1. Exploit the Lagrangian relaxation of the uncertainty set to obtain a tractable model for a class of RO min-max problems in which the function to be robustiﬁed is decomposable in the sum of the maximum of aﬃne functions. 2. Provide a better theoretical understanding of the relations between several approaches for RO problems with decomposable functions. In particular we show that our Lagrangian dual model coincides with the AARC model and that the classical dualization approach results from the Lagrangian dual approach with all the Lagrangian multipliers null. 3. Provide computational results for the lot-sizing problem with setups showing the impact of the setup costs on the several approaches considered. In particular, when the setup costs increase, the quality of the solutions obtained by the BT approach deteriorates rapidly. This behaviour was not observed when using the proposed Lagrangian dual model. For large setup costs, the BT approach provides a bound that is up to 28% larger than the 3 optimal solution provided by the BO approach, while an optimal choice of multipliers can reduce it to near 6%. A similar reduction on the gap can be quickly achieved by solving the Lagrangian dual model with the multipliers ﬁxed to their optimal value in the linear relaxation. 4. Design eﬃcient heuristic schemes. We propose a new Guided Iterated Local Search heuris- tic and a Subgradient Optimization method that explicitly uses the interpretation of the Lagrangian multipliers as penalties. Contributions Comparing with other heuristics, for large size in- stances, the Subgradient Optimization method runs in a shorter time, and ﬁnds solutions with true costs that are i) strictly better for 91.8% of the instances used and ii) up to 18.4% better than those obtained by the BT approach. The paper is organized as follows. In Section 2 a dual Lagrangian approach is presented for RO problems with decomposable functions and its relation with the known approaches is established. The dual Lagrangian approach is applied to the robust inventory problem in Section 3. Heuristics based on the interpretation of the Lagrangian multipliers, including a new Guided Iterated Local Search heuristic and a Subgradient Optimization method are also presented in Section 3. Computational tests are reported in Section 4 and ﬁnal conclusions are given in Section 5. 2 Lagrangian duality for RO problems with decomposable functions Consider the min-max robust model R∗= min u∈U R(u) R∗= min u∈U R(u) with R(u) = g(u) + max ξ∈Ω X t∈T ft(u, ξ) where U is a feasible set, Ω⊆Rn is some compact uncertainty set, T = {1, . . . , n} and each ft : U × Ω→R is an arbitrary continuous function. Variables u represent non-adjustable decisions. The decision maker chooses a vector u while an adversary determines the uncertain vector ξ ∈Ωthat is most unfavorable to the decision u ∈U. Problem R(u) is known as the adversarial problem (Bienstock and ¨Ozbay 2008) and it calculates what is called the true cost for the vector u. Problem R∗can be rewritten as a two-stage robust problem by using adjustable variables θt, such that θt(ξ) : Ω→R, t ∈T, as follows. R∗= min u,θ(·) max ξ∈Ωg(u) + X t∈T θt(ξ) s.t. θt(ξ) ≥ft(u, ξ), ∀ξ ∈Ω, t ∈T, (2.1) u ∈U. (2.1) When particular functions θt(ξ) are considered, conservative approaches of R∗are obtained. In particular, the usual non-adjustable approach examines the case where θt(ξ) = θt, t ∈T, that is, When particular functions θt(ξ) are considered, conservative approaches of R∗are obtained. In particular, the usual non-adjustable approach examines the case where θt(ξ) = θt, t ∈T, that is, C∗= min u,θ g(u) + X t∈T θt s.t. θt ≥ft(u, ξ), ∀ξ ∈Ω, t ∈T, (2.2) u ∈U. (2.2) 4 4 It is known (Bienstock and ¨Ozbay 2008) that the gap between the two approaches can be large. However, there are cases with no gap between these approaches, that is, R∗= C∗(see Ben-Tal et al. (2004), Marandi and den Hertog (2018) and El Housni and Goyal (2018)). In particular, this equality holds when the uncertainty region Ωis the Cartesian product of sets Ξt (that is Ω= Ξ1 × · · · × Ξn), and each function ft(u, ξ) in constraints (2.2) is only aﬀected by the terms of ξ which lie in Ξt: R∗= min u∈U max ξ∈Ωg(u) + X t∈T ft(u, ξt) = min u∈U g(u) + X t∈T max ξt∈Ξt ft(u, ξt) = C∗. Here we explore this property to derive a Lagrangian relaxation of the adversarial problem. First, for each constraint t ∈T, create a list of copies {ζt}t∈T of the variables ξ and a list of respective uncertainty sets {Ωt}t∈T , such that each Ω⊆Ωt and ∩t∈T Ωt = Ω(e.g. 2 Lagrangian duality for RO problems with decomposable functions for simplicity one can use Ωt := Ω). We further impose a set of constraints enforcing that all the copies must be equal. This leads to the following exact reformulation of R(u): R(u) = g(u) + max ζ1,...,ζn X t∈T ft(u, ζt) s.t. ζt = ζ1, ∀t ∈{2, ..., n}, (2.3) ζt ∈Ωt, ∀t ∈T. (2.3) Remark 1. In relation to the set of equalities (2.3), it is important to notice that one could impose additional redundant equalities ζt = ζℓfor t ̸= ℓor replace them with other equivalent sets of equations. For all those cases, the process derived next still holds. Remark 1. In relation to the set of equalities (2.3), it is important to notice that one could impose additional redundant equalities ζt = ζℓfor t ̸= ℓor replace them with other equivalent sets of equations. For all those cases, the process derived next still holds. Attaching Lagrangian multipliers λt ∈Rn to each constraint (2.3) and dualizing these con- straints in the usual Lagrangian way, the following Lagrangian relaxation of R(u) is obtained LR(u, λ) := g(u) + max ζ1,...,ζn X t∈T ft(u, ζt) − n X t=2 (λt)⊤(ζt −ζ1) s.t. ζt ∈Ωt, ∀t ∀t ∈T. The multipliers λ penalize the use of diﬀerent uncertainty vectors for diﬀerent constraints. Imposing that λ1 := −Pn t=2 λt this model is equivalent to The multipliers λ penalize the use of diﬀerent uncertainty vectors for diﬀerent constraints. Imposing that λ1 := −Pn t=2 λt this model is equivalent to LR(u, λ) := g(u) + max ζ1,...,ζn X t∈T ft(u, ζt) − X t∈T (λt)⊤ζt s.t. ζt ∈Ωt, ∀t By using the epigraph reformulation, model LR(u, λ) can be written as follows. LR(u, λ) = g(u) + min θ1,...,θn X t∈T θt s.t. θt ≥ft(u, ζt) −(λt)⊤ζt, ∀ζt ∈Ωt, t ∈T, For a given u and λ, the minimization problem in LR(u, λ) can be separated into n inde- pendent subproblems, one for each t ∈T : For a given u and λ, the minimization problem in LR(u, λ) can be separated into n inde- pendent subproblems, one for each t ∈T : LRt(u, λt) = min θt θt s.t. θt ≥ft(u, ζt) −(λt)⊤ζt, ∀ζt ∈Ωt LRt(u, λt) = min θt θt min θt θt s.t. θt ≥ft(u, ζt) −(λt)⊤ζt, ∀ζt ∈Ωt s.t. θt ≥ft(u, ζt) −(λt)⊤ζt, ∀ζt ∈Ωt and LR(u, λ) = g(u) + Pn t=1 LRt(u, λt). 2 Lagrangian duality for RO problems with decomposable functions 5 The Lagrangian dual problem is DLR(u) = min λ LR(u, λ). Hence we have R(u) ≤DLR(u). Denoting by D the problem D = min u∈U DLR(u), the following relation holds R∗= min u∈U R(u) ≤min u∈U DLR(u) = D. The Lagrangian dual model D can be written as follows: The Lagrangian dual model D can be written as follows: D = min u,λ,θ g(u) + n X t=1 θt s.t. θt ≥ft(u, ζt) −(λt)⊤ζt, ∀ζt ∈Ωt, t ∈T, λ1 = − n X t=2 λt, u ∈U. Deﬁne D(λ) = min u∈U LR(u, λ). Hence, D = min λ D(λ), and for given multipliers λ, Deﬁne D(λ) = min u∈U LR(u, λ). Hence, D = min λ D(λ), and for given multipliers λ, D(λ) =min u,θ g(u) + n X t=1 θt s.t. θt ≥ft(u, ζt) −(λt)⊤ζt, ∀ζt ∈Ωt, t ∈T, u ∈U. Noticing that C∗is obtained with λ = 0 and Ωt = Ω, we have the following relation. Theorem 2. When Ωt = Ω, we have R∗≤D≤C∗. Noticing that C∗is obtained with λ = 0 and Ωt = Ω, we have the following relation. Theorem 2. When Ωt = Ω, we have R∗≤D≤C∗. Proof. Proof. R∗= min u∈U R(u) ≤min u∈U min λ LR(u, λ) = D = min λ D(λ) ≤D(0) ≤C∗. Following the work of Ben-Tal et al. (2015), one can start identifying conditions under which D becomes tractable. In particular, Section 2.3 will focus on the case where each ft(u, ξ) captures the maximum of a ﬁnite set of aﬃne functions and Ωt is a famous polyhedral budgeted set. Theorem 3. Given that g(u) is a convex function, that ft(u, ξ) is the maximum of K functions htk(u, ξ) convex in u and concave in ξ, and that both U and each Ωt are compact convex sets, then D can be reformulated as the following ﬁnite dimensional convex program: D = min u,λ,θ,v g(u) + n X t=1 θt (2.4) s.t. θt ≥δ∗(vtk\|Ωt) −htk∗(u, vtk + λt) ∀k = 1, . . . , K, t ∈T, λ1 = − n X t=2 λt, u ∈U , (2.4) D = min u,λ,θ,v where δ∗(v\|Ωt) := supξ∈Ωt v⊤ξ is the support function for Ωt, while htk∗(u, v) := infξ{v⊤ξ − htk(u, ξ)} is the partial concave conjugate of htk(u, ξ). 2 Lagrangian duality for RO problems with decomposable functions Moreover, if the epigraph of functions g(u), δ∗(v\|Ωt), and htk∗(u, v) are polyhedral representable and set U is a polyhedron, then prob- lem (2.4) can be reduced to a linear program. where δ∗(v\|Ωt) := supξ∈Ωt v⊤ξ is the support function for Ωt, while htk∗(u, v) := infξ{v⊤ξ − htk(u, ξ)} is the partial concave conjugate of htk(u, ξ). Moreover, if the epigraph of functions g(u), δ∗(v\|Ωt), and htk∗(u, v) are polyhedral representable and set U is a polyhedron, then prob- lem (2.4) can be reduced to a linear program. The proof of this theorem is given in Appendix A. 6 and it holds R∗≤AARC ≤C∗. Next we establish the main result of this section stating that the Lagrangian dual bound D obtained with Ωt = Ω, t ∈T coincides with the aﬃne approximation AARC. Theorem 4. When Ωt = Ω, t ∈T we have that AARC = D. Theorem 4. When Ωt = Ω, t ∈T we have that AARC = D. Proof. Proof. When Ωt = Ω, model D can be obtained from model AARC by replacing νt 0 with θt and νt with λt, and adding the constraint Pn t=1 νt = 0, hence AARC ≤D. To prove that AARC ≥D, we can show that given any feasible solution (ν0, ν) of AARC that achieves a ﬁnite objective value, it is possible to construct a feasible solution for D that achieves the same objective value. To do so, set m = maxξ∈Ω Pn t=1(νt)⊤ξ, λ1 = −Pn t=2 νt, and θ1 = ν1 0 + m, and for each t ∈{2, . . . , n} we deﬁne λt = νt, and θt = νt 0. Clearly, the objective value for D is the same as achieved in AARC: g(u) + n X t=1 νt 0 + max ξ∈Ω n X t=1 (νt)⊤ξ = g(u) + n X t=1 νt 0 + m = g(u) + n X t=1 θt, and the solution (θ, λ) is feasible for D, since for the constraint t = 1 (the remaining constraints are easily shown to be equivalent) and for each ξ ∈Ω, we have and the solution (θ, λ) is feasible for D, since for the constraint t = 1 (the remaining constraints are easily shown to be equivalent) and for each ξ ∈Ω, we have θ1 + (λ1)⊤ξ = ν1 0 + m − n X t=2 (νt)⊤ξ ≥ν1 0 + (ν1)⊤ξ ≥f1(u, ξ) where the ﬁrst inequality follows from the deﬁnition of m since m = max ξ∈Ω n X t=1 (λt)⊤ξ ≥ n X t=1 (λt)⊤ξ, ∀ξ ∈Ω⇒m − n X t=2 (λt)⊤ξ ≥(λ1)⊤ξ, ∀ξ ∈Ω, and the second inequality follows from the feasibility of (ν0, ν) in AARC. and the second inequality follows from the feasibility of (ν0, ν) in AARC. and the second inequality follows from the feasibility of (ν0, ν) in AARC. 2.1 Dualization versus aﬃne approximation It is known that less conservative approaches than C∗to approximate R∗can be obtained assuming that θt(ξ) is the aﬃne approximation θt(ξ) = νt 0 + (νt)⊤ξ, with νt 0 ∈R and νt ∈Rn. The resulting model, called Aﬃnely Adjustable Robust Counterpart (AARC) model (Gorissen and den Hertog 2013) is AARC = min u,ν0,ν,s g(u) + s s.t. s ≥ n X t=1 νt 0 + (νt)⊤ξ , ∀ξ ∈Ω, νt 0 + (νt)⊤ξ ≥ft(u, ξ), ∀ξ ∈Ω, t ∈T, u ∈U and it holds R∗≤AARC ≤C∗. and it holds R∗≤AARC ≤C∗. 2.2 Extensions and related problems Next we discuss three extensions of Theorem 4. Next we discuss three extensions of Theorem 4. Two-stage robust linear programs with box uncertainty: First, we extended the result to two-stage robust linear programs where the uncertainty set is the box uncertainty set. Let us consider a two-stage robust linear program of the form min u∈U max ξ∈Ξ min y g(u) + c⊤y (2.5) s.t. Au + By ≤Dξ. min u∈U max ξ∈Ξ min y g(u) + c⊤y (2.5) (2.5) s.t. Au + By ≤Dξ. s.t. Au + By ≤Dξ. 7 where A ∈Rm×q, B ∈Rm×p, D ∈Rm×n and Ξ = {ξ ≥0 \| ξ ≤d}. A ∈Rm×q, B ∈Rm×p, D ∈Rm×n and Ξ = {ξ ≥0 \| ξ ≤d}. By dualizing model (2.5) over the second-stage variables y and then over the uncertain variables ξ, Bertsimas and de Ruiter (2016) obtain the Dual Reformulation (DR) of model (2.5) DR = min u∈U max ζ∈Ωmin z≥0 g(u) + ζ⊤Au + d⊤z (2.6) s.t. z ≥−D⊤ζ. DR = min u∈U max ζ∈Ωmin z≥0 g(u) + ζ⊤Au + d⊤z (2.6) (2.6) where Ω= {ζ ≥0 \| c + B⊤ζ = 0}. Bertsimas and de Ruiter (2016) proved that when aﬃne decisions rules are applied to both the primal model (2.5) and the dual model (2.6), the optimal values of the resulting models coincide. The DR model (2.6) can easily be rewritten as an instance of the general model R∗deﬁned in Section 2 as follows: DR = min u∈U,θ(·) g(u) + n X t=1 θt(ζ) (2.7) s.t. θt(ζ) ≥max(0, −dtD⊤ :,tζ) + (1/n)ζ⊤Au ∀ζ ∈Ω, t ∈T , (2.7) where D:,t refers to the t-th column of D (see Zhen et al. (2018) for elimination of the adap- tive variables). We can therefore state the following result as a consequence of Theorem 2 in Bertsimas and de Ruiter (2016). where D:,t refers to the t-th column of D (see Zhen et al. (2018) for elimination of the adap- tive variables). We can therefore state the following result as a consequence of Theorem 2 in Bertsimas and de Ruiter (2016). Corollary 1. The optimal value of the Lagrangian dual model of the dual reformulation model (2.7) coincides with the optimal value of the AARC applied either to the primal formulation (2.5) (y(ξ) = y0 + V ξ) or to the dual formulation (2.6) (θt(ζ) = νt 0 + (νt)⊤ζ). Corollary 1. 2.2 Extensions and related problems The optimal value of the Lagrangian dual model of the dual reformulation model (2.7) coincides with the optimal value of the AARC applied either to the primal formulation (2.5) (y(ξ) = y0 + V ξ) or to the dual formulation (2.6) (θt(ζ) = νt 0 + (νt)⊤ζ). Quadratic decision rules: In a diﬀerent direction, a similar result to the one proved in Theorem 4 can be derived for the case of the quadratic decision rules. By deﬁning for each t ∈T, θt(ξ) = µt 0 +(µt)⊤ξ +ξ⊤Πtξ, with Πt ∈Rn×n, the obtained Quadratic Adjustable Robust Counterpart (QARC) model can be written as follows: QARC = min u,ν0,ν,Π,s g(u) + s s.t. s ≥ n X t=1 νt 0 + (νt)⊤ξ + ξ⊤Πtξ , ∀ξ ∈Ω, νt 0 + (νt)⊤ξ + ξ⊤Πtξ ≥ft(u, ξ), ∀ξ ∈Ω, t ∈T, u ∈U. s.t. s ≥ n X t=1 νt 0 + (νt)⊤ξ + ξ⊤Πtξ , ∀ξ ∈Ω, νt 0 + (νt)⊤ξ + ξ⊤Πtξ ≥ft(u, ξ), ∀ξ ∈Ω, t ∈T, u ∈U. Following the idea of the proposed Lagrangian approach, in addition to the set of equality constraints (2.3), let us consider in model R(u) the new set of equalities ξt(ξt)⊤= ξ1(ξ1)⊤, t = 2, . . . , n (2.8) (2.8) Attaching a matrix Λt ∈Rn×n of Lagrangian multipliers to each one of constraints (2.8), the Lagrangian Dual Quadratic (DQ) model becomes DQ = min u,λ,Λθ g(u) + n X t=1 θt s.t. θt ≥ft(u, ξt) −(λt)⊤ξt −(ξt)⊤Λtξt, ∀ξt ∈Ωt, t ∈T, λ1 = − n X t=2 λt, Λ1 = − n X t=2 Λt, u ∈U. u ∈U. We can once again establish a connection between this dual model and the use of quadratic decision rules. 8 heorem 5. When Ωt = Ω, for all t ∈T, we have that QARC=DQ. Theorem 5. When Ωt = Ω, for all t ∈T, we have that QARC=DQ. The proof of this result follows straightforwardly the steps of the proof of Theorem 3. In particular, for showing that QARC≥DQ, one can deﬁne m = max ξ∈Ω{Pn t=1(vt)⊤ξ + ξ⊤Πtξ}, λ1 = −Pn t=2 νt, Λ1 = −Pn t=2 Πt, θ1 = ν1 0 + m and, for all t ∈{2, . . . , n}, λt = νt, Λt = Πt, and θt = νt 0. 2.2 Extensions and related problems Distributionally robust optimization: Finally, the form of problem R∗is not limited to classical robust optimization but also emerges quite naturally when handling distributionally robust optimization (DRO) problems. In particular, consider the following general moment- based DRO problem: min u∈U sup F∈D(U) EF [ X t ft(u, ξ)] , min u∈U sup F∈D(U) EF [ X t ft(u, ξ)] , where ξ is now considered to be drawn from a distribution F that lies in an ambiguity set D deﬁned as D(U) := ( F ∃µ ∈U, PF (ξ ∈Ω) = 1 EF [hk(ξ)] = µk , ∀k = 1, . . . , K ) , with each hk(ξ) deﬁning a moment function, and U ⊂RK deﬁning the set of possible mo- ments. Exploiting a famous reformulation for moment problems, one can, under fairly general conditions, reformulate the inner supremum as follows: sup F∈D(U) EF [ X t ft(u, ξ)] = sup µ∈U sup F∈D({µ}) EF [ X t ft(u, ξ)] = sup µ∈U inf q sup ξ∈Ω X t ft(u, ξ) + K X k=1 qk(hk(ξ) −µk) = inf q sup µ∈U,ξ∈Ω X t ft(u, ξ) + K X k=1 qk(hk(ξ) −µk) . where the ﬁrst step assumes that strong semi-inﬁnite conic duality holds (see Shapiro (2001) for more details), followed by an application of Sion’s minimax theorem as long as U is convex and bounded. This gives rise to the following reformulation of the DRO problem: min u∈U,q sup µ∈U,ξ∈Ω X t ft(u, ξ) + K X k=1 qk(hk(ξ) −µk) , (2.9) (2.9) One can directly see that this DRO reformulation takes the form of R∗. Theorem 4 therefore applies to the DRO problem reformulation (2.9). One can directly see that this DRO reformulation takes the form of R∗. Theorem 4 therefore applies to the DRO problem reformulation (2.9). 2.3 Duality for the B&T budgeted set and for the maximum of aﬃne function Here we consider the particular case that motivated our work, where functions ft(u, ζt) are given by the maximum of aﬃne functions. We consider the uncertainty set used by Bertsimas and Thiele (2006): t Ω= {ξ ∈[−1, 1]n \| t X j=1 \|ξj\| ≤Γt, t ∈T}, Ω= {ξ ∈[−1, 1]n \| t X j=1 \|ξj\| ≤Γt, t ∈T}, where a budget constraint is imposed for each time period and we refer to this set as the B&T budgeted set. We assume that ft(u, ζt) = max k∈K ˆfk t (u, ζt) where K is a ﬁnite set of indexes and ˆfk t (u, ζt) = Lk t (u) + X j∈T atk j ζt j, ∀t ∈T, k ∈K, 9 where atk j ∈R and Lk t (u) : U →R is an aﬃne function for all k ∈K and j, t ∈T. When Ωt = Ω, the Lagrangian dual problem takes the form where atk j ∈R and Lk t (u) : U →R is an aﬃne function for all k ∈K and j, t ∈T. When Ωt = Ω, the Lagrangian dual problem takes the form min u,θ,λ g(u) + n X t=1 θt s.t. θt ≥Lk t (u) +  X j∈T atk j ζt j − X j∈T ζt jλt j  , ∀ζt ∈Ωt, k ∈K, t ∈T, λ1 = − n X t=2 λt, u ∈U. u ∈U. The uncertainty sets Ωt are not sets of linear constraints due to the presence of the absolute value function in constraints Pj ℓ=1 \|ζt ℓ\| ≤Γj, j ∈T. There are several ways of converting sets Ωt into equivalent sets of linear constraints (see Ben-Tal et al. (2009)). Preliminary tests using diﬀerent forms of conversion indicate that the best results are obtained by replacing ζt with ζt+ −ζt−such that (ζt+, ζt−) ∈¯Ωt and ¯Ωt = n (ζt+ j , ζt− j ) ∈Rt × Rt \| j X ℓ=1 (ζt+ ℓ + ζt− ℓ) ≤Γj, j ∈T, (2.10) ζt+ j + ζt− j ≤1, j ∈T, (2.11) ζt+ j , ζt− j ≥0, j ∈T } . (2.10) For practical reasons, to reduce the size of the resulting model, we assume henceforward that ζt j = 0 for j > t, which implies that constraints (2.10) and (2.11) can be disregarded for j > t. 2.3 Duality for the B&T budgeted set and for the maximum of aﬃne function θt ≥Lk t (u) + Ak t (λ), ∀t ∈T, k ∈K, u ∈U, s.t. θt ≥Lk t (u) + Ak t (λ), ∀t ∈T, k ∈K, where Ak t (λ) = P⌊Γt⌋ ℓ=1 αtk j(ℓ)(λ)+(Γt −⌊Γt⌋)αtk j(⌈Γt⌉)(λ) and αtk j(ℓ)(λ) is the ℓth largest value among the values αtk 1 (λ), . . . , αtk n (λ). The proof is similar to the proof of Proposition 1 in Bertsimas and Sim (2004) so it is omitted. Remark 8. All the approximation models for R∗presented in this section overestimate the cost associated with each ﬁrst-stage solution u. Hence, those approaches may lead to poor bounds based on good solutions and the following relation holds R∗= R(u∗) ≤R(uJ) ≤J, where uJ denotes the ﬁrst-stage solution obtained with model J, and J ∈{D, ˆD, ˆD(λ)}. 2.3 Duality for the B&T budgeted set and for the maximum of aﬃne function In particular, the following form is a natural choice (see Remark 6): ˆΩt = {ζt ∈[−1, 1]n \| t X j=1 \|ζt j\| ≤Γt}, ˆΩt = {ζt ∈[−1, 1]n \| t X j=1 \|ζt j\| ≤Γt}, 10 and leads to the following relation: and leads to the following relation: R∗≤AARC = D ≤ˆD ≤ˆD(0), R∗≤AARC = D ≤ˆD ≤ˆD(0), here ˆD and ˆD(λ) denote, respectively, D and D(λ) when ˆΩt is considered instead of Ωt. where ˆD and ˆD(λ) denote, respectively, D and D(λ) when ˆΩt is considered instead of Ωt. Observe that since constraints (2.10) in set ¯Ωt were disregarded for j > t, the version of the AARC approach equivalent to model D consists of using the aﬃne policy θt(ξ) = νt 0 + (νt)⊤ξ with νt j = 0 for j > t. Observe that since constraints (2.10) in set ¯Ωt were disregarded for j > t, the version of the AARC approach equivalent to model D consists of using the aﬃne policy θt(ξ) = νt 0 + (νt)⊤ξ with νt j = 0 for j > t. Remark 6. In the case of the B&T budgeted set, the set of constraints in the adversarial problem is given by is given by n (ζ1, . . . , ζn) ∈[−1, 1]n \| ζt = ζℓ, t, ℓ∈T, t ̸= ℓ, (2.17) ℓ X j=1 \|ζt j\| ≤Γt, t, ℓ∈T} (2.18) (2.17) (2.18) where constraints (2.18) for ℓ< t are redundant in the presence of constraints (2.17). However, when constraints (2.17) are relaxed, constraints (2.18) are no longer redundant for ℓ< t and the corresponding Lagrangian relaxation may diﬀer. where constraints (2.18) for ℓ< t are redundant in the presence of constraints (2.17). However, when constraints (2.17) are relaxed, constraints (2.18) are no longer redundant for ℓ< t and the corresponding Lagrangian relaxation may diﬀer. Proposition 7. Given any ﬁxed λ such that P t∈T λt = 0 and letting αtk j (λ) =\| atk j −λt j \| for all j ∈T, the value of D is bounded above by Proposition 7. Given any ﬁxed λ such that P t∈T λt = 0 and letting αtk j (λ) =\| atk j −λt j \| for all j ∈T, the value of D is bounded above by ˆD(λ) = min u g(u) + n X t=1 θt s.t. 2.3 Duality for the B&T budgeted set and for the maximum of aﬃne function By considering the above linear transformation, one can apply linear programming duality to reformulate each robust constraint and obtain the following linear program: For practical reasons, to reduce the size of the resulting model, we assume henceforward that ζt j = 0 for j > t, which implies that constraints (2.10) and (2.11) can be disregarded for j > t. By considering the above linear transformation, one can apply linear programming duality to reformulate each robust constraint and obtain the following linear program: D = min u,λ,θ,q,r g(u) + n X t=1 θt s.t. θt ≥Lk t (u) + t X j=1 qtk j Γj + t X j=1 rtk j , ∀t ∈T, k ∈K, (2.12) t X ℓ=j qtk ℓ+ rtk j ≥(−1)i(atk j −λt j), ∀j, t ∈T : j ≤t, k ∈K, i ∈{1, 2} (2.13) qtk j , rtk j ≥0, ∀j, t ∈T : j ≤t, k ∈K, (2.14) λ1 = − n X t=2 λt, (2.15) u ∈U. (2.16) (2.12) ∀j, t ∈T : j ≤t, k ∈K, (2.15) u ∈U. u ∈U. (2.16) where the dual variables qtk j and rtk j are associated with constraints (2.10) and (2.11), respec- tively. In practice, when T is reasonably small, it can be interesting to rewrite D in a lower dimensional space by eliminating variables rtk j . The resulting model is called projected model and is given in Appendix B. Alternatively, one might improve numerical eﬃciency, albeit at the price of precision, by using a simpler set ˆΩt, such that Ωt ⊆ˆΩt. In particular, the following form is a natural choice (see Remark 6): where the dual variables qtk j and rtk j are associated with constraints (2.10) and (2.11), respec- tively. In practice, when T is reasonably small, it can be interesting to rewrite D in a lower dimensional space by eliminating variables rtk j . The resulting model is called projected model and is given in Appendix B. Alternatively, one might improve numerical eﬃciency, albeit at the price of precision, by using a simpler set ˆΩt, such that Ωt ⊆ˆΩt. 3 The case of a robust inventory problem A new set of constraints is also considered ut ≤Ptyt, yt ∈{0, 1}, ∀t ∈T, (3.2) (3.2) ut ≤Ptyt, yt ∈{0, 1}, ∀t ∈T, where Pt is an upper bound on the production quantity at period t. In order to keep the notation easy, and since all the theoretical results presented hold for both cases (with and without setups) hereafter in the derivation of the theoretical results, we consider only the simplest case where no setup costs (and no setup variables) are considered. For the computational aspects (Sections 3.3 and 4) the more general case with setups is considered. 3 The case of a robust inventory problem In this section we particularize the results of the previous section for the case of the robust inventory problem that motivated this study and relate them with those known from the lit- erature. We consider lot-sizing problems deﬁned over a ﬁnite time horizon of n periods and deﬁne T = {1, . . . , n}. For each time period t ∈T, consider the unit holding cost ht, the unit backlogging cost bt and the unit production cost ct. The demand in time period t is given by dt. Deﬁne xt as the inventory at the beginning of period t (x1 is the initial inventory level). In 11 case xt is negative it indicates a shortage. Variables ut ≥0 indicate the quantity to produce in time period t. When the demand dt is known and ﬁxed we obtain a basic deterministic lot-sizing problem that can be modelled as follows: min u,x n X t=1 (ctut + max{htxt+1, −btxt+1}) s.t. xt+1 = x1 + t X j=1 (uj −dj), ∀t ∈T, ut ≥0, ∀t ∈T. ut ≥0, If xt+1 ≥0, then max{htxt+1, −btxt+1} gives the holding cost htxt+1, otherwise it gives the backlogging cost −btxt+1 at the end of time period t. Here we consider the case where the demands dt are deﬁned by dt := µt + δtzt, for each t ∈T, where µt and δt are the nominal demand and the maximum allowed deviation in period t, respectively, and the uncertain variables zt belong to the B&T budgeted set: Ω= {z ∈[−1, 1]n \| t X j=1 \|zj\| ≤Γt, t ∈T}. sume that 0 ≤Γ1 ≤Γ2 ≤· · · ≤Γn, Γt ≤Γt−1 + 1 and 1 < t ≤n. and assume that 0 ≤Γ1 ≤Γ2 ≤· · · ≤Γn, Γt ≤Γt−1 + 1 and 1 < t ≤n. and assume that 0 ≤Γ1 ≤Γ2 ≤· · · ≤Γn, Γt ≤Γt−1 + 1 and 1 < t ≤n. The results presented in this section can easily be extended to accommodate other practical aspects such as setup costs and/or other production constraints. In that case, the objective function is n min u,y n X t=1 (ctut + Styt + max{htxt+1, −btxt+1}) (3.1) (3.1) where yt is the setup variable indicating whether there is a production setup in time period t, and St is the setup cost in time period t. 3.1 The Bienstock and ¨Ozbay and the Bertsimas and Thiele approaches θt ≥ht  x1 + t X j=1 (uj −µj) + At  , ∀t ∈T, θt ≥−bt  x1 + t X j=1 (uj −µj) −At  , ∀t ∈T, ut ≥0, ∀t ∈T, where, for all t ∈T, At = max z    t X j=1 δjzj \| t X j=1 \|zj\| ≤Γt, zt ∈[−1, 1]   . Notice that this approach is based on the supersets ˆΩt, t ∈T. Notice that this approach is based on the supersets ˆΩt, t ∈T. Notice that this approach is based on the supersets ˆΩt, t ∈T. 3.1 The Bienstock and ¨Ozbay and the Bertsimas and Thiele approaches First, we review two of the main approaches for robust inventory problems: the decom- position approach introduced by Bienstock and ¨Ozbay (2008) to solve the problem written as a min-max problem (BO approach) and the dualization approach employed by Bertsimas and Thiele (2006) (BT approach). Bienstock and ¨Ozbay (2008) consider the robust inventory prob- lem as a min-max problem where, for a given production vector u, the demand dt is picked by an adversary problem. The min-max formulation is the following: R∗= min u≥0 R(u) 12 where R(u) = max x,z n X t=1 (ctut + max{htxt+1, −btxt+1}) (3.3) s.t. xt+1 = x1 + t X j=1 (uj −µj −δjzj), ∀t ∈T, t X j=1 \|zj\| ≤Γt, ∀t ∈T, zt ∈[−1, 1], ∀t ∈T. R(u) = max x,z n X t=1 (ctut + max{htxt+1, −btxt+1}) (3.3) (3.3) s.t. xt+1 = x1 + t X j=1 (uj −µj −δjzj), ∀t ∈T, t X j=1 \|zj\| ≤Γt, ∀t ∈T, zt ∈[−1, 1], ∀t ∈T. j zt ∈[−1, 1], ∀t ∈T. Problem (3.3) corresponds to the general adversarial problem introduced in Section 2.3 with K = {1, 2}, L1 t (u) = ht x1 + Pt j=1(uj −µj) , L2 t (u) = −bt x1 + Pt j=1(uj −µj) , g(u) = P t∈T ctut and U = Rn +. Bienstock and ¨Ozbay (2008) solve the min-max problem using a decomposition approach where, in the master problem, a production planning problem is solved for a subset of demand scenarios, while in the subproblem (adversarial problem) the worst-case scenario is found for the current production plan and added to the master problem. A FPTAS is proposed in Agra et al. (2016b) where a similar decomposition approach is used and the adversarial problem is solved by dynamic programming. The dualization approach introduced by Bertsimas and Sim (2004) was developed for the robust inventory problem by Bertsimas and Thiele (2006). The formulation is as follows ˆC∗= min u,z n X t=1 (ctut + θt) (3.4) s.t. θt ≥ht  x1 + t X j=1 (uj −µj) + At  , ∀t ∈T, θt ≥−bt  x1 + t X j=1 (uj −µj) −At  , ∀t ∈T, ut ≥0, ∀t ∈T, (3.4) t=1 s.t. 3.2 Lagrangian relaxation based approaches θt ≥L1 t (u) + ht   t X j=1 qt1 j Γj + t X j=1 rt1 j  , ∀t ∈T, (3.7) θt ≥L2 t (u) + bt   t X j=1 qt2 j Γj + t X j=1 rt2 j  , ∀t ∈T, (3.8) ctut + θt) (3.6) (3.6) D = min u,λ,θ,q,p,r,s (3.7) θt ≥L2 t (u) + bt   t X j=1 qt2 j Γj + t X j=1 rt2 j  , ∀t ∈T, (3.8) (3.8) qt1 t + rt1 t ≥(−1)i  δt + n X j=t+1 λj t ht  , ∀i ∈{1, 2}, t ∈T, (3.9) (3.9) t X ℓ=j qt1 ℓ+ rt1 j ≥(−1)i δj −λt j ht ! , ∀i ∈{1, 2}, j, t ∈T : j < t, (3.10) The proof of this theorem is given in Appendix C and it directly follows from the application of the process described in Section 2 to the robust inventory problem. By replacing the sets Ωt with the supersets ˆΩt we obtain model ˆD, which is used in the heuristics proposed in Section 3.3. Model ˆD corresponds to model D by setting variables qtk j = 0 for all k ∈{1, 2}, j, t ∈T : j < t. Remark 10. The Bertsimas and Thiele model (3.4) is a model having the form of model C∗ where Ωis replaced in each constraint (2.2) by ˆΩt. Hence, we have the relations D ≤C∗≤ˆC∗ and ˆD ≤ˆD(0) ≤ˆC∗. The projected version of model D in a lower dimension space can be written as follows D = min u,λ,θ,q,p X t∈T (ctut + θt) (3.14) D = min u,λ,θ,q,p X t∈T (ctut + θt) (3.14) s.t. θt ≥L1 t (u) + ht   t X j=1 qt1 j Γj − t X j=1 \|πt j\| t X ℓ=j qt1 ℓ+ t−1 X j=1 πt j δj −λt j ht ! + πt t  δt + n X j=t+1 λj t ht    , D = min u,λ,θ,q,p X t∈T (ctut + θt) (3.14) D min u,λ,θ,q,p X t∈T (ctut + θt) (3.14) s.t. θt ≥L1 t (u) + ht   t X j=1 qt1 j Γj − t X j=1 \|πt j\| t X ℓ=j qt1 ℓ+ t−1 X j=1 πt j δj −λt j ht ! 3.2 Lagrangian relaxation based approaches To derive the Lagrangian relaxation of the adversarial problem (3.3) consider, for each time period t ∈T, a copy vt j of each variable zj with j ≤t. That is, consider new variables vt j ∈[−1, 1] which account for the deviation in period j aﬀecting period t, t ≥j, and impose the constraints vt t = vj t , ∀j, t ∈T, t < j. vt t = vj t , ∀j, t ∈T, t < j. (3.5) (3.5) With this set of equalities, constraints Pt j=1 \|zj\| ≤Γt, t ∈T are replaced by constraints Pℓ j=1 \|vt j\| ≤Γℓ, 1 ≤ℓ≤t ≤n and the following approximation for the problem R∗is obtained. 13 Theorem 9. Model D deﬁned below is a tractable approximation for the problem R∗. D = min u,λ,θ,q,p,r,s X t∈T (ctut + θt) (3.6) s.t. θt ≥L1 t (u) + ht   t X j=1 qt1 j Γj + t X j=1 rt1 j  , ∀t ∈T, (3.7) θt ≥L2 t (u) + bt   t X j=1 qt2 j Γj + t X j=1 rt2 j  , ∀t ∈T, (3.8) qt1 t + rt1 t ≥(−1)i  δt + n X j=t+1 λj t ht  , ∀i ∈{1, 2}, t ∈T, (3.9) t X ℓ=j qt1 ℓ+ rt1 j ≥(−1)i δj −λt j ht ! , ∀i ∈{1, 2}, j, t ∈T : j < t, (3.10) qt2 t + rt2 t ≥(−1)i   n X j=t+1 λj t bt −δt  , ∀i ∈{1, 2}, t ∈T, (3.11) t X ℓ=j qt2 ℓ+ rt2 j ≥(−1)i λt j bt + δj ! , ∀i ∈{1, 2}, j, t ∈T : j < t, (3.12) qt1 j , rt1 j , qt2 j , rt2 j ≥0, ∀j, t ∈T : j < t. (3.13) D = min u,λ,θ,q,p,r,s X t∈T (ctut + θt) (3.6) s.t. 3.2 Lagrangian relaxation based approaches + πt t  δt + n X j=t+1 λj t ht    , ∀πt i ∈{−1, 0, 1}, 1 ≤i ≤t, t ∈T, (3.15) θt ≥L2 t (u) + bt   t X j=1 qt2 j Γj − t X j=1 \|πt j\| t X ℓ=j qt2 ℓ+ t−1 X j=1 πt j δj + λt j bt ! + πt t  δt − n X j=t+1 λj t bt    , ∀πt i ∈{−1, 0, 1}, 1 ≤i ≤t, t ∈T, (3.16) qt1 j , qt2 j ≥0, ∀j, t ∈T : j ≤t. (3.17) D min u,λ,θ,q,p X t∈T (ctut + θt) (3.14) s.t. θt ≥L1 t (u) + ht   t X j=1 qt1 j Γj − t X j=1 \|πt j\| t X ℓ=j qt1 ℓ+ t−1 X j=1 πt j δj −λt j ht ! + πt t  δt + n X j=t+1 λj t ht    , ∀πt i ∈{−1, 0, 1}, 1 ≤i ≤t, t ∈T, (3.15) s.t. θt ≥L1 t (u) + ht   t X j=1 qt1 j Γj − t X j=1 \|πt j\| t X ℓ=j qt1 ℓ+ t−1 X j=1 πt j δj −λt j ht ! + πt t  δt + n X j=t+1 λj t ht    ,  j=1 j=1 ℓ=j j=1 t !  j=t+1 t  ∀πt i ∈{−1, 0, 1}, 1 ≤i ≤t, t ∈T, (3.15) θt ≥L2 t (u) + bt   t X j=1 qt2 j Γj − t X j=1 \|πt j\| t X ℓ=j qt2 ℓ+ t−1 X j=1 πt j δj + λt j bt ! + πt t  δt − n X j=t+1 λj t bt    , ∀πt i ∈{−1, 0, 1}, 1 ≤i ≤t, t ∈T, (3.16) qt1 j , qt2 j ≥0, ∀j, t ∈T : j ≤t. (3.17) (3.16) qt1 j , qt2 j ≥0, (3.17) Remark 11. The BT model can be obtained through the projected model (3.14)–(3.17) by setting λ = 0, qt1 j = qt2 j = 0, j, t ∈T : j < t and qt1 t = qt2 t . Although the number of constraints (3.15) and (3.16) in the projected model increases exponentially with the number n of time periods, most of these inequalities are redundant. 3.2 Lagrangian relaxation based approaches 14 In fact, for each k ∈{1, . . . , t} such that Pt j=1 \|πt j\| = k, only one inequality (3.15) and one inequality (3.16) are non dominated for each t ∈T. The projected model can be solved using a Benders decomposition approach together with a separation algorithm for the constraints (3.15) and (3.16) that can easily work by inspection. However, preliminary results reported in Section 4.1 show that many of such constraints need to be included. The next proposition provides an eﬃcient way to solve model ˆD when multipliers are ﬁxed. Such result will be used in the next section to design eﬃcient heuristics to ﬁnd solutions with lower true cost. Proposition 12. For ﬁxed multipliers λ, ˆD(λ) is given as follows Proposition 12. For ﬁxed multipliers λ, ˆD(λ) is given as follows ˆD(λ) = min u n X t=1 (ctut + θt) s.t. θt ≥ht  x1 + t X j=1 (uj −µj)  + A1 t (λ), ∀t ∈T, θt ≥−bt  x1 + t X j=1 (uj −µj)  + A2 t (λ), ∀t ∈T, ut ≥0, ∀t ∈T, ut ≥0, ∀t ∈T, with with A1 t (λ) = ⌊Γt⌋ X ℓ=1 αt j(ℓ) + (Γt −⌊Γt⌋)αt j(⌈Γt⌉) and A2 t (λ) = ⌊Γt⌋ X ℓ=1 βt j(ℓ) + (Γt −⌊Γt⌋)βt j(⌈Γt⌉), where αt j =\| −htδj +λt j \| for 1 ≤j < t, αt t =\| −htδt −Pn j=t+1 λj t \|, βt j =\| btδj +λt j \| for 1 ≤j < t, and βt t =\| btδt −Pn j=t+1 λj t \|, where αt j(ℓ) is the ℓth largest value among αt 1, . . . , αt t and βt j(ℓ) is the ℓth largest value among βt 1, . . . , βt t. where αt j =\| −htδj +λt j \| for 1 ≤j < t, αt t =\| −htδt −Pn j=t+1 λj t \|, βt j =\| btδj +λt j \| for 1 ≤j < t, and βt t =\| btδt −Pn j=t+1 λj t \|, where αt j(ℓ) is the ℓth largest value among αt 1, . . . , αt t and βt j(ℓ) is the ℓth largest value among βt 1, . . . , βt t. The proof is a direct application of Proposition 7 so it will be omitted. 3.3 Heuristic schemes to improve the quality of solutions Among all the models considered in this paper, model D, corresponding to the AARC ap- proach, is the one that provides bounds closer to R∗. Another important concern is related with obtaining solutions u such that R(u) is close to R∗, that is, solutions with the best possible true cost. From a practical perspective, obtaining such solutions ¯u is more relevant than obtaining good bounds. Taking into account this more practical orientation, in this section, we develop iterative heuristic solution approaches, based on the interpretation of the Lagrangian multipliers as penalties associated with constraints violation, to obtain solutions with a lower true cost. With that purpose, for a given vector of multipliers, the value of the uncertain variables vj t , 1 ≤t ≤j ≤n, must be computed at each iteration. Since such computation can easily be done by inspection in model ˆD but not in model D, we use model ˆD rather than model D. Besides, there are two more reasons to use model ˆD instead of model D. First, model ˆD is computationally easier to solve when the multipliers are ﬁxed (see Proposition 12) and second, the results presented in the computational section for the instances solved to optimality suggest that there are no signiﬁcant diﬀerences between the true cost of the solutions provided by both models ˆD and D. Given that model ˆD (and also model D) is a pure linear model, one would expect to solve it to optimality even for large size instances. However, when other aspects are included, the 15 model can quickly become very large and the direct use of such model can be prohibitive. In order to take advantage of model ˆD we derive heuristic schemes that iteratively ﬁx the value of the new variables (multipliers) leading to easier subproblems. The proposed heuristics are tested using the inventory problem when production setup costs are considered, that is, when the objective function is given by (3.1) and the set of constraints (3.2) is added. 3.3.1 Guided Iterated Local Search algorithm The ﬁrst heuristic approach that we propose is called Guided Iterated Local Search (GILS). The GILS heuristic can easily be used to solve other complex problems and it is inspired in the classical Iterated Local Search (ILS) heuristic based on the local branching scheme proposed by Fischetti and Lodi (2003). ILS heuristics have performed well in complex inventory problems with uncertainty, such as the Maritime Inventory Routing problem (Agra et al. 2016a, 2018a) and the Production Inventory problem (Agra et al. 2018b). The main idea of the ILS heuristic is to restrict the search space of some integer variables (setup variables in our case) to a neighbourhood of a given solution. For a given positive integer parameter ρ, deﬁne the neighborhood N(¯y, ρ) of ¯y as the set of feasible solutions of the model ˆD satisfying the additional local branching constraint (see Fischetti and Lodi (2003)): X t∈T\|yt=0 yt + X t∈T\|yt=1 (1 −yt) ≤ρ. (3.18) (3.18) The neighborhood N(¯y, ρ) is the set of solutions that diﬀer from the current solution ¯y by a maximum number of ρ values of the yt variables. The linear constraint (3.18) limits to ρ the total number of binary variables yt ﬂipping their value with respect to the solution ¯y, either from 1 to 0 or from 0 to 1. The GILS heuristic is a modiﬁed version of the ILS heuristic and can be seen as an improved version in which the search space is even more reduced through the inclusion of new constraints on the Lagrangian multipliers. Motivated by the fact that the Lagrangian multipliers are used to penalize the deviations between the copies of the uncertain variables of the adversarial problem, we impose, at each iteration, two types of constraints to guide the value of the multipliers as follows. Type I: Constraint λj t ≤0 if vt t −vj t < 0 or constraint λj t ≥0 if vt t −vj t > 0. Type II: Constraint λj t ≤λ j t if vt t −vj t < 0 or constraint λj t ≥λ j t if vt t −vj t > 0. Algorithm 1 Guided Iterated Local Search Algorithm 1 Guided Iterated Local Search 1: Solve the linear relaxation of model ˆD 1: Solve the linear relaxation of model D 2: Solve the integer model ˆD, with the Lagrangian multipliers ﬁxed to their values in the linear relaxation of model ˆD 3: Save the solution y obtained in the previous step 4: repeat 5: for all t, j ∈T such that t ≤j do 6: Compute the value of the uncertainty variables vj t and add either constraints of type I or of type II to the model according to a predeﬁned rule 7: end for 8: Add constraint (3.18) to the model ˆD and solve it for γ seconds 9: Update the solution y 10: Remove all the constraints added 11: until the time limit of β seconds or a maximum number of iterations is reached 2: Solve the integer model ˆD, with the Lagrangian multipliers ﬁxed to their values in the linear relaxation of model ˆD 2: Solve the integer model ˆD, with the Lagrangian multipliers ﬁxed to their values in the linear relaxation of model ˆD 6: Compute the value of the uncertainty variables vj t and add either constraints of type I or of type II to the model according to a predeﬁned rule 7: end for 8: Add constraint (3.18) to the model ˆD and solve it for γ seconds 9 U d t th l ti 8: Add constraint (3.18) to the model ˆD and solve it for γ seconds 9: Update the solution y 10: Remove all the constraints added 11: until the time limit of β seconds or a maximum number of iterations is reached Steps 5 to 7 are used to guide the values of the Lagrangian multipliers as penalties for variable deviations. By ignoring Steps 5 to 7, Algorithm 1 becomes the classic ILS heuristic, that will be also tested in the computational section. In Step 6, several speciﬁc rules can be used to choose in each iteration the type of constraints added to the problem. Some of those rules will be discussed in the computational section. It is important to notice that the purpose of Steps 5 to 7 is not to accelerate the algorithm. Additionally, we may also expect to obtain worse bounds (based on the value of model ˆD) using the GILS heuristic than using the ILS heuristic since we are restricting the search space. Algorithm 1 Guided Iterated Local Search By penalizing the diﬀerences between the copies of the uncertain variables, we aim to force the choice of a neighbor solution based on an estimation of the cost closer to the true one. With this technique we expect to obtain better quality solutions (with true cost close to the cost of the optimal solution). 3.3.2 Subgradient Optimization method Since model ˆD is based on a Lagrangian relaxation, we adapt the subgradient method, frequently used to solve the dual problem of a Lagrangian relaxation, to solve model ˆD heuris- tically. The Subgradient Optimization (SO) method that we propose depends on two given a priori parameters, parameter ItLim and parameter φ, and uses the following additional func- tions: R(u) : computes the true cost of a given production policy u. Cdeviations(λ) : given a vector λ , computes the value ¯v of the deviation variables v. Cdeviations(λ) : given a vector λ , computes the value ¯v of the deviation variables v. 3.3.1 Guided Iterated Local Search algorithm At each iteration, the current value of the uncertain variables vj t and the current value of the L i l i li d d b j d λ j f ll 1 j i l At each iteration, the current value of the uncertain variables vj t and the current value of the Lagrangian multipliers are denoted by vj t and λ j t, for all 1 ≤t ≤j ≤n, respectively. Lagrangian multipliers are denoted by vj t and λ j t, for all 1 ≤t ≤j ≤n, respectively. To start the GILS heuristic, an initial solution is required. Such solution can be found by solving the model ˆD and ﬁxing the Lagrangian multipliers to their value in the linear relaxation of model ˆD. The full algorithm is described in Algorithm 1. 16 16 Algorithm 1 Guided Iterated Local Search Cdeviations(λ) : given a vector λ , computes the value ¯v of the deviation variables v. Algorithm 2 Subgradient Optimization method 1: Initialization: NoImprove := 0, Bestbound := ∞, Bestvalue := ∞ 2: Solve the linear relaxation of model ˆD and store the multipliers λ 3: Set LB equal to the objective function value of the linear relaxation 4: repeat 5: if NoImprove < ItLim then 6: Impose constraints λj t = λ j t and make all the remaining variables free 7: else 8: Impose constraints yt = yt and make the Lagrangian multipliers free 9: NoImprove ←0 10: end if 11: Solve the integer model ˆD with the imposed constraints 12: Set Bound equal to the objective function value of model ˆD 13: if Bound < Bestbound then 14: Update Bestbound 15: NoImprove ←0 16: end if 17: if R(u) < Bestvalue then 18: Update Bestvalue 19: NoImprove ←0 20: end if 21: Compute vj t, the value of the deviation variables vj t , for all t, j ∈T such that t ≤j using function Cdeviations(λ) j t j Algorithm 2 Subgradient Optimization method 1: Initialization: NoImprove := 0, Bestbound := ∞, Bestvalue := ∞ 2: Solve the linear relaxation of model ˆD and store the multipliers λ 3: Set LB equal to the objective function value of the linear relaxation 4: repeat 5: if NoImprove < ItLim then 6: Impose constraints λj t = λ j t and make all the remaining variables free 7: else 8: Impose constraints yt = yt and make the Lagrangian multipliers free 9: NoImprove ←0 10: end if 11: Solve the integer model ˆD with the imposed constraints 12: Set Bound equal to the objective function value of model ˆD 13: if Bound < Bestbound then 14: Update Bestbound 15: NoImprove ←0 16: end if 17: if R(u) < Bestvalue then 18: Update Bestvalue 19: NoImprove ←0 20: end if 21: Compute vj t, the value of the deviation variables vj t , for all t, j ∈T such that t ≤j using function Cdeviations(λ) 22: Compute the subgradient sj t := vt t −vj t for all t, j ∈T such that t < j 23: Compute norm := PT t=1 PT j=t+1(sj t)2 24: Deﬁne stepsize := φBound−LB norm 25: Update multipliers λ j t ←λ j t + stepsize × sj t 26: Remove all the added constraints 27: NoImprove ←NoImprove + 1 Algorithm 2 Subgradient Optimization method 21: Compute vj t, the value of the deviation variables vj t , for all t, j ∈T such that t ≤j using function Cdeviations(λ) 28: until A time limit of β minutes is reached 28: until A time limit of β minutes is reached Cdeviations(λ) : given a vector λ , computes the value ¯v of the deviation variables v. The SO method starts by solving the linear relaxation of model ˆD to obtain the initial values for the Lagrangian multipliers λ. The optimal value of the linear relaxation is used to deﬁne a lower bound to the problem. In the loop (step 4 to step 28 of the Algorithm 2), model ˆD is solved with updated information and the corresponding bound as well as the true cost of the production policy are computed and compared with the current best values. The value of the Lagrangian multipliers is updated in steps 21 to 25 according to the interpretation of the multipliers as penalties associated with the violation of constraints (3.5), taking into account the value of the variables vj t and vt t. At each iteration, model ˆD is solved with the Lagrangian multipliers ﬁxed and all the remaining variables free, however, whenever a limit number of iteration (ItLim) is reached 17 without a better bound or a better solution (solution with lower true cost) is obtained, the multipliers are left free and the setup variables are ﬁxed. This strategy is used to escape from local minimums and hence explore new feasible regions of the search space. 4 Computational experiments This section reports the computational experiments carried out to compare the BO approach, the BT approach, the Lagrangian Dual approach based on model ˆD (that is named by LD), and the approach based on model D. Since we have proved that this last approach coincides with the aﬃnely adjustable robust counterpart approach, hereafter, it is denoted by AARC approach. A model equivalent to the AARC can be obtained by considering the dual reformulated model proposed in Bertsimas and de Ruiter (2016) solved through aﬃne decision rules. However, preliminary results not reported here showed that it is not beneﬁcial in our case to use such reformulation, since the computational times associated with this model are higher than the ones obtained by using the AARC model. In Section 4.1 we report the results for medium size lot-sizing instances with 30 time periods, 18 for which all the optimal solutions can be obtained, while in Section 4.2 larger size instances with at most 100 time periods are considered. for which all the optimal solutions can be obtained, while in Section 4.2 larger size instances with at most 100 time periods are considered. Table 1 displays the total number of constraints and the total number of non integer vari- ables of model D with 30 and 100 time periods. The reported results correspond to the cases where the setup costs are either considered or not (column Setup), and the cases where the La- grangian multipliers are either free or ﬁxed (column #Multipliers). In the column #Variables, the numbers in parenthesis indicate the total number of integer variables in model D associ- ated with the use of setup costs. Notice that the number of constraints for model ˆD is exactly the same as for model D and the number of variables is approximately 2/3 of the number of variables in model D. Table 1: Total number of variables and constraints of model D. n Setup #Multipliers #Constraints #V ariables No ﬁx 2850 990 30 free 2850 1425 Yes ﬁx 2910 990 (+30) free 2910 1425 (+30) No ﬁx 31100 10500 100 free 31100 15550 Yes ﬁx 31300 10500 (+100) free 31300 15550 (+100) Table 1: Total number of variables and constraints of model D. n Setup #Multipliers #Constraints #V ariables The computational experiments use instances generated as follows. 4 Computational experiments For each time period t ∈T, the nominal demand µt and the maximum allowed deviation δt are randomly generated in [0, 50] and [0, 0.2µt], respectively. The maximum number of deviations in period t is computed using the relation Γt = Γt−1+τ, with τ varying in {0, 1} and Γ0 is assumed to be zero. The initial stock level at the producer, x1, is randomly generated between 0 and 30 and the production capacity Pt is constant and equal to Pn t=1 µt. The production, holding and backlog costs are the same as those used by Bertsimas and Thiele (2006), i.e., ct = 1, ht = 4, bt = 6, respectively, for all t ∈T. Throughout this section, we consider two variants of the robust inventory problem (with and without setup costs). The production setup costs occur in many practical inventory problems. However, the main goal of using instances with setup costs is to get harder instances, since the inclusion of integer setup variables results in a non linear model. In order to compute the true cost R(u) of a given solution u, preliminary tests were conducted using four approaches: the dynamic program proposed by Bienstock and ¨Ozbay (2008), the dynamic program proposed by Agra et al. (2016b), the mixed integer formulation with big- M constraints presented by Gorissen and den Hertog (2013), and the decomposition approach proposed by Bienstock and ¨Ozbay (2008). The dynamic program proposed by Bienstock and ¨Ozbay (2008) provided, in general, better results and solved all the adversarial problems in less than one second for instances with 100 time periods. Hereafter, for all the approaches considered in the computational experiments, the true cost of a solution is computed using the dynamic program proposed by Bienstock and ¨Ozbay (2008). All tests were run using a computer with an Intel Core i7-4750HQ 2.00 GHz processor and 8 GB of RAM, and were conducted using the Xpress-Optimizer 28.01.04 solver with the default options. 19 4.1 Computational experiments for medium size instances upper bound provided by this approach degrades rapidly as the setup cost increases. This is not the case of both LD and AARC approaches where the obtained upper bounds are close to the cost of the solution obtained by the BO approach, even when the setup cost increases. In fact, for large setup costs, the BT approach provides an optimal bound that is up to 28% larger than the true cost of the solutions provided by the BO approach while the gaps associated to both the LD and AARC approach are up to 6% and 3%, respectively, for all the setup costs upper bound provided by this approach degrades rapidly as the setup cost increases. This is not the case of both LD and AARC approaches where the obtained upper bounds are close to the cost of the solution obtained by the BO approach, even when the setup cost increases. In fact, for large setup costs, the BT approach provides an optimal bound that is up to 28% larger than the true cost of the solutions provided by the BO approach while the gaps associated to both the LD and AARC approach are up to 6% and 3%, respectively, for all the setup costs upper bound provided by this approach degrades rapidly as the setup cost increases. This is not the case of both LD and AARC approaches where the obtained upper bounds are close to the cost of the solution obtained by the BO approach, even when the setup cost increases. In fact, for large setup costs, the BT approach provides an optimal bound that is up to 28% larger than the true cost of the solutions provided by the BO approach while the gaps associated to both the LD and AARC approach are up to 6% and 3%, respectively, for all the setup costs upper bound provided by this approach degrades rapidly as the setup cost increases. This is not the case of both LD and AARC approaches where the obtained upper bounds are close to the cost of the solution obtained by the BO approach, even when the setup cost increases. 4.1 Computational experiments for medium size instances In this subsection all the reported results are based on instances with 30 time periods. Preliminary experiments on a set of 10 instances were conducted to compare the performance of model (3.6)–(3.13) against the projected model (3.14)–(3.17). The second model is solved through a Benders decomposition procedure, having a separation scheme for constraints (3.15) and (3.16). The average running time was 721 seconds and the required average number of iterations was 552. Using the model (3.6)–(3.13) the average running time was lower than 1 second. Note that model D could also be solved using the decomposition procedure proposed by Ardestani-Jaafari and Delage (2018). However preliminary experiments indicate that its perfor- mance is similar to the one observed when Benders decomposition is used to solve the projected model (3.14)–(3.17), since a large number of iterations is needed. Therefore, henceforward, we consider only model (3.6)–(3.13). Now we analyse the impact of the setup cost in the presented approaches. Figures 1 to 4 report average results obtained for 16 diﬀerent setup costs with values in {0, 10, . . . , 150}. For each setup cost, one hundred instances were randomly generated considering diﬀerent samples of the nominal demand values. All obtained results are presented through their average values, therefore Mann-Whitney hypothesis tests are applied to ﬁnd signiﬁcant diﬀerences between the approaches. A signiﬁcance level of 1% is used in all tests. Figure 1 displays the average cost of the solutions obtained by the BO approach (optimal value) and the average objective function values corresponding to the LD, AARC and BT approaches (which are upper bounds for the value of the BO solution). The points marked with squares (LD(uBT )) represent the average cost of the solutions obtained by the LD approach for the production policy obtained by the BT approach, i.e., after obtaining the solution of the BT approach, the value of the production variables ut, t ∈T, is ﬁxed and model ˆD is solved. The obtained results suggest that the BT approach is too conservative, since the quality of the Setup Cost 0 50 100 150 Objective Value 8500 9500 10500 11500 12500 BO LD AARC BT LD(uBT) Figure 1: Cost of the solutions obtained by the diﬀerent approaches in terms of the setup cost. Figure 1: Cost of the solutions obtained by the diﬀerent approaches in terms of the setup cost. tested. When comparing the displayed lines associated with LD(uBT ) and BT we observe that, in general, there is a gap (that is up to 6%) between the corresponding bounds. This means that the optimal value of the Lagrangian multipliers for the production policy obtained by the BT approach is usually diﬀerent from zero (as considered in the BT approach). Hence, a better choice of the Lagrangian multipliers can be used to improve the quality of the upper bound provided by the BT approach. A prevailing conclusion for all the setup costs tested is that the LD, the AARC and the BT approaches lead to solutions with average upper bounds signiﬁcantly higher than the optimal value provided by the BO approach. Further, the average upper bounds obtained by the BT approach are signiﬁcantly higher than the ones obtained by both the LD and the AARC approaches for setup costs greater than 10, and signiﬁcantly greater than the ones obtained by the LD(uBT ) approach for high setup costs (greater than 110). Figure 2 reports the average computational time in seconds required by each approach to ﬁnd the solution, in terms of the setup cost. Setup Cost 0 50 100 150 Seconds 0 10 20 30 40 50 60 70 80 BO LD AARC BT Figure 2: Average computational time associated to each approach in terms of the setup cost. re 2: Average computational time associated to each approach in terms of the setup cost The computational time of the BT approach is always lower than one second. It can be observed that the exact BO approach is on average twice as faster as the LD approach. The computational time required by the AARC approach is approximately twice the computational time required by the LD approach. The average time required by the BO approach to solve each master problem ranges from 0 to 12 seconds while the computational time required to solve each adversarial problem is always lower than one second. Figure 3 displays the average true cost of the production policy determined by the approaches LD (R(uLD)), AARC (R(uAARC)), BT (R(uBT )), and compare them with the cost of the optimal production policy obtained by the BO approach. Note that these values are not the upper bounds obtained by the LD, AARC and BT approaches directly. 4.1 Computational experiments for medium size instances In fact, for large setup costs, the BT approach provides an optimal bound that is up to 28% larger than the true cost of the solutions provided by the BO approach while the gaps associated to both the LD and AARC approach are up to 6% and 3%, respectively, for all the setup costs 20 tested. tested. They are the true costs obtained by solving the adversarial problem for each solution obtained with the indicated approach. The behavior of the true cost of the production policy obtained by the LD, AARC and BT approaches resembles the trend observed for the upper bounds. However, when the setup costs are not considered, the true cost of the production policy obtained by the BT approach is, in general, lower than the one obtained by both the LD and AARC approaches. It is interesting to note that the true cost of the solutions determined by both the LD and AARC approaches are very close. In fact, the Mann-Whitney hypothesis tests reveal that, in 21 Setup Cost 0 50 100 150 Production Policy Cost 8500 9500 10500 11500 BO R(uLD) R(uAARC) R(uBT) Figure 3: True average cost of the solution obtained by both the LD and the BT approaches compared with the cost of the optimal production policy obtained by the BO approach. BO R(uLD) R(uAARC) R(uBT) Production Policy Cost Figure 3: True average cost of the solution obtained by both the LD and the BT approaches compared with the cost of the optimal production policy obtained by the BO approach. terms of the true cost of the production policy, the diﬀerences between both approaches are not signiﬁcant. Moreover, the average true costs of the production policies determined by both LD and AARC approaches are not signiﬁcantly diﬀerent from the average costs of the optimal production policies. However, the average true cost of the production policies determined by the BT approach is signiﬁcantly greater than the one determined by the LD and the AARC approaches for setup costs greater than 30. A key conclusion from Figure 3 is that in the case where the setup costs are not considered, the true average cost from the solutions obtained using the BT approach may give a fair approx- imation on the optimal value. However, when setup costs are high the BT approach can give poor bounds and, beyond that, it can also produce bad solutions (with costs up to 16% larger than the optimal true costs). This may indicate that for more complex inventory problems the overestimation of costs obtained by the BT approach may lead to poor decisions. tested. Figure 4 displays the average number of production periods associated with the production policy determined by the BO, the LD, the AARC and the BT approaches. 22 Setup Cost 0 50 100 150 Number of Porduction Periods 5 10 15 20 25 30 BO LD AARC BT Figure 4: Average number of production periods in the production policies obtained by the diﬀerent approaches in terms of the setup cost. BO LD AARC BT Figure 4: Average number of production periods in the production policies obtained by the diﬀerent approaches in terms of the setup cost. This ﬁgure can help to explain the results displayed in Figures 1 and 3, since the average numbers of production periods in the LD, AARC and BO approaches are similar. Notice that even when the setup cost is high, the number of production periods in the BT approach remains high, which may be justiﬁed by the fact that the BT approach tends to overestimate the contribution of the inventory costs in the objective function. The diﬀerences between the average number of production periods in the LD, AARC and BO approaches are not signiﬁcant for any setup costs used while such diﬀerences between the BT and BO approaches are signiﬁcant for all the setup cost tested. We also analyse the performance of the LD, the AARC and the BT approaches regarding the maximum number of deviations Γn in the last time period. The obtained results are reported in Appendix D. Table 2: Average optimality gaps obtained with model D with a time limit of two hours. Setup Cost 50 150 450 750 Gap (%) 0.14 1.39 3.61 5.51 4.2 Computational experiments for large size instances In this section we report the computational results for large size instances with up to 100 time periods. For these instances the exact BO approach cannot be solved to optimality within a reasonable time limit. Preliminary results showed that even for a small number of scenarios the master problem cannot be solved within eight hours. Similar diﬃculties were observed for a related lot-sizing problem in Attila et al. (2017). Furthermore, when the setup costs increase, model D (the one used in the AARC approach) becomes computationally harder to solve to optimality. For the instances with 100 time periods and setup costs greater than 70 we are not able to solve model D within a time limit of eight hours. Table 2 reports the average optimality gaps obtained with model D over a set of 10 instances with 100 time periods considering a time limit of two hours, for four diﬀerent setup costs. 23 23 Setup Cost 0 50 100 150 Upper Bound 92000 96000 100000 104000 LD(λLR) AARC(λLR) BT Figure 5: Average upper bound values obtained by the LD and AARC approaches considering the optimal Lagrangian multipliers of the linear relaxation of models ˆD and D, respectively, for diﬀerent setup costs. LD(λLR) AARC(λLR) BT Upper Bound Figure 5: Average upper bound values obtained by the LD and AARC approaches considering the optimal Lagrangian multipliers of the linear relaxation of models ˆD and D, respectively, for diﬀerent setup costs. Table 2 shows that the instances become more diﬃcult to solve when the setup cost increases. Results not reported here allow us to conclude that the optimal solution of the LD approach can be obtained in less than 2 hours for problem instances with up to 55 time periods while for the AARC approach only problem instances with up to 40 time periods can be solved to optimality within 2 hours. Hence, the main goal of this section is to test heuristic approaches that can be used on large size inventory models to obtain tight upper bounds as well as good solutions (with true cost close to the optimal value). When the Lagrangian multipliers are ﬁxed, models D and ˆD can be quickly solved, even if setup costs are considered. In particular, model ˆD with the multipliers ﬁxed to zero, that corresponds to the BT approach, can be solved in less than 5 seconds. Tuning of the parameters We consider two variants of the ILS heuristic, one based on model ˆD and other based on model D, denoted by ILS ˆD and ILSD, respectively. Both heuristics correspond to Algorithm 1 described in Section 3.3.1 without steps 5 to 8. However, instead of imposing a time limit or a maximum number of iterations, the algorithm stops when no improvement in the objective function value is observed. In both heuristics, the parameter ρ was set to 2 since with this parameter for the instances with 100 time periods, almost all the problems arising in each iteration of the ILS heuristics were solved to optimality in less than 150 seconds (the time limit imposed in each iteration). For both the GILS heuristic and the SO method, a set of 20 randomly generated instances with 30 time periods was used to tune the values of the parameters. Since in the GILS heuristic we are imposing additional constraints on the Lagrangian multipliers, the value of the objective function in a given iteration can be worse than the one obtained in the previous iteration. So it does not make sense to stop the algorithm when there is no improvement in the objective function value. Hence, the stopping criteria for the GILS heuristic is deﬁned using the number of iterations (that is limited to 15). Three rules were tested to choose the type of constraints added to the problem in each iteration: i) add only constraints of type I; ii) add only constraints of type II and iii) successively add constraints of type I k times and then add constraints of type II k times (with k = 1, 2, 3). Taking into account both the upper bounds and the true cost of the solutions, the best results were obtained when the third rule was used with k = 2, so this is the strategy used henceforward. To compare the GILS with both variants of the ILS heuristics we use the same time limit in each iteration (150 seconds) and also ρ = 2. For the SO method, diﬀerent values {0.25, 0.5, 1, 1.5, 2} of φ and diﬀerent values {5, 10, 15, 20} of parameter ItLim were tested. The best results where obtained when the values φ = 1 and ItLim = 10 were used. The time limit imposed in the SO method is 600 seconds. 4.2 Computational experiments for large size instances An initial value for the Lagrangian multipliers can easily be obtained by solving the linear relaxation of models D and ˆD, respectively. Figure 5 displays, for each setup cost in {0, 10, ..., 150}, the average upper bound values over 100 randomly generated instances with 100 time periods obtained by both LD and AARC approaches when all the multipliers are ﬁxed to their values in the optimal linear relaxations of models ˆD and D, respectively. The average upper bound values obtained by the BT approach are also displayed. Figure 5 shows opposite behavior of both LD and AARC approaches, when the Lagrangian multipliers are ﬁxed to their values in the linear relaxation, comparing with the BT approach. While the gap between the lines associated with the AARC and the BT approaches tend to decrease as the setup cost increases, the gap between the lines associated with the LD and BT approaches tends to increase as the setup cost increases. The ﬁrst gap varies between 2.6% and 3.0% while the second varies between 0.7% and 6.4%. These results show that, when the value of the setup cost increases, tighter upper bounds can be obtained by considering the Lagrangian multipliers ﬁxed to their values in the linear relaxation of model ˆD in the LD approach instead of considering all the multipliers equal to zero (as in the case of the BT approach). Furthermore, the diﬀerence between the computational time required to compute the upper bounds in both cases corresponds to the computational time required to solve the linear relaxation of model ˆD, which is always lower than 7 seconds for all the tested instances. This means that, in general, for large size instances, a better bound 24 than the one obtained by the BT approach can be quickly obtained by considering the optimal multipliers of the linear relaxation of model ˆD. From the theoretical study we know that the upper bound corresponding to the optimal solution of the AARC approach is lower than or equal to the upper bound obtained by the LD approach. Moreover, the value of the linear relaxation is lower in the AARC approach than in the LD approach. 4.2 Computational experiments for large size instances Nevertheless, Figure 5 shows that when the multipliers are ﬁxed to their value in the linear relaxation, the upper bounds provided by the LD approach tend to be better than the ones obtained with the AARC approach, when the value of the setup cost increases. 4.2.1 Evaluation of the proposed heuristics In this section we analyse the performance of both the GILS heuristic and the SO method presented in Section 3.3. It is important to remind that these two heuristics were speciﬁcally designed to generate better solutions and not necessarily better bounds resulting from the objective function values of the considered models. As reference methods we use the heuristic that consists on solving the full model D with a time limit of one hour, and the ILS heuristic. The ﬁrst heuristic will be called Full Model heuristic (FM heuristic). Tuning of the parameters Comparing upper bounds and true costs Here we compare the performance of the heuristics in terms of the setup cost (for instances with 100 time periods) and also in terms of the number of periods (for instances with a setup cost equal to 150). Tables 3 and 4 present the average upper bounds obtained for each heuristic tested as well as the corresponding average computational time in seconds. Each line of the 25 tables reports average values obtained for a set of 10 instances. The best average upper bounds obtained for each set of instances are marked in bold. Furthermore, the numbers in parenthesis next to the bounds indicate the number of best bounds obtained by the corresponding heuristic. : Average upper bounds obtained for the heuristics tested for instances with 100 time and setup costs varying from 25 to 150. periods and setup costs varying from 25 to 150. FM ILSD ILS ˆ D GILS SO Setup Cost Bound Sec. Bound Sec. Bound Sec. Bound Sec. Bound Sec. 25 92365(2) 3600 92338(8) 1364 94583 655 94620 525 94710 600 50 92756(5) 3600 92776(5) 1264 94943 762 95001 718 95097 600 75 93178(2) 3600 93120(8) 1206 95269 781 95365 801 95889 600 100 93474(4) 3600 93445(6) 1295 95594 836 95770 917 95893 600 125 93733(7) 3600 93789(3) 1011 95891 736 96080 1051 96188 600 150 94155(5) 3600 94131(5) 1032 96183 786 96286 1089 96592 600 Table 4: Average upper bounds obtained for the heuristics tested for instances with setup cost equal to 150 and time periods varying from 20 to 100. FM ILSD ILS ˆ D GILS SO n Bound Sec. Bound Sec. Bound Sec. Bound Sec. Bound Sec. 20 5380(10) 18 5385(6) 8 5526(1) 4 5539(1) 13 5524(1) 600 40 16673(9) 2309 16728(3) 58 17259 23 17294 52 17231 600 60 37273(4) 3600 37287(6) 192 38191 116 38258 188 38246 600 80 59383(1) 3600 59261(9) 644 61425 425 61606 774 61548 600 100 94155(5) 3600 94131(5) 1032 96183 786 96286 1089 96592 600 e 4: Average upper bounds obtained for the heuristics tested for instances with setup cos l to 150 and time periods varying from 20 to 100. The results presented in Tables 3 and 4 reveal that best upper bounds are obtained with both the FM and the ILSD heuristics. Tuning of the parameters These results agree with what was stated in the theoretical study since the best upper bounds are obtained by the heuristics based on model D. All the instances with 20 time periods and almost all the instances with 40 time periods are solved to optimality by the FM heuristic . This justiﬁes that the best results for the instances with these time periods are obtained with the FM heuristic. However, when the number of periods increases, best upper bounds are in general obtained by the ILSD heuristic. In Tables 5 and 6 we compare the heuristics in terms of the true cost of the obtained solutions. As in the previous tables, the best average results are marked in bold and the number of best solutions (with the best true cost) appears in parenthesis. At each iteration of the ILS heuristics, GILS heuristic and SO method, the true cost of the current solution is obtained and the best obtained value is reported. In the FM heuristic the true cost of all integer solutions found during the Branch-and-Bound process is computed and the best true cost is reported. Table 5: Average true cost of the solutions obtained for the heuristics tested for instances with 100 time periods and setup costs varying from 25 to 150. Table 5: Average true cost of the solutions obtained for the heuristics tested for instanc 100 time periods and setup costs varying from 25 to 150. Table 5: Average true cost of the solutions obtained for the heuristics tested for instances with 100 time periods and setup costs varying from 25 to 150. Setup Cost FM ILSD ILS ˆ D GILS SO 25 91875 92016 92445 92343 90729(10) 50 92223(2) 92428 92732 92582 91004(8) 75 92567(1) 92736 93012 92666 91320(9) 100 92890 93089 93299 92768 91223(10) 125 93147 93414 93520 92929 91607(10) 150 93479 93725 93829 93203 91840(10) 26 Table 6: Average true cost of the solutions obtained for the heuristics tested for instances with setup cost equal to 150 and time periods varying from 20 to 100. Tuning of the parameters n FM ILSD ILS ˆ D GILS SO 20 5319(3) 5353(1) 5359(1) 5354(1) 5301(5) 40 16534(1) 16619 16661(1) 16521 16155(9) 60 36988 37118 37210 36971 36066(10) 80 59061 59060 59518 59038 57728(10) 100 93479 93725 93829 93203 91840(10) The results presented in Tables 5 and 6 clearly suggest that the best average true costs are in general obtained by the SO method. Only for 9 out of the 110 instances presented in these two tables the best solutions were not found by the SO method. Furthermore, the computational time of the SO method (600 seconds) is much lower than the one required by the remaining heuristics. The SO method allows us to obtain solutions with true costs that are, on average, 1.8% lower than the ones obtained with the FM heuristic (which is the heuristic closer to the AARC approach). Hence, among all the heuristic solutions tested, the SO method is the most eﬃcient heuristic to obtain good solutions (with low true costs). As expected, the upper bound values obtained by both ILS heuristics are better than the ones obtained by the GILS heuristic. However, in terms of the true cost of the obtained production policies, the best results are in general obtained using the GILS heuristic. In fact, among all the 60 instances with 100 time periods considered in Table 5, 45 of the best solutions were found by the GILS heuristic while 9 and 6 were found by the ILSD and the ILS ˆD heuristics, respectively. Among all the 50 instances with a setup cost equal to 150 considered in Table 6, 38 best solutions were found by the GILS heuristic while 9 and 3 were found by the ILSD and the ILS ˆD heuristics, respectively. Looking deeper to the SO method Since the true cost of the solutions obtained with the BT approach is much higher than those obtained by all the heuristics tested, such results were not reported the Tables 5 and 6. However, in Table 7 we report some gaps showing the improvements on the true cost of the solutions obtained by the SO method compared to the true cost of the solutions obtained by the BT approach. Columns 2 to 7 refer to the instances presented in Table 5, those with 100 time periods and setup costs varying between 25 and 150, while columns 8 to 12 refer to the instances presented in Table 6, the ones with a setup cost equal to 100 and time periods varying between 20 and 100. Remember that the SO method starts from the solution obtained with model ˆD with the multipliers ﬁxed to their value in the linear relaxation of such model. Hence, the line Initial Solution reports the average gaps associated with the true cost of the initial solution used in the SO method comparing with the true cost of the solution obtained by the BT approach. The line Best Solution reports the average gaps associated with the true cost of the best solution found by the SO method comparing with the true cost of the solution obtained by the BT approach. Table 7: Average gaps (in percentage) between the SO method and the BT approach in terms of the true cost of the solutions. f f Instances from Table 5 Instances from Table 6 Setup Cost/Time periods 25 50 75 100 125 150 20 40 60 80 100 Initial Solution 0.3 1.2 2.2 2.8 3.3 3.8 7.3 5.1 4.5 4.2 3.8 Best Solution 2.4 3.8 5.1 6.5 7.3 7.9 18.4 15.4 11.8 9.9 7.9 27 We observe in Table 7 that the gap between the SO method and the BT approach, in terms of the true cost of the solutions, increases as the setup cost increases and decreases as the number of periods increases. For the hardest instances, the ones with 100 time periods and setup cost equal to 150, the BT approach provides solutions with true costs that are 7.9% larger than those obtained by the SO method. Looking deeper to the SO method Finally, in order to compare the quality of the solutions generated by the SO method with those resulting from the AARC method solved to optimality, we report in Table 8 the average optimality gaps associated with both the SO method and the AARC approach in terms of the true cost of the solutions, for instances with n = {10, 20, 30, 40} time periods (those instances where the AARC method can be solved to optimality within reasonable amount of time). For each number n, 25 instances were used. The numbers in parenthesis next to the gaps indicate the number of best solutions obtained by the corresponding method. The average gaps, in percentage, were computed according to the formula: Gap = R(uJ) −R(u∗) R(u∗) × 100, where u∗is the optimal solution (obtained by the BO approach) and uJ is the solution obtained by approach J, with J = AARC or J = SO. Table 8 suggests that for the instances solved to optimality the best solutions are on average obtained by the SO method since the gaps associated with this approach are lower than the ones associated with the AARC. where u∗is the optimal solution (obtained by the BO approach) and uJ is the solution obtained by approach J, with J = AARC or J = SO. Table 8 suggests that for the instances solved to optimality the best solutions are on average obtained by the SO method since the gaps associated with this approach are lower than the ones associated with the AARC. Table 8: Average optimality gaps associated to both the SO method and the AARC approach. n 10 20 30 40 SO 0.46(15) 0.68(23) 1.07(18) 1.03(23) AARC 0.80(10) 1.43(2) 2.06(7) 1.82(2) Table 8: Average optimality gaps associated to both the SO method and the AARC approach. n 10 20 30 40 SO 0.46(15) 0.68(23) 1.07(18) 1.03(23) AARC 0.80(10) 1.43(2) 2.06(7) 1.82(2) Furthermore, the number of best solutions found is greater in the SO method than in the AARC approach. Furthermore, the number of best solutions found is greater in the SO method than in the AARC approach. 5 Conclusion In this paper we consider RO min-max problems with decomposable functions. Based on the dual Lagrangian problem resulting from a Lagrangian relaxation of the reformulation of the adversarial problem, we provide a compact formulation to approximate the true min-max problem and show that the Bertsimas and Thiele dualization approach is a particular case of this approach with the multipliers equal to zero. Additionally, we show that the new dual Lagrangian formulation coincides with an aﬃne approximation. The theoretical results are applied to the robust inventory problem where the demands are uncertain and the uncertain variables belong to the B&T budgeted set. Computational results have shown that when other complicating aspects such as setup costs are present, by overes- timating the costs, the classical dualization approach from Bertsimas and Thiele (2006) can provide poor bounds and poor solutions. The dual Lagrangian formulation, which coincides with an aﬃne approximation model, leads to bounds closer to the true min-max value even for those instances where the dualization from Bertsimas and Thiele (2006) provide worst bounds. However, although the dual Lagrangian formulation leads to tractable models, their size can be too large to be solved to optimality for real size instances. Taking advantage of regarding such models from the perspective of Lagrangian duality theory, we propose heuristics approaches that 28 consider the new multipliers as penalties for violation of the constraints of the adversarial prob- lem. Thus, such penalties penalize the overestimation of the true cost of each feasible solution. Using such idea, we introduce a Guided Iterated Local Search heuristic and a Subgradient Op- timization method to solve large size inventory models. The Subgradient Optimization method proved to be eﬃcient to obtain better solutions than those obtained using other approximation approaches including the dual Lagrangian formulation. ACKNOWLEDGMENT The research of the ﬁrst three authors is supported by the Center for Research and Devel- opment in Mathematics and Applications (CIDMA) through the Portuguese Foundation for Sci- ence and Technology (FCT - Funda¸c˜ao para a Ciˆencia e a Tecnologia), references UIDB/04106/2020 and UIDP/04106/2020. 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Zhen J, den Hertog D, Sim M (2018) Adjustable robust optimization via fourier-motzkin elimination. Operations Research 66(4):1086–1100. 30 Appendix B. Projected Model Here we present the projected version of model D mention in Section 2.3 as well as the steps followed in its derivation. Proposition 13. Projecting out variables rtk j , j, t ∈T : j ≤t, k ∈K model D can be written as follows. D = min u,λ,θ,q,r g(u) + n X t=1 θt s.t. θt ≥Lk t (u) + t X j=1 qtk j Γj − t X j=1 \|πt j\| t X ℓ=j qtk ℓ+ t X j=1 πt j(atk j −λt j), ∀πt i ∈ {−1, 0, 1}, t ∈T, k ∈K, (5.1) qtk j ≥0, ∀j, t ∈T : j ≤t, k ∈K. D = min u,λ,θ,q,r g(u) + n X t=1 θt s.t. θt ≥Lk t (u) + t X j=1 qtk j Γj − t X j=1 \|πt j\| t X ℓ=j qtk ℓ+ t X j=1 πt j(atk j −λt j), qtk j ≥0, qtk j ≥0, ∀j, t ∈T : j ≤t, k Proof. Proof.Using Fourier-Motzkin elimination, we ﬁrst project out variables rtk t . For each k ∈K, we have from (2.13) and (2.14), rtk t ≥−qtk t + atk t −λt t, rtk t ≥−qtk t −atk t + λt t, rtk t ≥−qtk t + atk t −λt t, rtk t ≥−qtk t −atk t + λt t, rtk t ≥0. Combining (2.12) with these inequalities we obtain Combining (2.12) with these inequalities we obtain θt ≥Lk t (u) + t X j=1 qtk j Γj + t−1 X j=1 rtk j −\|πt t\|qtk t + πt t(atk t −λt t), ∀πt t ∈{−1, 0, 1}, t ∈T, k ∈K. By iteratively eliminating rtk j from j = t −1 until j = 1 and by using (2.13) and (2.14) we obtain (5.1). By iteratively eliminating rtk j from j = t −1 until j = 1 and by using (2.13) and (2.14) we obtain (5.1). By iteratively eliminating rtk j from j = t −1 until j = 1 and by using (2.13) and (2.14) we obtain (5.1). 31 Proof. Proof. Proof. Proof. Proof. Proof. We start by writing model R(u) with the new variables vt j, for j, t ∈T : j ≤t as follows: Proof. Proof. We start by writing model R(u) with the new variables vt j, for j, t ∈T : j ≤t as follows: We start by writing model R(u) with the new variables vt j, for j, t ∈T : j ≤t as follows: R(u) = max x,v n X t=1 (ctut + max{htxt+1, −btxt+1}) s.t. xt+1 = x1 + t X j=1 (uj −µj −δjvt j), ∀t ∈T, ℓ X j=1 \|vt j\| ≤Γℓ, ∀ℓ, t ∈T : ℓ≤t, vj t = vt t, ∀t, j ∈T : t < j, (5.2) vj t ∈[−1, 1], ∀t, j ∈T : t ≤j. R(u) = max x,v n X t=1 (ctut + max{htxt+1, −btxt+1}) s.t. xt+1 = x1 + t X j=1 (uj −µj −δjvt j), ∀t ∈T, j ℓ X j=1 \|vt j\| ≤Γℓ, ∀ℓ, t ∈T : ℓ≤t, vj t = vt t, ∀t, j ∈T : t < j, (5.2) vj t ∈[−1, 1], ∀t, j ∈T : t ≤j. ∀ℓ, t ∈T : ℓ≤t, (5.2) Following the process described in Section 2, we attach a Lagrangian multiplier λj t to each constraint (5.2) for 1 ≤t < j ≤n, and dualize these constraints in the usual Lagrangian way. This leads to the following relaxed problem LR(u, λ) = max x,v n X t=1  ctut + max{htxt+1, −btxt+1} − n X j=t+1 λj t(vt t −vj t )   s.t. xt+1 = x1 + t X j=1 (uj −µj −δjvt j), ∀t ∈T, ℓ X j=1 \|vt j\| ≤Γℓ, ∀ℓ, t ∈T : ℓ≤ vj t ∈[−1, 1], ∀t, j ∈T : t ≤ s.t. xt+1 = x1 + t X j=1 (uj −µj −δjvt j), s.t. xt+1 = x1 + t X j=1 (uj −µj −δjvt j), ∀t ∈T, ∀t ∈T, ∀ℓ, t ∈T : ℓ≤t, ∀t, j ∈T : t ≤j. Appendix C. Proof of Theorem 9 Here we provide the proof of Theorem 9 presented in Section 3.2. Proof. Proof. ∀ℓ, t ∈T : ℓ≤t, ∀t, j ∈T : t ≤j. Proof. Proof. θt ≥L1 t (u) + ht max (vt+ j ,vt− j )∈¯Ωt   −  δt + n X j=t+1 λj t ht  (vt+ t −vt− t ) + t−1 X j=1 λt j ht −δj ! (vt+ j −vt− j )   , (5.3) θt ≥L2 t (u) + bt max (vt+ j ,vt− j )∈¯Ωt     δt − n X j=t+1 λj t bt  (vt+ t −vt− t ) + t−1 X j=1 λt j bt + δj ! (vt+ j −vt− j )   , (5 4) where ¯Ωt :=   (vt+ j , vt− j ) ∈Rt × Rt \| j X ℓ=1 (vt+ ℓ + vt− ℓ) ≤Γj; vt+ j + vt− j ≤1; vt+ j , vt− j ≥0; 1 ≤j ≤t   . Associating the dual variables qt1 j and rt1 j to the constraints of the inner problem in the RHS of constraints (5.3) and the dual variables qt2 j and rt2 j to the constraints of the inner problem in the RHS of constraints (5.4) we obtain model D. Proof. Proof. Rearranging the terms in the objective function by noticing that n X t=1 n X j=t+1 λj tvj t = n X t=2 t−1 X j=1 λt jvt j, and eliminating variables xt, t > 1, the relaxed problem can be written as follows Rearranging the terms in the objective function by noticing that n X t=1 n X j=t+1 λj tvj t = n X t=2 t−1 X j=1 λt jvt j, and eliminating variables xt, t > 1, the relaxed problem can be written as follows LR(u, λ) = max v n X t=1  ctut + max   L1 t (u) −ht t X j=1 δjvt j, L2 t (u) + bt t X j=1 δjvt j   −vt t n X j=t+1 λj t + t−1 X j=1 λt jvt j   s.t. ℓ X j=1 \|vt j\| ≤Γℓ, ∀ℓ, t ∈T : ℓ≤t, vt j ∈[−1, 1], ∀j, t ∈T : j ≤t. LR(u, λ) = max v n X t=1  ctut + max   L1 t (u) −ht t X j=1 δjvt j, L2 t (u) + bt t X j=1 δjvt j   −vt t n X j=t+1 λj t + t−1 X j=1 λt jvt j   s.t. ℓ X j=1 \|vt j\| ≤Γℓ, ∀ℓ, t ∈T : ℓ≤t, vt j ∈[−1, 1], ∀j, t ∈T : j ≤t. where L1 t (u) = ht  x1 + t X j=1 (uj −µj)  and L2 t (u) = −bt  x1 + t X j=1 (uj −µj)  . where L1 t (u) = ht  x1 + t X j=1 (uj −µj)  and L2 t (u) = −bt  x1 + t X j=1 (uj −µj)  . ( ) For a given u and λ, the problem LR(u, λ) can be separated into n independent subproblems, one for each time period. Hence, for a ﬁxed time period t ∈T, the corresponding subproblem 32 can be written as follows can be written as follows LRt(u, λt) = min θt ctut + θt s.t. Proof. Proof. θt ≥L1 t (u) + max vt∈Ωt   −ht t X j=1 δjvt j −vt t n X j=t+1 λj t + t−1 X j=1 λt jvt j   , θt ≥L2 t (u) + max vt∈Ωt   bt t X j=1 δjvt j −vt t n X j=t+1 λj t + t−1 X j=1 λt jvt j   , LRt(u, λt) = min θt ctut + θt LRt(u, λt) = min θt ctut + θt θt s.t. θt ≥L1 t (u) + max vt∈Ωt   −ht t X j=1 δjvt j −vt t n X j=t+1 λj t + t−1 X j=1 λt jvt j   , θt ≥L2 t (u) + max vt∈Ωt   bt t X j=1 δjvt j −vt t n X j=t+1 λj t + t−1 X j=1 λt jvt j   , where Ωt = {vt ∈[−1, 1]t \| Pj ℓ=1 \|vt ℓ\| ≤Γj, 1 ≤j ≤t}. where Ωt = {vt ∈[−1, 1]t \| Pj ℓ=1 \|vt ℓ\| ≤Γj, 1 ≤j ≤t}. where Ωt = {vt ∈[−1, 1]t \| Pj ℓ=1 \|vt ℓ\| ≤Γj, 1 ≤j ≤t}. Linearizing LRt(u, λt) by writing variables vt j as vt j = vt+ j −vt− j , for all j, t ∈T, j ≤t and rearranging the terms in the set of constraints, model LRt(u, λt) becomes Linearizing LRt(u, λt) by writing variables vt j as vt j = vt+ j −vt− j , for all j, t ∈T, j ≤t and rearranging the terms in the set of constraints, model LRt(u, λt) becomes LRt(u, λt) = min θt ctut + θt s.t. θt ≥L1 t (u) + ht max (vt+ j ,vt− j )∈¯Ωt   −  δt + n X j=t+1 λj t ht  (vt+ t −vt− t ) + t−1 X j=1 λt j ht −δj ! (vt+ j −vt− j )   , (5.3) θt ≥L2 t (u) + bt max (vt+ j ,vt− j )∈¯Ωt     δt − n X j=t+1 λj t bt  (vt+ t −vt− t ) + t−1 X j=1 λt j bt + δj ! (vt+ j −vt− j )   , (5 4) LRt(u, λt) = min θt ctut + θt LRt(u, λt) = min θt ctut + θt LRt(u, λt) = min θt ctut + θt s.t. Appendix D. Computational results with respect to Γn Here we analyse the performance of the LD, the AARC and the BT approaches regarding the maximum number of deviations Γn in the last time period (see section 4.1). The maximum number of deviations in the previous periods is taken as follows. Given a value k ∈{1, . . . , Γn}, the time periods t ∈T such that Γt = k are the ones in the range t = q(k −1) + k−1 X ℓ=1 αℓ+ 1, . . . , qk + k X ℓ=1 αℓ, where q = j n Γn k , and αℓ= 1 if ℓ≤n −qΓn and αℓ= 0, otherwise. This rule ensures that given two values k1, k2 ∈{1, . . . , Γn} the diﬀerence between the number of periods having at most k1 and k2 deviations is either zero or one. Figures 6 and 7 display the results for the case where Γ30 ranges from 0 (nominal case) to 30 (box-constrained case). For each value of Γ30, we consider 100 randomly generated instances and the average gap Gap = UB −BO BO × 100 Gap = UB −BO BO × 100 33 is displayed, where UB is a given upper bound and BO is the optimal value obtained by using the BO approach.The lines associated with LD, AARC and BT represent the average gap cor- responding to the upper bounds obtained by the LD, AARC and BT approaches, respectively. The lines associated with R(uLD), R(uAARC) and R(uBT ) represent the average gap corre- sponding to the true cost of the solutions obtained by the LD, AARC and the BT approaches, respectively. In Figure 6 the setup costs are not considered, while in Figure 7 a setup cost of value 150 is considered. Γ30 0 5 10 15 20 25 30 Gap (%) 0 4 8 12 LD AARC BT R(uLD) R(uAARC) R(uBT) Figure 6: Average gaps in terms of the maximum number of deviations considering no setup costs. Γ30 0 5 10 15 20 25 30 Gap (%) 0 4 8 12 LD AARC BT R(uLD) R(uAARC) R(uBT) Figure 6: Average gaps in terms of the maximum number of deviations considering no setup costs. Appendix D. Computational results with respect to Γn Γ30 0 5 10 15 20 25 30 Gap (%) 0 5 10 15 20 25 30 LD AARC BT R(uLD) R(uAARC) R(uBT) Figure 7: Average gaps in terms of the maximum number of deviations considering a setup cost equal to 150. Γ30 0 5 10 15 20 25 30 Gap (%) 0 5 10 15 20 25 30 LD AARC BT R(uLD) R(uAARC) R(uBT) Figure 7: Average gaps in terms of the maximum number of deviations considering a setup cost equal to 150. For both cases, the average gap associated with the LD and AARC approaches is always lower than 10% and 7%, respectively, while for the BT approach such gap can reach 28%. In particular, in our experiments, for the box-constrained case, there is no gap associated with the upper bounds obtained by both the LD and AARC approaches. In general, the average gap associated with the true cost of the solutions determined by both LD and AARC approaches tends to decrease as the number of deviations increases and it is zero for the box-constrained case. For both cases, the average gap associated with the LD and AARC approaches is always lower than 10% and 7%, respectively, while for the BT approach such gap can reach 28%. In particular, in our experiments, for the box-constrained case, there is no gap associated with the upper bounds obtained by both the LD and AARC approaches. In general, the average gap associated with the true cost of the solutions determined by both LD and AARC approaches tends to decrease as the number of deviations increases and it is zero for the box-constrained case. 34
https://openalex.org/W2585214471	https://link.springer.com/content/pdf/10.1007%2Fs00894-017-3234-y.pdf	English	null	Solvatochromic effect in absorption and emission spectra of star-shaped bipolar derivatives of 1,3,5-triazine and carbazole. A time-dependent density functional study	Journal of molecular modeling	2,017	cc-by	5,745	Introduction Abstract A series of three star-shaped compounds containing both donor (carbazole) and acceptor (2,4,6-triphenyl-1,3,5- triazine) moieties linked through various linking bridges was studied theoretically at the linear response TD-DFT level of theory to describe their absorption and fluorescence spectra. The concept of a localized charge-transfer excited state has been applied successfully to explain the observed strong solvatochromic effect in the emission spectra of the studied molecules, which can be utilized for the fabrication of color tunable solution-processable OLEDs. The concept is in particu- larly applicable to donor–acceptor species with a C3 symmetry point group where the static dipole moment changes dramati- cally upon electronic excitation. An important peculiarity of the studied molecules is that they are characterized by non-zero values of the HOMO and LUMO orbitals in the same common part of molecular space that provides a large electric dipole transition moment for both light absorption and emission. In recent years, star-shaped organic materials have attracted a great deal of attention due to their promising applications in organic light-emitting devices (OLEDs) [1–6]. One of the main challenges in this field is the realization of ambipolar transporting properties within single-type molecules, together with the strong photoluminescence that allows improvement of the luminance characteristics of OLEDs [5]. Another im- portant feature of star-shaped organic luminophores is that they tend to form exciplexes with a wide range of organic materials, which is very useful for the emission-color tuning ability of OLEDs [7–9]. The general strategy to create ambipolar star-shaped emit- ters is to combine donor (D) and acceptor (A) fragments with- in the same molecule [5, 6], which (1) facilitates injection and transport properties of both charge-carriers-holes and elec- trons, and (2) activates intermolecular charge-transfer (CT) excited states in the photoluminescence spectra. Such CT states usually are the lowest-lying excited states of the star- shaped molecules, i.e., they are responsible for the fluores- cence process. Moreover, the nonzero values of the HOMO and LUMO wave-functions in the same common part of the molecular space is a very important property of efficient star- shaped emitters [10, 11], providing a large electric dipole tran- sition moment for both light absorption and emission [12]. Keywords Star-shaped compounds . OLEDs . TDDFT . Solvatochromic effect . Dipole moment Electronic supplementary material The online version of this article (doi:10.1007/s00894-017-3234-y) contains supplementary material, which is available to authorized users. J Mol Model (2017) 23: 55 DOI 10.1007/s00894-017-3234-y J Mol Model (2017) 23: 55 DOI 10.1007/s00894-017-3234-y ORIGINAL PAPER Solvatochromic effect in absorption and emission spectra of star-shaped bipolar derivatives of 1,3,5-triazine and carbazole. A time-dependent density functional study Gleb V. Baryshnikov1,2 & Sergey V. Bondarchuk2 & Valentina A. Minaeva2 & Hans Ågren1 & Boris F. Minaev1,2 Received: 11 November 2016 /Accepted: 13 January 2017 /Published online: 4 February 2017 # The Author(s) 2017. This article is published with open access at Springerlink.com * Gleb V. Baryshnikov glebchem@rambler.ru 1 Division of Theoretical Chemistry and Biology, School of Biotechnology, KTH Royal Institute of Technology, 10691 Stockholm, Sweden 2 Department of Chemistry and Nanomaterials Science, Bogdan Khmelnitsky Cherkasy National University, blvd. Shevchenko 81, 18031 Cherkasy, Ukraine Introduction It is well-known that D-A molecules usually demonstrate solvent-dependent behavior in their absorption spectra due to the high polarization of the ground-state molecular structure [12–17]. Strong positive solvatochromism in absorption spec- tra is observed frequently for molecules with a ππ* nature of band-productive electronic state. The absolute value of the red shift depends, usually linearly, on the solvent polarity (the higher the solvent polarity, the stronger the red shift) [6]. This is because the more polar solvent species polarizes * Gleb V. Baryshnikov glebchem@rambler.ru 1 Division of Theoretical Chemistry and Biology, School of Biotechnology, KTH Royal Institute of Technology, 10691 Stockholm, Sweden 2 Department of Chemistry and Nanomaterials Science, Bogdan Khmelnitsky Cherkasy National University, blvd. Shevchenko 81, 18031 Cherkasy, Ukraine 55 Page 2 of 8 55 Page 2 of 8 J Mol Model (2017) 23: 55 molecules with a higher static dipole moment more strongly (particularly for D-A systems). But, in the case of symmetrical star-shaped molecules (3D-A, for example), the static dipole moment of the whole system is equal to zero because of the C3 symmetry point group restriction. Therefore, these systems should not demonstrate strong solvatochromism in the absorp- tion spectra. However, if the excited state of the star-shaped molecule corresponds to the local CT ππ-state, the structure of the excited-state geometry should be significantly distorted and the dipole moment of the excited state should differ mark- edly from zero. This means that clear solvatochromic behavior should be observed in the emission (fluorescence) spectra of D-A star-shaped molecules with local CT excited states rather than in absorption spectra where the vertical excitation pre- vails. In this way we can vary the emission color of the star- shaped compound in various solvents using the same excita- tion energy, which is very useful for applications of C3 sym- metry point group in photovoltaic cells, OLEDs and bioimaging technologies [6]. continuum model (PCM) using integral equation formalism (IEFPCM) [31]. To define cavities, the universal force field (UFF) radii were used. The overlap index and minimum radi- us of the spheres were specified as 0.8 and 0.5 Å, respectively. We calculated the energies and oscillator strength values for 15 vertical electronic transition using the same 6-31 G basis set as that used for geometry optimization. The fluorescence energies were computed, taking into account relaxation of the excited state geometry (TD-DFT optimization of the S1 state geometry), including the state-specific equilibrium solvation correction. Introduction The electronic absorption spectra curves were fitted using the Gauss distribution function and a half-width of 3000 cm−1 with the SWizard 5.0 program package [32]. Molecular visu- alizations were performed with Chemcraft 1.6 [33]. Effect of exact HFE on UV-vis spectra prediction In the present work, we focused on the three recently syn- thesized star-shaped compounds containing both D (carbazole) and A (2,4,6-triphenyl-1,3,5-triazine) moieties connected through various linking bridges [6]. We describe the results of quantum-chemical calculations carried out in order to study the Bstructure–optical properties relationship^ of these D-A mate- rials. Such compounds demonstrate clear solvent-dependent fluorescence, but the solvent effect is less observable in the absorption spectra. We think that the selected star-shaped com- pounds are really good candidates to prove the theory of local CT excited states in organic fluorofores of the C3 symmetry point group. The optimized structures of the studied propeller-shaped bi- polar derivatives of 1,3,5-triazine and carbazole, namely, 2,4,6-tris(4-(3-tert-butyl-carbazol-9-yl)phenyl)-1,3,5-triazine (TR1), 2,4,6-tris(4-(3,6-di-tert-butyl-carbazol-9-yl)phenyl)- 1,3,5-triazine (TR2) and 2,4,6-tris(4-((9-hexyl-carbazol-3- yl)ethynyl)phenyl)-1,3,5-triazine (TR3) are illustrated in Fig. 1, and the calculated IR spectra (except of TR2) are listed in Table S1 and Fig. S1. The ground state of these structures belongs to the C3 point group symmetry. As one can see in Table S1, the stationary points are characterized by the ab- sence of imaginary frequencies, and are vibrationally stable. To find the most appropriate functional for the absorption spectra calculation, we have performed a series of trials using hybrid functionals with different amounts of the exact HFE [34]. The results obtained are collected in Table 1. A strong correlation between the HFE (%) and the S0→S1 transition energy and intensity (oscillator strength) was found (Fig. S2). Except for the ωB97XD and CAM-B3LYP func- tionals, the following correlation coefficients were found: TR1 and TR2 (R2 = 0.9817), TR3 (R2 = 0.9866) for transition energy; TR1 and TR2 (R2 = 0.9995), TR3 (R2 = 0.9690) for oscillator strength. Note that the aforementioned correlations are presented for the hybrid functionals only. The range- separated functionals used in this study, namely, CAM- B3LYP and ωB97XD, were not involved in the correlation because they provide a different scheme for electron transition energy. As one can see in Table 1, the increased amount of HFE causes a rise in the transition energy. The range- separated functionals provide the same trend. Table 1 Energy and intensity of the S0→S1 transition [in hexane, the polarizable continuum model (PCM) using integral equation formalism (IEFPCM) model] in the absorption spectra of the species TR1–TR3 as functions of the Hartree-Fock exchange (HFE) contribution in the exchange- correlation functional Computational details The calculations presented in this paper were performed in terms of density functional theory (DFT) using the Gaussian09 suite of programs [18, 19]. Geometry optimizations were carried out by the hybrid exchange-correlation functional B3LYP [20, 21] with the Pople’s split-valence basis set (almost double-ζ in the va- lence shell, 6-31 G) and addition of polarization (d, p) functions [22, 23]. The optimized structures were checked for absence of imaginary frequencies in the vibrational spectra, and the geom- etries obtained were justified as global minima. The UV-vis spectra were obtained in terms of time- dependent density functional theory (TD-DFT) [24]. For this purpose, we applied the conventional B3LYP scheme as well as the modified B3LYP functional with the changed contribu- tion of the exact Hartree-Fock exchange (HFE) part, which was increased up to 30%. Furthermore, we used the PBE0 [25], mPW1PBE [26], CAM-B3LYP [27], BMK [28], ωB97XD [29], and M062X [30] functionals. Polar media simulations were performed in terms of the polarizable Herein, the custom-defined scheme (B3LYP-30) [18, 20, 21] provides the best fit with the experimental data (3.19 eV) [17], and overestimates the transition energy by only 0.032 eV Page 3 of 8 55 Page 3 of 8 55 b Experimental data for n-hexane solution [6] a For the range-separated functionals the values correspond to the short-range exchange b Experimental data for n-hexane solution [6] a For the range-separated functionals the values correspond to the short-range exchange b Experimental data for n-hexane solution [6] J Mol Model (2017) 23: 55 As one can see in Fig. 2, the HOMO orbital for TR1–TR3 molecules is localized mainly on the carbazole moieties. Fig. 2 Frontier molecular orbitals—the highest occupied (HOMO) and the lowest unoccupied (LUMO)—of the studied dyes TR1–TR3 Table 2 Calculated wave lengths (eV), oscillator strength ( f ) and orbital assignment for the quasi-degenerate S0→S1 and S0→S2 electronic transitions of TR1–TR3 molecules by the B3LYP-30/6-31G(d,p) method within IEFPCM approach (n-hexane) Transition E (eV) f Assignment TR1 S1 3.22 0.730 HOMO→LUMO+1 (+48%) HOMO–1→LUMO (23%) HOMO–2→LUMO+1 (20%) S2 3.22 0.731 HOMO→LUMO (+44%) HOMO–2→LUMO (+24%) HOMO–1→LUMO+1 (23%) TR2 S1 3.18 0.793 HOMO→LUMO+1 (+50%) HOMO–2→LUMO (+18%) HOMO–1→LUMO+1 (+17%) S2 3.18 0.794 HOMO→LUMO (+45%) HOMO–1→LUMO (21%) HOMO–2→LUMO+1 (+19%) TR3 S1 3.10 2.311 HOMO→LUMO+1 (+40%) HOMO–1→LUMO (21%) HOMO–2→LUMO+1 (20%) S2 3.10 2.311 HOMO→LUMO (+39%) HOMO–2→LUMO (+21%) HOMO–1→LUMO+1 (21%) 55 Page 4 of 8 J Mol Model (2017) 23: 55 TR1–TR3 compounds can be recommended not only as emit- ters for OLEDs [6, 35, 36] but also as light-harvesting mate- rials for photovoltaic solar cells. Note that compounds TR1– TR3 demonstrate high photoluminescence quantum yields reaching 0.85 [6]. TR1–TR3 compounds can be recommended not only as emit- ters for OLEDs [6, 35, 36] but also as light-harvesting mate- rials for photovoltaic solar cells. Note that compounds TR1– TR3 demonstrate high photoluminescence quantum yields reaching 0.85 [6]. Table 2 Calculated wave lengths (eV), oscillator strength ( f ) and orbital assignment for the quasi-degenerate S0→S1 and S0→S2 electronic transitions of TR1–TR3 molecules by the B3LYP-30/6-31G(d,p) method within IEFPCM approach (n-hexane) Table 2 Calculated wave lengths (eV), oscillator strength ( f ) and orbital assignment for the quasi-degenerate S0→S1 and S0→S2 electronic transitions of TR1–TR3 molecules by the B3LYP-30/6-31G(d,p) method within IEFPCM approach (n-hexane) Transition E (eV) f Assignment TR1 S1 3.22 0.730 HOMO→LUMO+1 (+48%) HOMO–1→LUMO (23%) HOMO–2→LUMO+1 (20%) S2 3.22 0.731 HOMO→LUMO (+44%) HOMO–2→LUMO (+24%) HOMO–1→LUMO+1 (23%) TR2 S1 3.18 0.793 HOMO→LUMO+1 (+50%) HOMO–2→LUMO (+18%) HOMO–1→LUMO+1 (+17%) S2 3.18 0.794 HOMO→LUMO (+45%) HOMO–1→LUMO (21%) HOMO–2→LUMO+1 (+19%) TR3 S1 3.10 2.311 HOMO→LUMO+1 (+40%) HOMO–1→LUMO (21%) HOMO–2→LUMO+1 (20%) S2 3.10 2.311 HOMO→LUMO (+39%) HOMO–2→LUMO (+21%) HOMO–1→LUMO+1 (21%) Nature of the absorption spectra In this section, we discuss the calculated data on the absorp- tion spectra of TR1–TR3 molecules that are characterized by the close similar spectral properties (Table 2) due to the similar star-shaped structure (i.e., the same symmetry selection rules) and also the same D and A fragments for each of the three molecules. In Table 1, we selected only transitions with oscil- lator strengths >0.01. The first long-wavelength most intense absorption band for TR1–TR3 molecules corresponds to the quasi-degenerate electronic transitions to the S1 and S2 states; the main configurations for both S1 and S2 states are the HOMO→LUMO+1 and HOMO→LUMO, respectively. Thus, here we account for the fact that LUMO and LUMO+ 1 are almost degenerate. The HOMO, LUMO and LUMO+1 wavefunctions for the TR1–TR3 dyes are illustrated in Fig. 2, and the complete set of MOs involved in the electron transi- tions are presented in Figs. S3–S5. As one can see in Fig. 2, the HOMO orbital for TR1–TR3 molecules is localized mainly on the carbazole moieties. TR1 S1 3.22 0.730 HOMO→LUMO+1 (+48%) HOMO–1→LUMO (23%) HOMO–2→LUMO+1 (20%) S2 3.22 0.731 HOMO→LUMO (+44%) HOMO–2→LUMO (+24%) HOMO–1→LUMO+1 (23%) TR2 S1 3.18 0.793 HOMO→LUMO+1 (+50%) HOMO–2→LUMO (+18%) HOMO–1→LUMO+1 (+17%) S2 3.18 0.794 HOMO→LUMO (+45%) HOMO–1→LUMO (21%) HOMO–2→LUMO+1 (+19%) TR3 S1 3.10 2.311 HOMO→LUMO+1 (+40%) HOMO–1→LUMO (21%) HOMO–2→LUMO+1 (20%) S2 3.10 2.311 HOMO→LUMO (+39%) HOMO–2→LUMO (+21%) HOMO–1→LUMO+1 (21%) In this section, we discuss the calculated data on the absorp- tion spectra of TR1–TR3 molecules that are characterized by the close similar spectral properties (Table 2) due to the similar star-shaped structure (i.e., the same symmetry selection rules) and also the same D and A fragments for each of the three molecules. In Table 1, we selected only transitions with oscil- lator strengths >0.01. The first long-wavelength most intense absorption band for TR1–TR3 molecules corresponds to the quasi-degenerate electronic transitions to the S1 and S2 states; the main configurations for both S1 and S2 states are the HOMO→LUMO+1 and HOMO→LUMO, respectively. Thus, here we account for the fact that LUMO and LUMO+ 1 are almost degenerate. The HOMO, LUMO and LUMO+1 wavefunctions for the TR1–TR3 dyes are illustrated in Fig. 2, and the complete set of MOs involved in the electron transi- tions are presented in Figs. S3–S5. As one can see in Fig. 2, the HOMO orbital for TR1–TR3 molecules is localized mainly on the carbazole moieties. Fig. 2 Frontier molecular orbitals—the highest occupied (HOMO) and the lowest unoccupied (LUMO)—of the studied dyes TR1–TR3 J Mol Model (2017) 23: 55 Fig. 1 Structure of the species TR1–TR3 optimized by the DFT(B3LYP)/6-31G(d,p) method in the n-hexane [polarizable continuum model (PCM) using integral equation formalism (IEFPCM)] solvent LUMO wave-functions are characterized by nonzero expan- sion coefficients in the same common part of the molecular space (phenyl ring nearest to the triazine core). This is a very important property of efficient light-harvesting end light- emissive materials. Such HOMO–LUMO Boverlapping^ pro- vides a large electric dipole transition moment for light ab- sorption and light emission processes (S0↔S1) [12], and, therefore, all the studied molecules are characterized by high values of oscillator strength for S0→S1 absorption as well as high fluorescence quantum yields for the S1→S0 emission channel (about 80% in non-polar media). For this reason, (Table 1). The regular B3LYP functional gave a strong under- estimation of the transition energy (about 0.330 eV), which is a known limitation of this functional for the CTstates [34]. All other hybrid functionals demonstrate the same underestima- tion trend (Table 1). The use of range-separated functionals like CAM-B3LYP usually ensures more adequate energies for the CT states, but in our case this type of functional strongly overestimated the fluorescence-responsible S1 state energy (Table 1). This is due to the fact that the first S1 state for the TR1–TR3 molecules combines the CT nature with local π→ π-excitation. As can be seen from Fig. J Mol Model (2017) 23: 55 2, the HOMO and Functional HFE (%)a TR1 TR2 TR3 λ (nm) E (eV) f λ (nm) E (eV) f λ (nm) E (eV) f Exp.b 389 3.19 397 3.12 394 3.14 B3LYP-30 30 385 3.22 0.730 390 3.18 0.793 400 3.10 2.311 B3LYP 20 434 2.86 0.589 439 2.82 0.643 440 2.82 1.837 PBE0 25 410 3.03 0.655 415 2.99 0.713 419 2.96 2.065 mPW1PBE 25 410 3.03 0.655 415 2.99 0.713 419 2.96 2.067 CAM-B3LYP 19 327 3.79 1.151 331 3.75 1.234 352 3.52 2.936 BMK 42 355 3.49 0.898 360 3.45 0.971 374 3.32 2.673 ωB97XD 22 315 3.93 1.314 318 3.89 1.403 343 3.62 3.081 M062X 54 334 3.71 1.054 338 3.67 1.137 354 3.51 2.892 a For the range-separated functionals the values correspond to the short-range exchange b Experimental data for n-hexane solution [6] nded not only as emit- ight-harvesting mate- hat compounds TR1– ence quantum yields Table 2 Calculated wave lengths (eV), oscillator strength ( f ) and orbital assignment for the quasi-degenerate S0→S1 and S0→S2 electronic transitions of TR1–TR3 molecules by the B3LYP-30/6-31G(d,p) method within IEFPCM approach (n-hexane) J Mol Model (2017) 23: 55 55 Page 4 of 8 J Mol Model (2017) 23: 55 55 Page 4 of 8 TR1–TR3 compounds can be recommended not only as emit- ters for OLEDs [6, 35, 36] but also as light-harvesting mate- rials for photovoltaic solar cells. Note that compounds TR1– TR3 demonstrate high photoluminescence quantum yields reaching 0.85 [6]. Nature of the absorption spectra In this section, we discuss the calculated data on the absorp- tion spectra of TR1–TR3 molecules that are characterized by the close similar spectral properties (Table 2) due to the similar star-shaped structure (i.e., the same symmetry selection rules) and also the same D and A fragments for each of the three molecules. In Table 1, we selected only transitions with oscil- lator strengths >0.01. The first long-wavelength most intense absorption band for TR1–TR3 molecules corresponds to the quasi-degenerate electronic transitions to the S1 and S2 states; the main configurations for both S1 and S2 states are the HOMO→LUMO+1 and HOMO→LUMO, respectively. Thus, here we account for the fact that LUMO and LUMO+ 1 are almost degenerate. The HOMO, LUMO and LUMO+1 wavefunctions for the TR1–TR3 dyes are illustrated in Fig. 2, and the complete set of MOs involved in the electron transi- tions are presented in Figs. S3–S5. Solvatochromic effect An interesting experimental observation is that molecules TR1–TR3 demonstrate a strong solvatochromic effect only in the fluorescence spectra, but not in the absorption spectra. The authors of a previous study [6] explained this fact by the strong differences in electronic structure of the ground and first singlet excited states for the studied TR1–TR3 molecules. Therefore, we tracked the effect of solvent on molecular features such as permanent dipole moment of S0 and S1 electronic states, and also on the energy of S1 state, since the latter has a CT nature. The calculated data of the dipole moment dependence are pre- sented in Fig. 4. Despite the symmetrical core of the molecular graph ©3 symmetry point group), the presence of carbazole ligands causes occurrence of a weak permanent dipole moment in the ground state of TR1–TR3 molecules (Fig. 4). The corresponding dipole moment vector is oriented along the main molecular axis perpendicular to the triazine core plain (Fig. 5). As one can see in Fig. 4, the values of the ground state dipole moment (μ) are relatively small, and vary- ing in the range of 0.1–1.0 D. This is because of the quasi-C3 symmetry, which almost excludes the existence of a perma- nent dipole moment for the TR1–TR3 molecules in the ground state. As expected, the dipole moment values correlate with the dielectric permittivity of the solvents (Fig. 4). With a rise in solvent polarity, the μ value reaches a maximum in the We should stress that the experimental absorption spectrum of TR1 (Fig. 3) exhibits three blue-shifted bands that are not reproduced by the vertical TD-DFT calculation. This is a pro- gression of C–C vibrations (ν = 1276 cm−1), which corre- spond mostly to the triazine-phenyl link. Upon HOMO– LUMO excitation, this C–C bond becomes much stronger. The HOMO is a non-bonding orbital with respect to such a link, but the LUMO is a bonding orbital (Fig. 3); thus, the force constant of this mode is higher in the excited state. Such a strong change in the force field, and of the mode displacement upon excitation, leads to the occurrence of a long progression of the νC–C mode (1276 cm−1) in the first absorption band. Nature of the absorption spectra As one can see in Fig. 2, the HOMO orbital for TR1–TR3 molecules is localized mainly on the carbazole moieties. Page 5 of 8 55 J Mol Model (2017) 23: 55 Meanwhile, the LUMOs display electron densities primarily on the triazine core, and also on the phenyl bridges. Actually, both S1 and S2 states are characterized by their CT nature, but, at the same time, both HOMO and LUMO/LUMO+1 orbitals make large contributions to the common atoms of the linker (phenyl ring in the case of TR1, TR2 molecules and phenyl-ethynyl fragment in TR3). This provides a large transition dipole mo- ment for the S0–S1 and S0–S2 transitions. In fact, the larger the common area for the HOMO and LUMO/LUMO+1 wave- function, the higher the transition dipole moment and its oscil- lator strength. It can be seen from Table 2 that S0–S1 and S0–S2 transitions for the TR1 and TR2 molecules are characterized by almost the same oscillator strength values due to the same linker fragment. For the phenyl-ethynyl containing TR3 com- pound, the intensity of the S0–S1 and S0–S2 transitions is three times higher than for the TR1 and TR2 compounds due to longer linker fragment in the TR3 molecule. This fact is in good agreement with the experimental spectra regarding the first absorption band (εmax 0 −0 = 7 × 104 M−1cm−1 for the TR1 and TR2 compounds, while for the TR3 compound, εmax 0 −0 = 3.4 × 105 M−1cm−1) [6]. Solvatochromic effect The higher energy band in the absorption spectrum of TR1 (at 4.16 eV), TR2 (at 4.13 eV) and TR3 (at 4.07 eV) corresponds to a local excitation of the ππ-type in the carbazole moieties (Tables S3, S4). Fig. 4 Dipole moment μ (D) for the ground (S0) and first excited (S1, ) states of the TR1–TR3 dyes as a function of dielectric permittivity of solvent The HOMO is a non-bonding orbital with respect to such a link, but the LUMO is a bonding orbital (Fig. 3); thus, the force constant of this mode is higher in the excited state. Such a strong change in the force field, and of the mode displacement upon excitation, leads to the occurrence of a long progression of the νC–C mode (1276 cm−1) in the first absorption band. The higher energy band in the absorption spectrum of TR1 (at 4.16 eV), TR2 (at 4.13 eV) and TR3 (at 4.07 eV) corresponds to a local excitation of the ππ-type in the carbazole moieties (Tables S3, S4). Fig. 3 Plot of the calculated absorption spectra of the studied dyes TR1– TR3 Fig. 4 Dipole moment μ (D) for the ground (S0) and first excited (S1, ) states of the TR1–TR3 dyes as a function of dielectric permittivity of solvent (at 4.07 eV) corresponds to a local excitation of the ππ-type in the carbazole moieties (Tables S3, S4). Fig. 3 Plot of the calculated absorption spectra of the studied dyes TR1– TR3 Fig. 4 Dipole moment μ (D) for the ground (S0) and first excited (S1, ) states of the TR1–TR3 dyes as a function of dielectric permittivity of solvent Fig. 3 Plot of the calculated absorption spectra of the studied dyes TR1– TR3 Fig. 4 Dipole moment μ (D) for the ground (S0) and first excited (S1, *) states of the TR1–TR3 dyes as a function of dielectric permittivity of solvent Fig. 3 Plot of the calculated absorption spectra of the studied dyes TR1– TR3 J Mol Model (2017) 23: 55 55 Page 6 of 8 Fig. 5 Orientation of the permanent dipole moment vector for the ground (S0) and first excited (S1) singlet state of molecules TR1–TR3 anent dipole moment vector for the ground (S0) and first excited (S1) singlet state of molecules TR1–TR Fig. Solvatochromic effect 5 Orientation of the permanent dipole moment vector for the ground (S0) and first excited (S1) singlet state of molecules TR1–TR3 A strong electric polarization of the TR1–TR3 molecules in the S1 excited state provides strong stabilization of the ex- cited molecule in polar solvents in comparison with nonpolar media. This means that more polar solvents provide stronger solvation of the excited-state molecule, which leads to a de- crease in S1 state energy. This statement is in perfect qualita- tive agreement with experimental observations: the fluores- cence wavelength increases strongly with the rise of solvent polarity. At the same time, the energy of the vertical S0→S1 transition (in absorption) is almost insensitive for the solvent effect, which means a strong increase in Stokes shift (Δν) with the rise in solvent polarity (experimentally, the Δν values change from the 615 cm−1 in nonpolar hexane to 9842 cm−1 in highly polar acetonitrile). Though our IEFPCM-B3LYP-30/6- 31G(d) calculations strongly overestimate quantitatively the solvation energy of the S1 state of TR1–TR3, the overall case of TR1 and TR2. At the same time, the corresponding μ values for TR3 tend towards a minimum (Fig. 4). But, in all cases, the variation in the μ value is not more than 0.25 D between the non-polar cyclohexane solvent and the strongly polar acetonitrile. A similar correlation occurs for the S0–S1 vertical transition energies vs. dielectric permittivity. Both the theoretical and experimental energies decrease towards the rise in solvent polarity (Table 3). The calculated transition energies, however, are more robust to a change of solvent; in practice, this effect is much more pronounced [6]. The μ value and vector orientation change crucially upon electronic excitation of TR1–TR3 molecules into the S1 state (Figs. 4, 5). We predicted an increase of >30 times the perma- nent dipole moment for the S1 exited state of molecules TR1– TR3, which is caused by the strong charge separation upon the S0–S1 electronic transition of CT nature. Table 3 The vertical (vert.) and adiabatic (ad.) S0–S1 transition energies (eV) for the TR1–TR3 molecules as a function of dielectric permittivity of solvent (theoretical values calculated using the B3LYP-30 scheme) Conclusions 3. Michaleviciute A, Gurskyte E, Volyniuk DYU, Cherpak VV, Sini G, Stakhira PY, Grazulevicius JV (2012) J Phys Chem C 116: 20769–20778 In this work, we have presented a computational study of the absorption spectra for a series of star-shaped compounds con- taining both D (carbazole) and A (2,4,6-triphenyl-1,3,5-tri- azine) moieties bound through various linking bridges. These compounds demonstrate solvent-sensitive absorption in the whole visible range depending on solvent polarity. This is due to the strong charge-polarization of the studied molecules upon excitation into the S1 excited state of CT na- ture. It has been confirmed by the very high permanent dipole moment for the S1 excited state (>30 D) rather than for the S0 state (≤1 D). As a result, the solvatochromic effect should be observed only in fluorescence spectra, but not in absorption spectra, in complete agreement with experimental data. 4. Kreger K, Bäte M, Neuber C, Schmidt H-W, Strohrieg P (2007) Adv Funct Mater 17:3456–3461 5. Jin R (2015) J Mol Model 21:219–228 6. Matulaitis T, Kostiv N, Grazulevicius JV, Peciulyte L, Simokaitiene J, Jankauskas V, Luszczynska B, Ulanski J (2016) Dyes Pigments 127:45–58 7. Hung W-Y, Chiang P-Y, Lin S-W, Tang W-C, Chen Y-T, Liu S-H, Chou P-T, Hung Y-T, Wong K-T (2016) ACS Appl Mater Interfaces 8:4811–4818 8. Cherpak V, Stakhira P, Minaev B, Baryshnikov G, Stromylo E, Helzhynskyy I, Chapran M, Volyniuk D, Hotra Z, Dabuliene A, Tomkeviciene A, Voznyak L, Grazulevicius JV (2015) ACS Appl Mater Interfaces 7:1219–1225 9. Angioni E, Chapran M, Ivaniuk K, Kostiv N, Cherpak V, Stakhira P, Lazauskas A, Tamulevičius S, Volyniuk D, Findlay NJ, Tuttle T, Grazulevicius JV, Skabara PJ (2016) J Mater Chem C 4:3851–3856 An interesting feature of the S0-S1 transition for the studied compounds is a high intensity in the absorption spectrum, which is unusual for the CT transition. This result can be ex- plained by the fact that the corresponding HOMO and LUMO+ 1 orbitals possess large contributions at the common atoms of the linker, which provides a large transition dipole moment for the S0-S1 transition. Actually, the solvent-dependent fluores- cence for the studied compounds can be well explained by the localized CT excited state concept, which should be appli- cable to related D-A star-shaped systems. 10. Baryshnikov GV, Minaev BF, Minaeva VA, Ning Z, Zhang Q (2012) Opt Spectrosc 112:168–174 11. References 1. Detert H, Lehmann M, Meier H (2010) Materials 3:3218–3330 2. Ren S, Zeng D, Zhong H, Wang Y, Qian S, Fang Q (2010) J Phys Chem B 114:10374–10383 Solvatochromic effect ( g ) Dye n-Hexane (ε = 1.88) Toluene (ε = 2.37) CHCl3 (ε = 4.71) THF (ε = 7.43) CH2Cl2 (ε = 8.93) Acetone (ε = 20.49) CH3CN (ε = 35.69) Exp Theor Exp Theor Exp Theor Exp Theor Exp Theor Exp Theor Exp Theor TR1 (vert.) 3.12 3.22 3.22 3.18 3.27 3.19 3.28 3.2 3.3 3.2 3.33 3.2 3.35 3.2 TR1 (ad.) 3.19 2.17 2.86 2.01 2.58 1.64 2.55 1.49 2.48 1.44 2.38 1.29 2.26 1.24 TR2 (vert.) 3.12 3.18 3.15 3.18 3.2 3.19 3.24 3.2 3.24 3.2 3.26 3.2 3.35 3.2 TR2 (ad.) 3.05 2.05 2.79 1.9 2.53 1.55 2.52 1.4 2.42 1.36 2.32 1.22 2.13 1.17 TR3 (vert.) 3.15 3.1 3.15 3.05 3.18 3.06 3.17 3.05 3.19 3.05 3.23 3.05 3.24 3.05 TR3 (ad.) 3.1 2.79 2.74 2.68 2.54 2.37 2.45 2.23 2.4 2.18 2.23 2.06 2.13 2.02 J Mol Model (2017) 23: 55 Page 7 of 8 Page 7 of 8 55 by the Swedish National Infrastructure for Computing (SNIC) at the Parallel Computer Center (PDC) through the project BMultiphysics Modeling of Molecular Materials^, SNIC 020/11-23. tendency of the S1 energy vs. solvent polarity dependence is in good agreement with the experimental data. 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For the TR3 molecule, the excited state conforma- tional structure is the same as the ground state structure 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. 24. Stratmann RE, Scuseria GE, Frisch MJ (1998) J Chem Phys 109: 8218–8224 25. Adamo C, Barone V (1999) J Chem Phys 110:6158–6170 27. Yanai T, Tew D, Handy N (2004) Chem Phys Lett 393:51–57 28. Boese AD, Martin JML (2004) J Chem Phys 121:3405–3416 Conclusions Baryshnikov GV, Minaev BF, Myshenko EV, Minaeva VA (2013) Opt Spectrosc 115:484–490 12. Minaev BF, Baryshnikov GV, Minaeva VA (2011) Dyes Pigments 92:531–536 13. 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https://openalex.org/W3174029343	https://csimq-journals.rtu.lv/article/download/csimq.2021-29.01/2915	English	null	Virtual Prototyping: Evaluating the Digital Twin Based Virtual Factory for New Product Introduction	Complex Systems Informatics and Modeling Quarterly	2,021	cc-by	9,074	Complex Systems Informatics and Modeling Quarterly (CSIMQ) eISSN: 2255-9922 Published online by RTU Press, https://csimq-journals.rtu.lv Article 163, Issue 29, December 2021/January 2022, Pages 1–16 https://doi.org/10.7250/csimq.2021-29.01 Complex Systems Informatics and Modeling Quarterly (CSIMQ) eISSN: 2255-9922 Published online by RTU Press, https://csimq-journals.rtu.lv Article 163, Issue 29, December 2021/January 2022, Pages 1–16 https://doi.org/10.7250/csimq.2021-29.01 Complex Systems Informatics and Modeling Quarterly (CSIMQ) eISSN: 2255-9922 Published online by RTU Press, https://csimq-journals.rtu.lv Article 163, Issue 29, December 2021/January 2022, Pages 1–16 https://doi.org/10.7250/csimq.2021-29.01 Additional information. Author ORCID iD: E. Yildiz – orcid.org/0000-0003-3002-0873, Ch. Møller – orcid.org/0000-0003- 0251-3419, A. Bilberg – orcid.org/0000-0002-4780-8459, and J. K. Rask – orcid.org/0000-0002-8926-1826. PII S225599222100163X. Received: 19 July 2021. Accepted: 13 December 2021. Available online: 31 December 2021. © 2021 Emre Yildiz, Charles Møller, Arne Bilberg, and Jonas Kjær Rask et al. This is an open access article licensed under the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0). Reference: E. Yildiz, C. Møller, A. Bilberg, and J. K. Rask “Virtual Prototyping: Evaluating the Digital Twin Based Virtual Factory for New Product Introduction” Complex Systems Informatics and Modeling Quarterly, CSIMQ, no. 29, pp. 1–16, 2021. Available: https://doi.org/10.7250/csimq.2021-29.01 * Corresponding author © 2021 Emre Yildiz, Charles Møller, Arne Bilberg, and Jonas Kjær Rask et al. This is an open access article licensed under the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0). Reference: E. Yildiz, C. Møller, A. Bilberg, and J. K. Rask “Virtual Prototyping: Evaluating the Digital Twin Based Virtual Factory for New Product Introduction” Complex Systems Informatics and Modeling Quarterly, CSIMQ, no. 29, pp. 1–16, 2021. Available: https://doi.org/10.7250/csimq.2021-29.01 Additional information. Author ORCID iD: E. Yildiz – orcid.org/0000-0003-3002-0873, Ch. Møller – orcid.org/0000-0003- 0251-3419, A. Bilberg – orcid.org/0000-0002-4780-8459, and J. K. Rask – orcid.org/0000-0002-8926-1826. PII S225599222100163X. Received: 19 July 2021. Accepted: 13 December 2021. Available online: 31 December 2021. Keywords: Virtual Factory, Digital Twin, Virtual Prototyping, Virtual Reality, Simulation and Modeling, Industry 4.0. 1 Introduction Forces like innovation and technology, competition, changing demands, and regulations are among the main dynamics that shape the evolution of industries [1]. The specific rhythm of the evolution in each manufacturing industry occurs in three domains: products, processes, and systems [2]. Therefore, companies need to handle concurrent evolution (co-evolution) of product, process, and system models to achieve the capability to adapt to their respective industrial environments [3]. However, the increasing frequency of changes results in shorter product and production lifecycles and shifting of decision-making from companies to customers, which results in higher complexity. Thus, achieving architectural isomorphism across the product, process and organisation (system) architecture required to maintain effective alignment of an organisation with its evolving environment [4] is becoming a significant challenge for manufacturing organisations. Physical prototype building activities during the introduction of a new product can be considered among the most critical activities to achieve and ensure architectural isomorphism. However, physical prototype builds are often highly time-consuming, costly, and complex due to the uncertain and genuine nature of models and operations. When it comes to the wind industry, wind turbine generator (WTG) manufacturing covers a wide variety of production and manufacturing operations, such as heavy metal manufacturing (towers), large-size fiberglass composite material production (blades), complex and heavy parts assembly (gearbox, nacelle, generator, etc.), and electrical and electronic systems manufacturing (control and grid infeed systems). Therefore, physical prototype builds during the New Product Introduction (NPI) are becoming much more challenging for companies such as Vestas Wind Systems A/S (later Vestas). Moreover, despite the particularities of the WTG manufacturing operations, there are significant similarities with various other industries, which can provide a sound basis for the generalisability of the knowledge discovered in this study. WTG tower manufacturing, for instance, incorporates noteworthy similarities with the heavy steel fabrication operations of the aerospace, construction, maritime, and oil and gas industries. WTG blades manufacturing contains unique characteristics, reaching 107 meters in length for a single piece of fiberglass composite product, which is one of the largest in the world [5]. In terms of size, WTG blade production has some similarities with maritime production, such as superyachts [6]. Meanwhile, the global fiberglass market value reached 13 billion USD as of 2020 and is expected to reach 18.6 billion USD by 2027, mainly driven by the automotive and construction industry [7]. Virtual Prototyping: Evaluating the Digital Twin Based Virtual Factory for New Product Introduction Emre Yildiz1*, Charles Møller1, Arne Bilberg2, and Jonas Kjær Rask3 1 Center for Industrial Production, Aalborg University, Fibigerstraede 16, 9220 Aalborg, Denmark 2 Department of Technology and Innovation, University of Southern Denmark, 6400 Sønderborg, Denmark 3 DIGIT, Aarhus University, Department of Electrical and Computer Engineering, Finlandsgade 22, 8200 Aarhus N, Denmark ey@mp.aau.dk, charles@mp.aau.dk, abi@iti.sdu.dk, jkr@ece.au.dk Abstract. Shortening lifecycles and increasing complexity make product and production lifecycle processes more challenging than ever for manufacturing enterprises. Virtual Prototyping (VP) technologies promise a viable solution to handle such challenges in reducing time and physical builds as well as increasing quality. In previous studies, the Digital Twin (DT) based Virtual Factory (VF) concept showed significant potential to handle co-evolution by integrating 3D factory and product models with immersive and interactive 3D Virtual Reality (VR) simulation technology as well as real-time bidirectional data synchronisation between virtual and physical production systems. In this article, we present an extension to the paper “Demonstrating and Evaluating the Digital Twin Based Virtual Factory for Virtual Prototyping” presented at CARV2021. The study presents an evaluation by industry experts of the DT based VF concept for VP in the context of New Product Introduction (NPI) processes. The concept is demonstrated in two cases: wind turbine blade manufacturing and nacelle assembly operations at Vestas Wind Systems A/S. The study shows that the VF provides an immersive virtual environment, which allows the users to reduce the time needed for prototyping. The industry experts propose several business cases for the introduced solution and find that the phases that would have the most gain are the later ones (production) where the product design is more mature. Keywords: Virtual Factory, Digital Twin, Virtual Prototyping, Virtual Reality, Simulation and Modeling, Industry 4.0. Keywords: Virtual Factory, Digital Twin, Virtual Prototyping, Virtual Reality, Simulation and Modeling, Industry 4.0. 1 Introduction While gearbox, generator, and nacelle manufacturing shares similar genetics with the automotive and heavy machinery industries, the converter and control systems of WTGs incorporate similar approaches to those of various electrical and electronic manufacturing industries. pp g Although there are significant affinities between WTG manufacturing operations and other industries like the automotive, aerospace and maritime industries, WTGs are still not mature products and are continuing to evolve rapidly, together with the associated manufacturing systems and processes. In terms of size and weight, for instance, modern cars, ships, and planes are not substantially different from the same products manufactured in the 1980s. WTGs, however, have now reached rotor sizes of over 200 meters from 10 meters in the 1980s, and over 400 tons of nacelle weight from 5 tons in the 1980s [8], [9]. Despite the tremendous increase in the sizes and weights of WTGs during the last four decades, the (onshore) wind energy cost per kilowatt/hour decreased to 0.05 USD from 0.4 USD during the same period [10]. Thus, the cost per megawatt oriented tendering approach results in significant pressure on WTG manufacturing companies to improve the performance of their turbines by redesigning their products, together with the associated processes and manufacturing systems. In this regard, the Digital Twin (DT) based Virtual Factory (VF) concept [11] is becoming a highly relevant solution by enabling integration, interoperability, and interaction capabilities across product and production lifecycle processes in virtual environments [12]. 2 In recent studies, the DT based VF is considered a promising solution to deal with co- evolution problems with its potential to achieve dynamic, open, holistic, and cognitive system capabilities [12], [13]. Moreover, industry experts considered Virtual Prototyping (VP) among the highest value promising industrial use cases for the DT based VF concept [12]. Although a virtual prototype as a computer simulation of a physical product covers all product lifecycle aspects, including service and maintenance [8], building and testing, the virtual prototype of NPI processes is particularly challenging due to the need for concurrent engineering and complex and ambiguous models and operations. However, due to the same challenges, VP maintains a significant potential for high value by enabling (1) early testing, (2) expensive or impossible tests, (3) fewer physical builds, (4) safer builds, (5) increased agility, (6) reduced cost, (7) complexity handling, and (8) reduced time to market [14]. 1 Introduction Thus, the need for evaluating the DT based VF concept in more particular VP use cases was raised in [12]. It should be noted that this article is an extension to the conference paper titled “Demonstrating and Evaluating the Digital Twin Based Virtual Factory for Virtual Prototyping” presented at CARV2021†[15]. This article is distinguished from the previous one by extending the evaluation and discussion, amongst other additions. Following the next section, which frames the scope and objectives of the work, Section 3 summarises related works on VP and VF. Section 4 describes the research methodology. Section 5 presents the results and evaluations of the industry experts, followed by the discussion and implications in Section 6 and conclusions in Section 7. † https://carv2020.com/ 2 Research Scope and Objectives In this study, we respond to the need addressed in [12] for evaluation of the DT based VF concept (introduced in [5]) in particular VP cases and thus present an evaluation of the concept in the context of NPI processes. The evaluation is established on the data gathered during group interviews with industry experts. In order to support the rigour and novelty of the work in hand, there is a need to clarify the differences between the scopes and objectives of the previous works [12] and the present work. The study in [12] was conducted to evaluate the DT based VF concept in terms of (1) achieving the cornerstones of the competence-based strategic management concept (dynamic, open, cognitive, and holistic), (2) enabling concurrent engineering of products, processes, and systems, (3) supporting shorter product and production lifecycles, (4) the usefulness, effectiveness, and consistency of artefacts [3]. Therefore, the essential premise for the arguments in [12] “is that DT based VF can support competence-based strategic management of manufacturing organisations during their adaptation to dynamic and complex environments.” While performing the evaluation of the concept broadly in the scope of competence theory, knowledge about the particular aspects of the concept, including implications of collaborative VR and VP, was discovered. Discussions on VP in [12] provided some pieces of evidence for the potentially high value of using DT based VF during the NPI processes. However, there was a need for in-depth discussions and data on “how” the concept could be utilised during NPI and contribute to each phase of prototyping. Moreover, due to the comprehensive and complex nature of coordinated engineering operations during the NPI, none of the individual interviewees in [12] was confident enough about their judgements on extensive utilisation of the concept for VP operations. Therefore, the present study is conducted by narrowing down the scope of the DT based VF evaluation and discussions on the VP context with a particular focus on NPI activities. Due to (1) the high level of coordinated engineering during NPI, which causes a limited view for an individual engineer, and (2) the diversity of manufacturing operations of WTGs, it was decided to conduct the interviews with focus groups with distinct engineering (tooling, training, design, etc.) and manufacturing (tower, blade, assembly, etc.) backgrounds. 3.1 Virtual Prototyping With the advances in Computer-Aided Design (CAD), Computer-Aided Manufacturing (CAM) and visualisation and interaction capabilities, the development of virtual environments and realistic virtual representations of product models has gained more attention from scholars and industry experts. Thus, development and interaction with such virtual models become viable solutions promising significant advantages for the industrial processes in terms of reducing time, decreasing costs, and increasing quality [18]. As a result of this, VP, a key aspect from the application point of view, is starting to get attention in both the application and knowledge domains. However, there have been many different interpretations of VP techniques, which have caused some confusion. To prevent such confusion, Wang defined a virtual prototype as “a computer simulation of a physical product that can be presented, analysed, and tested from concerned product lifecycle aspects such as design/engineering, manufacturing, service, and recycling as if on a real physical model. The construction and testing of a virtual prototype is called virtual prototyping (VP)” [19]. Wang also addressed the needs for concurrent design, analysis, optimisation, and integration of simulation tools. Some studies consider VP to alleviate the shortcomings of rapid prototyping (RP) [20], while others stress the difference between RP and VP [21]. Alongside early adoptions in the aerospace and automotive industries [14], VP technologies are also promising significant value in different industries such as construction [22], the maritime industry [23], and heavy machinery industries [24]. Studies about VP techniques focus on different product lifecycle aspects, including product design, analysis, testing and assembly process design [25]–[28]. However, there are limited studies focusing on the VP of products from a manufacturing aspect [29]. Although scholars concentrate on various VP simulations such as structural material and structural behaviour simulations [28], [30], recent studies show more attention to immersive VR integrated simulation tools [31]. Recent review studies show that advances in simulation technologies, including real- time data integration, realistic visual representations and embedded VR and augmented reality (AR) capabilities, can make simulations a proven enabler for digital integration and access to data across the product and production life cycles [32]. In this respect, the VF concept, as high-fidelity integrated factory simulations representing factories as a whole, can provide viable virtual environments for constructing and testing virtual prototypes, since they enable the experimentation and validation of the various product, process, and system models concurrently [11]. 2 Research Scope and Objectives Group interviews were 3 established to instigate richer arguments and discussions on an issue or a proposition by enabling the collision of diverse experiences and approaches on the same subject. Thus, the objective of the study is to conduct an exploratory work to discover knowledge on (1) how the DT based VF can be utilised in VP operations of WTG manufacturing, and (2) what benefits can be gained from such industrial implementation. Therefore, the concept is demonstrated in wind turbine blade manufacturing and nacelle assembly cases at Vestas, as in the previous work [12]. However, the evaluation was performed by a more specialised and experienced group of interviewees. In contrast to the previous evaluation, which included both higher- and lower-level experts, the interviewees in this work were mostly medium-level and technical experts with more (14.8) years of experience (compared to 11 years in [12]). Therefore, the study draws upon previous research, including concept design and development of the VF [16], its extension with DT [11], [12] and collaborative VR capabilities [17]. Thus, we spare the reader from prolonged discussions on the concept and its design and development methodologies. However, we would strongly recommend the reader to refer to the subject studies for in-depth discussions on practical and theoretical aspects of the work. 3.1 Virtual Prototyping Therefore, we will briefly present some studies focusing on VF in the next section. 4 3.2 Virtual Factory Since Onosato and Iwata [33], [34] introduced the integration of product and factory models as an integral aspect of VF and virtual manufacturing, various definitions have been given for VF, including emulation facility, integrated simulation and virtual organisations [35]. Jain et al. defined VF “as an integrated simulation model of major subsystems in a factory that considers the factory as a whole and provides an advanced decision support capability” [35]. An integrated VF framework concept which can synchronise the real factory and VF was introduced by Sacco, Pedrazzoli, and Terkaj [36]. While Jain et al. [37] stressed the multi-resolution capabilities of VF simulation models, Yang et al. [38] introduced the VF concept for collaborative design and analysis of manufacturing systems. There is also a higher level of utilisation of the VF concept as a collaborative business process monitoring environment for achieving business goals [39]. Yildiz and Møller [16] presented the VF concept as a more dynamic and open system by integrating VF into actual manufacturing execution and product lifecycle systems, as illustrated in Figure 1. A more distinct conceptualisation of the product, process and system domains in between the product and production lifecycle processes enables better interpretation of the link between these domains. Such representation is also considered a better way to handle complexity and improve efficiency for DT development and the fidelity of simulation models. The utilisation of collaborative VR training simulations in the VF concept, together with DT capabilities, was also studied by Yildiz et al. [11], [17]. They also considered VF as “an immersive virtual environment wherein digital twins of all factory entities can be created, related, simulated, manipulated and communicate with each other in an intelligent way” [16]. A comprehensive demonstration of the DT based VF concept in industrial cases [12] showed significant potential for the concept to handle co-evolution and called for more particular evaluation in VP cases. Figure 1. Digital Twin based Virtual Factory concept [12] Figure 1. Digital Twin based Virtual Factory concept [12] In this regard, we further discuss the methodology for the demonstration of the DT based VF concept in wind turbine blade manufacturing and nacelle assembly cases, as well as its evaluation in the context of VP in NPI processes. 5 5 4 Methodology There are two exercises of the knowledge, which are (1) creation of the knowledge and (2) applying the knowledge [40, p. 3]. Knowledge about natural phenomena is discovered/created by applying scientific methods like experimentation, observation, and empirical evidence collection. Such descriptive knowledge about nature enables the manipulation of the same nature by utilising purposefully designed physical (tools, machines, etc.) or conceptual artefacts (models, methods, etc.) [44]. When such artefacts take physical forms, it is usually called technology. The consequence of manipulating natural phenomena is the emergence of new scientific methods, discoveries and thus new knowledge [45]. Since it is not possible to provide an in-depth discussion on science‒technology dualism in this work, it is important to stress that the subject study aims to discover prescriptive knowledge, which is demonstrated in the utility, effectiveness, reliability, consistency, etc. of the designed artefact. Since this study aims to explore the practical usefulness of a conceptual design in empirical cases, Design Science Research Methodology (DSRM) is considered a well-suited methodology. Thus, the research demands instrumental knowledge to uncover the effects of interventions on a manufacturing enterprise’s specific (NPI) operations. Various research works which have been carried out over the last three years were conducted on the trails of guidelines, methods, and frameworks of DSRM [41]–[44]. However, this article covers only the demonstration and evaluation activities of six DSRM activities. Since the designed artefacts should have an impact on the practice, the design science research objective of the present paper is to report the kinds of impact of the IT artefact and design theories. Four demonstration and evaluation sessions were conducted, each with five participants, with a total of 20 participants. A session covered (1) presentation of the artifacts, tools, and capabilities, (2) live demonstration of DT based VF, including VR interaction, and (3) semi- structured group interviews. Therefore, the demonstration and evaluation methods are briefly articulated in the following sub-sections. 4.2 Group Interviews The group interviews aim to collect data to evaluate the DT based VF concept in the context of VP during the NPI processes with well-grounded pieces of evidence and arguments by exploring the interpretations and perspectives of industry experts. During the group discussions, the interpretations of the experts were critically examined by instigating a process of reflection to gain more specific, accurate and grounded pieces of evidence. Moreover, group discussions with diverse technical expertise on similar operations in different manufacturing domains allowed the experts to reach more reliable judgements or demonstrate independence through slight disagreements. Since the purpose of Design Science Research (DSR) is to discover the practical usefulness of a solution to deal with empirical challenges with practical methods, DSR focuses on the pragmatic validity and practical relevance of a generic design [43]. Therefore, justification of a solution concerns, including but not limited to, the effectiveness, usefulness, and consistency. In this regard, group interviews were initially planned as physical meetings to enable interviewees to experience first-hand a DT based VF demo with immersive and interactive VR environments. Unfortunately, due to the COVID-19 pandemic, live demonstrations had to be performed during an online meeting which allowed the interviewees to see the physical and virtual environments but not fully experience VR. The interviews were recorded with the Microsoft Teams software tool and transcribed with services provided by the Microsoft Stream platform. The guidelines for designing and conducting interviews by Kvale [47] were followed. The number of interviewees was limited, with five in each session to avoid uneven participation in discussions, and to acquire more valuable knowledge with more intensive interviews and penetrating interpretations. Participants were intentionally mixed for each session based on their background and department (NPI, Blade, Manufacturing, Nacelle, etc.) to increase the diversity of expertise in each session. A list of the expert interviewees is available in the appendix. 4.1 Demonstration Demonstration of general artefacts (concepts, architectures, methods, demo) delineates how to use the developed artefacts effectively in particular contexts. Therefore, although the discovered knowledge will be about the particular context, it should be able to transfer into various contexts within the same application domain without losing its necessary effectiveness [43]. In this study, DT based VF is demonstrated in two diverse cases, including large composite manufacturing (blade) and complex and heavy parts assembly (nacelle) at Vestas. Although such cases are unique to the wind turbine manufacturing industry, there are significant common aspects with various industries such as shipbuilding, automotive, and aviation. Thus, the knowledge can provide guidelines for experienced professionals in the industry and enable the evaluation of deviations in the form of “if‒then” specific to each context. Since the DT based VF is a generic concept, and the capabilities and implications of the implemented concept depend on the context-specific tools, data and IT infrastructure, a short presentation was made to explain and distinguish the concept, tools, technologies, demo, and methods. Some parts of the data about the demonstration are subject to the intellectual and financial interest of Vestas. Thus, a significant part of the data is covered by the provisions given to Vestas by the research collaboration agreement. However, we strongly advise the readers to access the part of the demonstration video that is publicly available via [46]. The video shows examples of DT based VF simulations that are synchronised with process data from the real factory via the manufacturing execution system. The synchronisation allows the reflection of changes in VFs to produce manufacturing data analysis based on timings from the real factory. Furthermore, the video shows that you can manipulate the VFs in a VR environment and even have multiple users in the same VF simulation environment to perform collaborative and 6 coordinated manufacturing (assembly) operations. This enables operators to train together in the virtual environment on products that may not be in production yet. coordinated manufacturing (assembly) operations. This enables operators to train together in the virtual environment on products that may not be in production yet. 5.1 Mock-up Builds A mock-up is an early build of the concept design, which aims to verify the functionality of the early design before the detailed design is finalised. A mock-up can be a physical or non-physical build, including a 3D model, computer simulation, hardware in the test, or a mock-up in the factory. However, if the difference in the new design is not minor compared to previous product variants, mock-up activities usually involve a significant number of physical builds. Shifting to the containerised products under the scope of modularisation, for example, could require significant changes in product design, production processes and, thus, the shop floor systems. g g p g p p p y Most of the participants agreed that the DT based VF simulations could solve many problems and errors before early builds, but could not eliminate physical mock-up builds completely. This is mainly because NPI procedures dictate some early tests, such as a fatigue test on the physical build and a turbine test for legal certifications, requiring a minimum of three blades. Therefore, for the blade production case, it is considered that a minimum of four mock-up builds are inevitable under the present circumstances. However, 3D model simulations in factory environments were considered significantly beneficial due to increased confidence in the models (Int. 2). In addition, the majority of the experts considered that reducing the time spent on mock- up builds and errors found while using VF simulations would provide sufficient value for the business case. Some stated that the significance of the time spent on design and process changes is due to the high number of issues found during the mock-up builds (Int. 10). Quoting one of the experts: “Even in early stages, this (DT based VF) could be beneficial to at least anticipate some issues that might occur in production due to design mistakes or miscalculations in design. I think that would be a great help in the decision making (on design.)” (Int. 14). Moreover, extending the tools with a detailed and specific process and material simulations, such as material behaviours and resin injection, is considered highly valuable for the blade manufacturing case. Thus, one expert stated, “There are 1800 pieces of plies in each blade. If VF could be able to predict the tolerances, that would be a great business case.” (Int. 18). 5 Results and Evaluation In total, 20 interviewees participated in the demonstration and evaluation of the DT based VF concept in VP. Four sessions each included five experts and covered the introduction of the concept, demonstration, and evaluation/discussion sections, which took approximately 4 hours. Only the evaluation part of each session was recorded and took 50 minutes on average. The average years of experience of the experts were 14.8 and ranged from a minimum of 3 years to a maximum of 24 years. As anticipated, the expert discussions on the evaluation of DT based VF roamed around four terms, representing some of the main activities of NPI. These terms are (1) mock-ups, (2) design prototypes, (3) process prototypes, and (4) 0-series production. Therefore, the results and evaluations are organised and presented under the respective headlines. One of the recent product introduction operations at the case company comprised four mock- up builds, five design prototype builds, and four process prototypes. Although the cost of the prototyping depends significantly on the magnitude of the design modifications, the cost of prototype activities of a new wind turbine on such a scale can easily reach over 10 million €. Some of the comments, critiques, and arguments from the industry experts are referenced to the respective participant number in the appendix. For instance, interviewee number 5 is referenced as (Int. 5). 7 7 5.1 Mock-up Builds Simulating the resin injection process during the WTG blade production is conventionally considered significant for the design and performance of the product. However, experts highlight that such high-resolution production processes and low-resolution factory operations have significant mutual impacts. Heat and humidity originated at the geographical location, for instance, can have a significant impact on such processes as resin injection or torque processes and, eventually overall factory operations. Thus, integrated VF simulations could enable both analyses with high-resolution simulations and synthesis with low-resolution simulations. 5.2 Design Prototypes A design prototype is a physical build that represents the final design documentation and aims to verify functionality and requirements at the system, product and component level. Ideally, the design prototype should finalise and freeze the Engineering Bill of Materials (eBOM), but most likely, some corrections on the design will be made later on. DT based VF simulations were considered more beneficial for design prototypes than mock- up builds by the majority of experts since design prototypes are focused less on specific/critical materials behaviours and performances and more on the overall design integrity. It was stated by (Int. 18) that the majority of the issues (failures, corrections, improvements) faced during one of the late blade introduction processes were design-related (drawing corrections 40%, cutting file corrections 24%, bill of materials corrections 14%, among others). Therefore, aiming to find such issues in DT based VF simulations is considered a better approach (Int. 18). On the other hand, one expert stated that “Replacing the (physical) model is not something we should consider for the near or medium future. But I think that if we could go that way, we could put a good CAD model of the product into the VF environment to do the same prototyping. So, we can play with it and test it in the weeks before the design prototype. Then I think we can lower the 8 time that we spend on making the design prototype. But avoiding the design prototype completely is a bit optimistic as it is now. Considering the last design prototype, I guess VF would possibly help to decrease the time to market. We probably have several 100 issues in a design prototype. They are mostly product-related. My guess is around 10% is the space issues (which can be solved by VF).” (Int. 11). Furthermore, the commonly agreed statement made by (Int. 17) was, “I do not think we should reduce the number of blades that we built (in prototyping processes). Yes, I know they are expensive, but I think we can improve the quality and perhaps improve the speed.” Such stress on increasing quality could link to the increasing cost of downtime due to quality problems in WTGs in recent years [48]. q y p y In the nacelle assembly case, cable routing and measurements in 3D CAD models are considered a highly challenging process. 5.2 Design Prototypes “It is supercritical, especially when we try to design a cable bundle from point A to point B, and it goes above the four different cable trays and bins. It is very difficult to draw them laying correctly on the cable tray and all the way, and that is where we see the most of the errors.” (Int. 20). The impact of such challenges on the production process on the shop floor can also be considered a sign of the importance of multi-resolution simulations of the factory operations. 5.3 Process Prototypes The process prototype is a physical representation that has the full functionality of the final design. The purpose of the process prototypes is to verify the various documentations of the design concerning the Health, Safety and Environment (HSE), manufacturability, production process (mBOM; Manufacturing Bill of Materials), production setup, transportation, installation, and service (sBOM; Service Bill of Materials). DT based VF is considered highly useful for process prototypes. “Designing and modifying the production layout to find out: How to execute? How big a part of a nacelle we build and try to join them with the crane at the states. That is something we could definitely figure out in (VF) for new products.” (Int. 20). However, most experts also considered the output of physical process prototypes and previous prototypes as very useful for simulating what-if scenarios and optimising 0-series and serial production processes (Int. 2). One expert stressed the importance of specific/detailed process simulations with high-level simulations by stating, “if we were be able to integrate other simulations like the infusion process with VF simulations, then we could run the blade production process right from lay-up till the curing process. The infusion process includes the material properties, the curing properties, the heating, and the moulds conditions. So, if these applications could talk to each other, then we could run the entire process within a single platform.” (Int. 18). 5.5 Other Reflections DT based VF is also considered for various capabilities besides simulating various prototype activities during NPI. The VR capabilities of the DT based VF were considered valuable for pre- training for large scale production roll-outs: “what if we have a new factory starting up where 1000 people are in the organisation (facility). Then we can do pre-training before we actually put them out on the mould.” (Int. 17). Moreover, collaborative VR shows a sign of higher value for communication with suppliers, and it is considered very “useful to have VF during technology transfer between factories.” (Int. 1). However, one expert argued that “when implementing VR, we need to be very focused on who will use it, because not everyone can work with it and that requires change management ‒ a change of mindset.” (Int. 11). One interviewee also addressed the risk of a rapidly increasing volume of data that needs to be input to the models for achieving close-to-reality models and stressed the importance of DT technology (Int. 20). Another argued that “Approximately every 6 to 10 years we have a new generation nacelle. In between these years, the designs are very similar, and of course, it is easy to create a digital twin. But if we are introducing a new generation nacelle, it is more difficult to have a digital twin of the production.” (Int. 1). The possibility of disrupting the product development process based on the VF concept was discussed during the evaluation. Here it was mentioned that the development process follows a strict gate/tier process, where the product design is locked by the time it reaches production development. This means that tools and processes are not defined and might not be available. To reduce this issue, one expert stated that “it would be beneficial if we went as soon as the design is finished or maybe even before that, when we have the model of the product, we could start simulating the process. Maybe we should change the way we look at it and start with the tools earlier.” (Int. 10). It was also mentioned that the concept could provide safety benefits in that “there are so many safety aspects within a nacelle building, a blade building, a tower where we could utilise the safety aspects in VF as well. Also, the system could capture if VR trainees had the right safety behaviour.” (Int. 17). 5.5 Other Reflections This was further reinforced by the expert: “I have seen examples (…) where workers did the safety training through the virtual reality headset. (…) They reduced from 32 days to seven or eight days by having virtual reality training sessions.” (Int. 17). 5.4 0-Series Production 0-series production is nominally the same as the serial production on the line. However, it represents the design in the line to verify the production capacity in terms of resources, tools, space, etc., and the processes capabilities to achieve the expected takt time with the proper quality requirements. The number of physical builds in 0-series production generally depends on accelerating the learning curve and the ability to reach the target takt time. 0-series production is considered the most effective use case for DT based VF since the design and processes are more mature in this phase. Thus, DT based VF promises high value by enabling the Vestas experts “to start up with the right sequence, the right staffing, right factory layout, and have a shorter time to market,” (Int. 5) since the “use and distribution of labour throughout the blade production is extremely time-consuming.” (Int. 4). Accelerating the learning curve, which represents how to produce faster, is considered highly possible. One expert stated that reaching the actual takt time for a nacelle may take three to six months and added, “VF would help to save 25% of the time of 0-series.” (Int. 11). The criticality of this issue is that 9 “as soon as we get into 0-series and very short takt times, then we will notice (for example) the lack of crane capacity very quickly. The lead time for getting a new crane from the day we realise it – if we are very lucky – is four to six weeks. So that can be a very serious limiting factor. It is very valuable to see if the crane capacity is a limiting factor and it would be very useful to try out various scenarios in VF simulations.” (Int. 11). Thus, DT based VF is considered very useful “to understand what the bottlenecks are to identify the risk of moving the paths or fixing the assembly sequences,” (Int. 13), as well as “lean optimisations on the operators.” (Int. 17). 6 Discussion and Implications The research work presented in this paper achieved the demonstration and evaluation of the DT based VF concept in the context of VP cases, particularly NPI processes. While the previous works evaluated the concept in terms of supporting the co-evolution of manufacturing enterprises, the present work focuses on the potential implications of the concept, particularly in the mock-up build, design prototype, process prototype and 0-series production processes, which are articulated based on expert comments. The expert comments provide valuable pieces of evidence and data on how and to what extent DT based VF can support complex manufacturing operations during the NPI processes. 10 Since the introduction of a new WTG into the renewable energy market is critical to compete for market share, reducing the time to market is considered strategically vital for WTG manufacturers. Therefore, there is significant pressure on coordinated engineering operations during NPI. However, the expert comments give signs that such pressure also creates significant quality problems later in the manufacturing operations and product performance. NPI engineers are simply aiming to mature their product, process, and system/organisation models from mock- up build to 0-series production. Physical prototypes provide better evidence and greater confidence to make decisions during the NPI process yet lead to significant time and cost issues. Thus, the majority of the experts assert that reducing the time to market should not be a performance indicator for utilising a solution like the DT based VF in early NPI, but instead reducing the number of issues/problems during the NPI operations. Moreover, the limitations on decreasing the physical prototype builds mean that taking this as a basis for fully eliminating physical prototypes in the near future is not realistic. p y p yp Since the bidirectional real-time data integration is inherent in the DT based VF concept, questions are raised on how to utilise the historical data of a digital twin technology for a fast- changing model. For example, the historical data of a DT could simply be irrelevant in a totally new production setup. Therefore, DT based VF is considered more valuable for later phases of NPI operations due to the larger volume of data and more mature models. 6 Discussion and Implications Moreover, this may support the view that easy-to-use DES tools without advanced integration (with MES and PLM), and the DT capability could still be more valuable for early concept/idea simulations by enabling rapid iterations of lightweight model development and simulations. Overall, the integration of multi-resolution simulations to represent a manufacturing system including its subsystems stands as the core capability of VF to handle evolving complexity. Such integration enables greater confidence in the impacts of major changes in overall operations from both the bottom-up and top-down approaches. Changing the WTG towers from steel to wood [49], for instance, could cause significant changes in material processing and thus the rest of operations assembly, transportation, etc. A dedicated simulation of wood processing (high resolution) could give reliable results to model and simulate the operations at the respective production line, factory, transportation, or installation (low resolution) level. Thus, the chain of reactions triggered by changes at various levels could be modelled and simulated to handle complexity in a flexible and agile way. Funding The Manufacturing Academy of Denmark (MADE) and Vestas Wind Systems A/S funded the research presented in this article, including equipment support (Grant: 6151-00006B). The authors of this article would also like to thank FlexSim for supporting our research by providing the license free of charge. 7 Conclusion This article addresses the need for thorough case evaluations of the DT based VF concept in the context of VP during NPI activities. DT based VF can enable the coordinated engineering of product and production lifecycle processes by enabling the integrated representation of product, process, and systems (organisation) models for designing, verifying, optimising and interacting with such models. Although the VF is not a brand-new concept, recent developments in state-of- the-art technologies like IoT, DT and VR and their integration into modelling and simulation tools are exploiting the potential of the VF concept exponentially. The majority of the studies on VF focus on either very specific cases and technologies or high-level conceptual work. Therefore, the present study aims to close the gap between theory and practice by exploring (1) the implications of the DT based VF in VP operations of WTG manufacturing and (2) the benefits that can be gained from industrial implementation of this concept. The concept was demonstrated in the wind turbine blade and nacelle production facilities of Vestas and evaluated by industry experts during semi-structured group interviews. The results of the evaluation indicate that the DT based VF concept can have significant value in multiple phases of prototyping. However, the experts agree that the most value would be added in the later phases of product introduction, where this concept provides an integrated representation of the products, processes and system (factory) models. In these phases, the product design will be more mature, but the process is still at a preliminary stage. By providing the integration, the experts can discover design errors in the product and process without creating real-life prototypes. This works towards reducing the prototyping time and increasing the initial quality of the product and process. Finally, the experts address a need for an extension of the demo with more specific simulations focused on material design and behaviour (with higher resolution). 6.1 Limitations and Future Work The work presented in this article covers only the demonstration and evaluation activities of the DSRM. The other DSRM activities, including the problem identification, definition of the objectives of the solution, design, and development activities, that were performed during the previous three years, were covered in previous studies. Thus, the reader should refer to those studies for more information to close the gap between the initial claim, which is the design and development of the DT based VF solution [11], [16], [17], and demonstration and evaluation. Moreover, conducting a study to evaluate such a comprehensive solution in dynamic, social, and complex organisations carries the risk of internal validity confusions [50]. This is mainly rooted in the difficulty of isolating dynamic and complex manufacturing organisations that inherit a large number of interdependent variables. Such challenges limit us to performing experimental or quasi-experimental studies. Therefore, it should be kept in mind that the data in terms of articulating the capabilities of the solution and other interpretations provided by experts during the interviews are context-specific, relying on the demonstration. It is observed that the participants were finding it hard to articulate the implications of DT based VF in high resolution manufacturing operations like specific machining, drilling, welding, and resin injection simulations. Therefore, increasing the scale of resolution in the DT based VF concept in future works could make it possible to capture more quantitative data and cases promising tangible value for the concept. 11 Moreover, due to increasing size and weight, together with regulation and localisation requirements in the wind energy market, more radical solutions like moveable/containerised factories or containerised WTGs are attracting attention. Therefore, implementing the DT based VF concept in a moveable factory concept while extending the simulations with transportation, on-site assembly operations, etc. to simulate the concurrent evolution of the product, process and system/organisation as well as its impact on the value chain, promises a significant potential. 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References Available: https://doi.org/10.1016/j.jom.2018.10.003 15 Appendix: List of Interviewees List of Expert Interviewees Interview Date & Time No Interviewee Title Years of experience Department 18.03.2021 & 14:30 1 Industrialisation Lead 12 New Product Master (NPM) 2 Manufacturing Intelligence 13 Blade Launch & Execution Centre (BLEC), Manufacturing Intelligence 3 Manager 15 BLEC Blade Launch Team 4 Continuous Improvement Specialist 15 BLEC, Continuous Improvement 5 Industrialisation Lead 20 Manufacturing Readiness – Blade (BLA) 22.03.2021 & 10:00 6 Senior Specialist 19 Functional Excellence 7 Senior Specialist, Performance & Execution 24 BLEC, Performance & Execution 8 Design for Excellence- Lead Professional 11 Design for Manufacturing 9 Lead Health, Safety, Environment Specialist 22 BLEC 10 NPM Lead (Electrical) 12 Manufacturing Readiness – Assembly and Towers (ASSY/TOW) 23.03.2021 & 10:00 11 Specialist 11 Manufacturing Readiness - ASSY/TOW 12 NPM Specialist 21 Manufacturing Readiness - BLA 13 Specialist 13 Manufacturing Readiness - ASSY/TOW 14 Design for Excellence Engineer 3 Design for Manufacturing 15 Tooling Professional Quality Tools 15 Manufacturing Readiness - BLA 29.03.2021 & 14:00 16 NPM Lead (Blades) 14 Manufacturing Readiness - BLA 17 Sr. Manager, Training & Global Transfer 10 Global Training & Knowledge Transfer 18 NC Tech Continuous Improvement lead 13 Manufacturing Readiness - NC TECH 19 NPM Lead (Towers) 13 Manufacturing Readiness - ASSY/TOW 20 Specialist 21 Manufacturing Readiness - ASSY/TOW Appendix: List of Interviewees 16
https://openalex.org/W4309660170	https://iris.unimore.it/bitstream/11380/1313149/2/keac661.pdf	English	null	Safety and tolerance of vaccines against SARS-CoV-2 infection in systemic lupus erythematosus: results from the COVAD study	Rheumatology	2,022	cc-by	11,001	Safety and tolerance of vaccines against SARS-CoV-2 infection in systemic lupus erythematosus: results from the COVAD study Safety and tolerance of vaccines against SARS-CoV-2 infection in systemic lupus erythematosus: results from the COVAD study Naveen R. 1, Elena Nikiphorou2,3, Mrudula Joshi4, Parikshit Sen5, Julius Lindblom6, Vishwesh Agarwal7, James B. Lilleker 8,9, Ai Lyn Tan 10,11, Babur Salim12, Nelly Ziade 13,14, Tsvetelina Velikova15, Abraham Edgar Gracia-Ramos 16, Masataka Kuwana 17, Jessica Day 18,19,20, Ashima Makol21, Oliver Distler 22, Hector Chinoy 8,23,24, Lisa S. Traboco25, Suryo Anggoro Kusumo Wibowo26, Erick Adrian Zamora Tehozol27, Jorge Rojas Serrano28, Ignacio Garcıa-De La Torre29, COVAD Study Group§, Rohit Aggarwal30, Latika Gupta 1,8,31,32, Vikas Agarwal1†, Ioannis Parodis 6,33†* 1Department of Clinical Immunology and Rheumatology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, India 2Centre for Rheumatic Diseases, King’s College London, London, UK 3 1Department of Clinical Immunology and Rheumatology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, India 2Centre for Rheumatic Diseases, King’s College London, London, UK 3 , g g , , 3Rheumatology Department, King’s College Hospital, London, UK , g g , , 3Rheumatology Department, King’s College Hospital, London, UK gy p g g p 4Byramjee Jeejeebhoy Government Medical College and Sassoon General Hospitals, Pune, India 5 g , , , 6Division of Rheumatology, Department of Medicine Solna, Karolinska Institutet and Karolinska University Ho 7 gy p 7Mahatma Gandhi Mission Medical College, Navi Mumbai, Maharashtra, India g 8Centre for Musculoskeletal Research, Division of Musculoskeletal and Dermatological Sciences, School of Biological Sciences, Faculty Biology, Medicine and Health, Manchester Academic Health Science Centre, The University of Manchester, Manchester, UK 9 entre for Musculoskeletal Research, Division of Musculoskeletal and Dermatological Sciences, School of Bio ology, Medicine and Health, Manchester Academic Health Science Centre, The University of Manchester, Ma Centre for Musculoskeletal Research, Division of Musculoskeletal and Dermatological Sciences, School of Biological Sciences, Fac Biology, Medicine and Health, Manchester Academic Health Science Centre, The University of Manchester, Manchester, UK 9 y gy, Manchester Centre for Clinical Neurosciences, Northern Care Alliance NHS Foundation Trust, Salford, UK 10NIHR Leeds Biomedical Research Centre, Leeds Teaching Hospitals Trust, Leeds, UK 11 11Leeds Institute of Rheumatic and Musculoskeletal Medicine, University of Leeds, Leeds, UK 12 gy p j p p 13Rheumatology Department, Saint-Joseph University, Beirut, Lebanon 14 13Rheumatology Department, Saint-Joseph University, Beirut, Lebanon 14 gy p p y 14Rheumatology Department, Hotel-Dieu de France Hospital, Beirut, Lebanon 15 14Rheumatology Department, Hotel-Dieu de France Hospital, Beirut, Lebano 15 15Department of Clinical Immunology, Medical Faculty, University Hospital ‘Lozenetz’, Soﬁa University St. Rheumatology, 2023, 62, 2453–2463 https://doi.org/10.1093/rheumatology/keac661 Advance access publication 22 November 2022 Original article Rheumatology Rheumatology, 2023, 62, 2453–2463 https://doi.org/10.1093/rheumatology/keac661 Advance access publication 22 November 2022 Original article Rheumatology Rheumatology, 2023, 62, 2453–2463 https://doi.org/10.1093/rheumatology/keac661 Advance access publication 22 November 2022 Original article Rheumatology Rheumatology, 2023, 62, 2453–2463 https://doi.org/10.1093/rheumatology/keac661 Advance access publication 22 November 2022 Original article Rheumatology, 2023, 62, 2453–2463 https://doi.org/10.1093/rheumatology/keac661 Advance access publication 22 November 2022 Original article 63 ogy/keac661 November 2022 Rheumatology Clinical science Luke’s Medical Center-Global City, Taguig, Philippines 26Division of Rheumatology, Department of Internal Medicine, Faculty of Medicine, Universitas Indonesia/Dr Cipto Mangunkusumo General Hospital, Jakarta, Indonesia 27Autoimmunity Division, Centro Me´dico Pensiones, Me´rida, Yucata´n, Mexico 28Rheumatologist and Clinical Investigator, Interstitial Lung Disease and Rheumatology Unit, Instituto Nacional de Enfermedades Respiratorias, Mexico City, Mexico 29Departamento de Inmunologıa y Reumatologıa, Hospital General de Occidente and University of Guadalajara, Guadalajara, Jalisco, Mexico 30Division of Rheumatology and Clinical Immunology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA 31Department of Rheumatology, Royal Wolverhampton Hospitals NHS Trust, Wolverhampton, UK 32City Hospital, Sandwell and West Birmingham Hospitals NHS Trust, Birmingham, UK 33Department of Rheumatology, Faculty of Medicine and Health, O¨ rebro University, O¨ rebro, Sweden ownloaded from https://academic.oup.com/rheumatology/article/62/7/2453/6839954 by Universita degli Studi di Modena e Reggi user on 31 August 2023 V C The Author(s) 2022. Published by Oxford University Press on behalf of the British Society for Rheumatology. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (https://creativecommons.org/ licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Received: 16 September 2022. Accepted: 10 November 2022 Safety and tolerance of vaccines against SARS-CoV-2 infection in systemic lupus erythematosus: results from the COVAD study Kliment Ohridski, Soﬁa, Bulgaria 16Department of Internal Medicine, General Hospital, National Medical Center, ‘La Raza’, Instituto Mexicano del Seguro Social, Mexico City, Mexico Department of Allergy and Rheumatology, Nippon Medical School Graduate School of Medicine, Tokyo, Japan 17Department of Allergy and Rheumatology, Nippon Medical School Graduate School 18 p gy gy, pp 18Department of Rheumatology, Royal Melbourne Hospital, Parkville, VIC, Australia 18Department of Rheumatology, Royal Melbourne Hospital, Parkville, VIC, Australia 20Department of Medical Biology, University of Melbourne, Parkville, VIC, Australia 21 p gy y 21Division of Rheumatology, Mayo Clinic, Rochester, MN, USA 22 21Division of Rheumatology, Mayo Clinic, Rochester, MN, USA 22 gy y 22Department of Rheumatology, University Hospital Zu¨rich, University of Zu¨rich, Zu¨rich, Switzerland 23 i i i i C i 23National Institute for Health Research Manchester Biomedical Research Centre, Manchester University NHS Foundation Tr 23National Institute for Health Research Manchester Biomedical Research Centre, Manchester University NHS Foundation Trust, The University of Manchester, Manchester, UK niversity of Manchester, Manchester, UK y Department of Rheumatology, Salford Royal Hospital, Northern Care Alliance NHS Foundation Trust, Salford, U y 24Department of Rheumatology, Salford Royal Hospital, Northern Care Alliance NHS Foundation Trust, Salford, UK 25Department of Medicine Section of Rheumatology St Luke’s Medical Center-Global City Taguig Philippines Department of Rheumatology, Salford Royal Hospital, Northern Care Alliance NHS Foundation Trust, Salford, U Department of Medicine, Section of Rheumatology, St. Luke’s Medical Center-Global City, Taguig, Philippines gy, y p , , , Medicine, Section of Rheumatology, St. The Author(s) 2022. Published by Oxford University Press on behalf of the British Society for Rheumatology. h l d b d d h f h b l Clinical science Clinical science Safety and tolerance of vaccines against SARS-CoV-2 infection in systemic lupus erythematosus: results from the COVAD study Naveen R. 1, Elena Nikiphorou2,3, Mrudula Joshi4, Parikshit Sen5, Julius Lindblom6, Vishwesh Agarwal7, James B. Lilleker 8,9, Ai Lyn Tan 10,11, Babur Salim12, Nelly Ziade 13,14, Tsvetelina Velikova15, Abraham Edgar Gracia-Ramos 16, Masataka Kuwana 17, Jessica Day 18,19,20, Ashima Makol21, Oliver Distler 22, Hector Chinoy 8,23,24, Lisa S. Traboco25, Suryo Anggoro Kusumo Wibowo26, Erick Adrian Zamora Tehozol27, Jorge Rojas Serrano28, Ignacio Garcıa-De La Torre29, COVAD Study Group§, Rohit Aggarwal30, Latika Gupta 1,8,31,32, Vikas Agarwal1†, Ioannis Parodis 6,33†* 1Department of Clinical Immunology and Rheumatology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, India 2Centre for Rheumatic Diseases, King’s College London, London, UK 3Rheumatology Department, King’s College Hospital, London, UK 4Byramjee Jeejeebhoy Government Medical College and Sassoon General Hospitals, Pune, India 5Maulana Azad Medical College, New Delhi, Delhi, India 6Division of Rheumatology, Department of Medicine Solna, Karolinska Institutet and Karolinska University Hospital, Stockholm, Sweden 7Mahatma Gandhi Mission Medical College, Navi Mumbai, Maharashtra, India 8Centre for Musculoskeletal Research, Division of Musculoskeletal and Dermatological Sciences, School of Biological Sciences, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, The University of Manchester, Manchester, UK 9Neurology, Manchester Centre for Clinical Neurosciences, Northern Care Alliance NHS Foundation Trust, Salford, UK 10NIHR Leeds Biomedical Research Centre, Leeds Teaching Hospitals Trust, Leeds, UK 11Leeds Institute of Rheumatic and Musculoskeletal Medicine, University of Leeds, Leeds, UK 12Rheumatology Department, Fauji Foundation Hospital, Rawalpindi, Pakistan 13Rheumatology Department, Saint-Joseph University, Beirut, Lebanon 14Rheumatology Department, Hotel-Dieu de France Hospital, Beirut, Lebanon 15Department of Clinical Immunology, Medical Faculty, University Hospital ‘Lozenetz’, Soﬁa University St. Kliment Ohridski, Soﬁa, Bulgaria 16Department of Internal Medicine, General Hospital, National Medical Center, ‘La Raza’, Instituto Mexicano del Seguro Social, Mexico City, Mexico 17Department of Allergy and Rheumatology, Nippon Medical School Graduate School of Medicine, Tokyo, Japan 18Department of Rheumatology, Royal Melbourne Hospital, Parkville, VIC, Australia 19Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia 20Department of Medical Biology, University of Melbourne, Parkville, VIC, Australia 21Division of Rheumatology, Mayo Clinic, Rochester, MN, USA 22Department of Rheumatology, University Hospital Zu¨rich, University of Zu¨rich, Zu¨rich, Switzerland 23National Institute for Health Research Manchester Biomedical Research Centre, Manchester University NHS Foundation Trust, The University of Manchester, Manchester, UK 24Department of Rheumatology, Salford Royal Hospital, Northern Care Alliance NHS Foundation Trust, Salford, UK 25Department of Medicine, Section of Rheumatology, St. enses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the origin mmercial re-use, please contact journals.permissions@oup.com his is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (htt Clinical science Luke’s Medical Center-Global City, Taguig, Philippines 26Division of Rheumatology, Department of Internal Medicine, Faculty of Medicine, Universitas Indonesia/Dr Cipto Mangunkusumo General Hospital, Jakarta, Indonesia 27Autoimmunity Division, Centro Me´dico Pensiones, Me´rida, Yucata´n, Mexico 28Rheumatologist and Clinical Investigator, Interstitial Lung Disease and Rheumatology Unit, Instituto Nacional de Enfermedades Respiratorias, Mexico City, Mexico 29Departamento de Inmunologıa y Reumatologıa, Hospital General de Occidente and University of Guadalajara, Guadalajara, Jalisco, Mexico 30Division of Rheumatology and Clinical Immunology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA 31Department of Rheumatology, Royal Wolverhampton Hospitals NHS Trust, Wolverhampton, UK 32City Hospital, Sandwell and West Birmingham Hospitals NHS Trust, Birmingham, UK 33Department of Rheumatology, Faculty of Medicine and Health, O¨ rebro University, O¨ rebro, Sweden Downloaded from https://academic.oup.com/rheumatology/article/62/7/2453/6839954 by Universita degli Studi di Modena e Reggi user on 31 August 2023 Safety and tolerance of vaccines against SARS-CoV-2 infection in systemic lupus erythematosus: results from the COVAD study Naveen R. 1, Elena Nikiphorou2,3, Mrudula Joshi4, Parikshit Sen5, Julius Lindblom6, Vishwesh Agarwal7, James B. Lilleker 8,9, Ai Lyn Tan 10,11, Babur Salim12, Nelly Ziade 13,14, Tsvetelina Velikova15, Abraham Edgar Gracia-Ramos 16, Masataka Kuwana 17, Jessica Day 18,19,20, Ashima Makol21, Oliver Distler 22, Hector Chinoy 8,23,24, Lisa S. Clinical science Traboco25, Suryo Anggoro Kusumo Wibowo26, Erick Adrian Zamora Tehozol27, Jorge Rojas Serrano28, Ignacio Garcıa-De La Torre29, COVAD Study Group§, Rohit Aggarwal30, Latika Gupta 1,8,31,32, Vikas Agarwal1†, Ioannis Parodis 6,33†* 1Department of Clinical Immunology and Rheumatology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, India 2Centre for Rheumatic Diseases, King’s College London, London, UK 3Rheumatology Department, King’s College Hospital, London, UK 4Byramjee Jeejeebhoy Government Medical College and Sassoon General Hospitals, Pune, India 5Maulana Azad Medical College, New Delhi, Delhi, India 6Division of Rheumatology, Department of Medicine Solna, Karolinska Institutet and Karolinska University Hospital, Stockholm, Sweden 7Mahatma Gandhi Mission Medical College, Navi Mumbai, Maharashtra, India 8Centre for Musculoskeletal Research, Division of Musculoskeletal and Dermatological Sciences, School of Biological Sciences, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, The University of Manchester, Manchester, UK 9Neurology, Manchester Centre for Clinical Neurosciences, Northern Care Alliance NHS Foundation Trust, Salford, UK 10NIHR Leeds Biomedical Research Centre, Leeds Teaching Hospitals Trust, Leeds, UK 11Leeds Institute of Rheumatic and Musculoskeletal Medicine, University of Leeds, Leeds, UK 12Rheumatology Department, Fauji Foundation Hospital, Rawalpindi, Pakistan 13Rheumatology Department, Saint-Joseph University, Beirut, Lebanon 14Rheumatology Department, Hotel-Dieu de France Hospital, Beirut, Lebanon 15Department of Clinical Immunology, Medical Faculty, University Hospital ‘Lozenetz’, Soﬁa University St. Kliment Ohridski, Soﬁa, Bulgaria 16Department of Internal Medicine, General Hospital, National Medical Center, ‘La Raza’, Instituto Mexicano del Seguro Social, Mexico City, Mexico 17Department of Allergy and Rheumatology, Nippon Medical School Graduate School of Medicine, Tokyo, Japan 18Department of Rheumatology, Royal Melbourne Hospital, Parkville, VIC, Australia 19Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia 20Department of Medical Biology, University of Melbourne, Parkville, VIC, Australia 21Division of Rheumatology, Mayo Clinic, Rochester, MN, USA 22Department of Rheumatology, University Hospital Zu¨rich, University of Zu¨rich, Zu¨rich, Switzerland 23National Institute for Health Research Manchester Biomedical Research Centre, Manchester University NHS Foundation Trust, The University of Manchester, Manchester, UK 24Department of Rheumatology, Salford Royal Hospital, Northern Care Alliance NHS Foundation Trust, Salford, UK 25Department of Medicine, Section of Rheumatology, St. cess article distributed under the terms of the Creative Commons Attribution-NonCommercial License (https://creativecomm Received: 16 September 2022. Accepted: 10 November 2022 V C The Author(s) 2022. Published by Oxford University Press on behalf of the British Society for Rheumatology. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (https://creativecommons.org/ licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Introduction post-vaccination compared with RA patients on other drugs [12]. The international vaccination against COVID in sys- temic lupus (VACOLUP) study that comprised 696 patients with SLE showed an adequate safety and tolerability profile for COVID-19 vaccination, with a minimal risk for disease flares, including following mRNA vaccines [13]. However, as- sessment of relative risks was not possible in that study due to the absence of a control group [13]. Vaccination against SARS-CoV-2 infection (COVID-19) has at large been proven efficacious and safe for the healthy popu- lation [1]. However, data concerning populations considered vulnerable due to background diseases or ongoing immuno- suppressant therapy, such as patients with SLE, are limited. Increasing knowledge and experience on the management of COVID-19 has been pivotal in addressing the controversies surrounding the use of or discontinuation of immunosuppres- sion prior to COVID-19 vaccination [2–6]. Current recom- mendations encourage COVID-19 vaccination prior to B-cell depleting therapy and withholding immunosuppression post- vaccination in some circumstances, depending on agent and dose [7]. Furthermore, hesitancy for vaccination remains a concern, especially among patients with autoimmune rheu- matic diseases (AIRDs) [8, 9]. Interestingly, toll-like receptor agonists used as COVID-19 vaccine adjuvants have the poten- tial to induce SLE flares via upregulation of the type I inter- feron pathway [10]. There is a dearth of data in terms of the impact of disease activity or use of immunosuppressant or immunomodulatory therapies on vaccine-specific adverse events (AEs) in patients with SLE. Moreover, the paucity of data on COVID-19 vac- cine safety in SLE from diverse populations makes impossible their generalizability to the global population. The present study from the COVAD initiative addresses the COVID-19 vaccine safety and tolerance in the short term, that is, within seven days post-vaccination among patients with SLE com- pared with patients with other AIRDs, non-rheumatic autoim- mune diseases (nrAIRDs), or healthy controls (HC), through a multicentre patient-reported electronic survey. p y We recently reported vaccine safety data from patients with idiopathic inflammatory myopathies (IIMs) from the global COVID-19 Vaccination in Autoimmune Diseases (COVAD) study initiative, which demonstrated self-reported safety and tolerance for COVID-19 vaccination, yet a higher frequency of skin rashes post-vaccination among dermatomyositis patients, especially those with active disease during vaccina- tion [11]. Safety and tolerance of vaccines against SARS-CoV-2 infection in systemic lupus erythematosus: results from the COVAD study Luke’s Medical Center-Global City, Taguig, Philippines p , gy, y, g g, pp 26Division of Rheumatology, Department of Internal Medicine, Faculty of Medicine, Universitas Indonesia/Dr Cipto Mangunkusumo Gener Hospital, Jakarta, Indonesia p , , 27Autoimmunity Division, Centro Me´dico Pensiones, Me´rida, Yucata´n, Mexico 28 Autoimmunity Division, Centro Medico Pensiones, Merida, Yucatan, Mexico 28Rheumatologist and Clinical Investigator, Interstitial Lung Disease and Rheumatology Unit, Instituto Nacional de Enfermedades Respiratorias Mexico City Mexico t and Clinical Investigator, Interstitial Lung Disease and Rheumatology Unit, Instituto Nacional de Enfermedad exico City, Mexico y Rheumatologist and Clinical Investigator, Interstitial Lung Disease and Rheumatology Unit, Instituto Nacional d espiratorias Mexico City Mexico y 28Rheumatologist and Clinical Investigator, Interstitial Lung Disease and Rheumatology Unit, Instituto Na Rheumatologist and Clinical Investigator, Interstitial Lung Disease and Rheumatology Unit, Instituto Nacional d espiratorias, Mexico City, Mexico Respiratorias, Mexico City, Mexico 29 Respiratorias, Mexico City, Mexico Departamento de Inmunologıa y Reumatologıa, Hospital General de Occidente and University of Guadalajara, Guadalajara, Jalisco, Mexic 30Division of Rheumatology and Clinical Immunology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA p g y g p y j j 30Division of Rheumatology and Clinical Immunology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA 31 Division of Rheumatology and Clinical Immunology, University of Pittsburgh School of Medicine, Department of Rheumatology, Royal Wolverhampton Hospitals NHS Trust, Wolverhampton, UK p gy, y p p , 32City Hospital, Sandwell and West Birmingham Hospitals NHS Trust, Birmingham, UK 33 ¨ ¨ 33Department of Rheumatology, Faculty of Medicine and Health, Orebro University, Orebro, Sweden 2454 Naveen R. et al. Correspondence to: Ioannis Parodis, Division of Rheumatology, Department of Medicine Solna, Karolinska University Hospital Solna, 176 76 Stockholm, Sweden. E-mail: ioannis.parodis@ki.se †Vikas Agarwal and Ioannis Parodis contributed equally to this study. g q y y §The complete list of contributors from the COVAD Study Group as well as their afﬁliations are provided in the supplementary data available at Rheumatology online. §The complete list of contributors from the COVAD Study Group as well as their afﬁliations are provided in the supplementary data §The complete list of contributors from the COVAD Study Group as well as their afﬁliations are provided in the supplementary data available at Rheumatology online. Rheumatology key messages gy y g • While four-ﬁfths of SLE patients reported COVID-19 vaccination-related adverse events (AEs), the majority were minor. • Frequencies of major AEs (2.6%) and hospitalizations (0.2%) were similar to those in healthy individuals. • Active SLE yielded increased post-vaccine fever, fatigue and tachycardia frequencies. Background therapies had no inﬂuence. E yielded increased post-vaccine fever, fatigue and tachycardia frequencies. Background therapies had no inﬂuen Abstract Downloaded from https://academic.oup.com/rheumatology/article/62/7/2453/6839954 by Universita degli Studi di Modena e Reggi user on 31 August 2023 Objective: To determine COVID-19 vaccine-related adverse events (AEs) in the seven-day post-vaccination peri immune rheumatic diseases (AIRDs), non-rheumatic autoimmune diseases (nrAIDs), and healthy controls (HC). Methods: Data were captured through the COVID-19 Vaccination in Autoimmune Diseases (COVAD) question Multivariable regression models accounted for age, gender, ethnicity, vaccine type and background treatment. Results: Among 9462 complete respondents, 583 (6.2%) were SLE patients (mean age: 40.1 years; 94.5% females; 40.5% Asian; 42.9% Pﬁzer- recipients). Minor AEs were reported by 83.0% of SLE patients, major by 2.6%, hospitalization by 0.2%. AE and hospitalization frequencies were similar between patients with active and inactive SLE. Rashes were more frequent in SLE patients vs HC (OR; 95% CI: 1.2; 1.0, 1.5), chills less frequent in SLE vs AIRDs (0.6; 0.4, 0.8) and nrAIDs (0.5; 0.3, 0.8), and fatigue less frequent in SLE vs nrAIDs (0.6; 0.4, 0.9). Pﬁzer-recipients reported higher overall AE (2.2; 1.1, 4.2) and injection site pain (2.9; 1.6, 5.0) frequencies than recipients of other vaccines, Oxford/AstraZeneca- recipients more body ache, fever, chills (OR: 2.5, 3.0), Moderna-recipients more body ache, fever, chills, rashes (OR: 2.6, 4.3). Hospitalization frequencies were similar across vaccine types. AE frequencies were similar across treatment groups, although chills were less frequent in antimalarial users vs non-users (0.5; 0.3, 0.9). Conclusion: While COVID-19 vaccination-related AEs were reported by four-ﬁfths of SLE patients, those were mostly minor and comparable to AEs reported by healthy individuals, providing reassurance regarding COVID-19 vaccination safety in SLE. AEs reported by healthy individuals, providing reassurance regarding COVID 19 vaccination safety in SLE. Keywords: COVID-19, vaccine, adverse events, systemic lupus erythematosus, rheumatology Rheumatology key messages • While four-ﬁfths of SLE patients reported COVID-19 vaccination-related adverse events (AEs), the majority were minor. • Frequencies of major AEs (2.6%) and hospitalizations (0.2%) were similar to those in healthy individuals. • Active SLE yielded increased post-vaccine fever, fatigue and tachycardia frequencies. Background therapies had no inﬂuence. Keywords: COVID-19, vaccine, adverse events, systemic lupus erythematosus, rheumatology Introduction Similarly, we recently reported data on COVID-19 vaccine safety in patients with RA from the same survey, wherein most of the adverse events were minor and compara- ble to those reported by healthy individuals. In that work, patients on methotrexate and antimalarial agents were reported to have experienced fewer minor adverse events Study design and data collection We conducted an international, online, cross-sectional, multi- centre survey-based study, as a part of the COVAD initiative [14]. A comprehensive patient self-reporting electronic survey was developed, consisting of questions related to COVID-19 and AIRDs. This questionnaire included demographic details, AIRD-specific diagnosis, treatment details, current symptom status, COVID-19 infection history including symptoms, du- ration and complications (hospitalization and requirement of COVID-19 vaccine safety in lupus 2455 oxygen therapy), COVID-19 vaccination details, short-term (seven days) post-vaccination AEs (based on the Centre for Disease Control and Prevention criteria) and patient-reported outcome measures as per the Patient Reported Outcomes Measurement Information System (PROMIS) tool [15]. After vetting by international experts, pilot testing, revisions, vali- dation and translation into 18 languages, the survey was hosted on an online platform (http://surveymonkey.com) and was circulated by the international COVAD study group (106 physicians) across 94 countries (Supplementary Table S1, available at Rheumatology online), as well as through numer- ous social media platforms and online patient support groups. Convenience sampling was used and all participants over the age of 18 years were included. Duplicate responses were re- moved manually. Methods have been detailed at length in the published COVAD study protocol [14]. had completed the survey in full formed the study population that was deemed eligible for analysis. Multiple relevant varia- bles were extracted from the survey responses, including AEs during the seven-day post-vaccination period. Adverse events post vaccination Seven-day AEs were categorized into injection site pain or re- action, minor AEs, major AEs, and hospitalizations. Minor AEs included myalgia, body aches, fever, chills, nausea and vomiting, headache, rashes, fatigue, diarrhoea, abdominal pain, high pulse rate or palpitations, rise in blood pressure, fainting, difficulty in breathing, dizziness, and chest pain. Major AEs consisted of serious reactions to vaccination, re- quiring urgent medical attention, including anaphylaxis, a marked difficulty in breathing, throat closure (choking) and severe rashes. AEs not listed above were reported as ‘others’ in an open-ended question. Active and inactive disease Active and inactive disease state four weeks prior to vaccina- tion was assessed by the patient’s response to the question ‘What was the status of your autoimmune disease in the four weeks (prior to) before the first dose of COVID-19 vaccine?’. Responses of active and worsening/static/improving disease were grouped together to designate ‘active disease’. Patients who indicated ‘inactive’ as response formed the inactive dis- ease group. Those who responded ‘I don’t know’ or ‘Other’ were assessed for activity at an individual basis based on answers to the following questions: (i) What were your symp- toms four weeks prior to vaccination? (ii) If you have any swelling of your joints, how many joints are swollen? (iii) Did you require an increase in the dose of any of these [referring to previous answers] immunosuppressant medications (IS), or start a new immunosuppressant medicine within the six months prior to the first COVID-19 vaccine? Downloaded from https://academic.oup.com/rheumatology/article/62/7/2453/6839954 by Universita degli Studi di Modena e Reggi user on 31 August 2023 Informed consent of the participants was obtained electron- ically through an initial question in the online survey, prior to the main study questionnaire. No incentives were offered for survey completion. Central approval was obtained from the Sanjay Gandhi Postgraduate Institute of Medical Sciences (SGPGIMS) ethics committee as per local guidelines [Institutional Ethics Committee of Sanjay Gandhi Postgraduate Institute of Medical Sciences, Raebareli Road, Lucknow, 226014 (IEC Code: 2021–143-IP-EXP-39)] and the Checklist for Reporting results of the Internet E-Surveys (CHERRIES) was adhered to when reporting results [16, 17]. Statistical analysis ancestry was Asian (40.5%), followed by Caucasian (37,9%). Seventy-one per cent had taken two doses of COVID-19 vac- cine. Pfizer-BioNTech (BNT162b2) (42.9%) and Oxford/ AstraZeneca (ChAdOx1 nCoV-19) (11.7%) were the most common vaccines received. The most common co-existing other AIDs within SLE patients was thyroid disorder (n ¼ 46; 7.8%), type 1 diabetes (n ¼ 5; 0.8%) and haemolytic anaemia (n ¼ 8; 1.3%). ancestry was Asian (40.5%), followed by Caucasian (37,9%). Seventy-one per cent had taken two doses of COVID-19 vac- cine. Pfizer-BioNTech (BNT162b2) (42.9%) and Oxford/ AstraZeneca (ChAdOx1 nCoV-19) (11.7%) were the most common vaccines received. The most common co-existing other AIDs within SLE patients was thyroid disorder (n ¼ 46; 7.8%), type 1 diabetes (n ¼ 5; 0.8%) and haemolytic anaemia (n ¼ 8; 1.3%). As the survey was originally designed for patients with IIMs and distributed to IIM patient groups, patients with IIM formed a disproportionately large subgroup and were there- fore excluded from the other AIRDs group prior to analysis to avoid selection bias. Patients with SLE were excluded from the other AIRDs group. Comparisons were performed for vaccination-related AEs in patients with active vs inactive SLE. Also, comparisons between patients with SLE and patients with other AIRDs (IIM and SLE patients excluded) were conducted, as well as between patients with SLE and patients with nrAIRDs, and between patients with SLE and HC. Patients with SLE and autoimmune disease comorbidities were compared with those without autoimmune disease comorbidities. Autoimmune disease comorbidities included other AIRDs and nrAIDs reported by SLE patients. Chi- squared (v2) and Mann–Whitney U tests were used for com- parisons between groups for categorical and continuous vari- ables, respectively. The variables that differed across SLE, other AIRDs, nrAIDs and HC in univariable analysis were in- cluded in multivariable binary logistic regression analysis (BLR) with adjustment for baseline factors defined a priori, that is, age, gender, ethnicity, country group defined by hu- man development index (HDI), as well as factors with P < 0.200 in the univariable analyses. Stepwise forward re- gression methodology was used to build the regression models starting with age, gender, ethnicity and country group by HDI first, followed by addition of other covariates. The results for continuous variables were expressed as median (IQR) in view of the non-normal distribution of the data (by Kolmogorov–Smirnov test and Shapiro–Wilk test). In multi- variable models, P < 0.05 was considered statistically signifi- cant. Characteristics of study participants Of 10679 survey participants with complete responses, 1227 patients with IIM (11.4%) were excluded (Fig. 1; Table 1). Of 9462 respondents forming the population under study, 583 were patients with SLE (6.2%), 3069 patients with other AIRDs (32.4%), 1079 patients with nrAIDs (11.4%) and 4731 were HC (50.0%). Within the total study population, 7028 (74.3%) respondents were females, the respondents’ mean age was 42.26 14.8years, and the most frequently reported ethnicity was Caucasian (49.3%). The three most represented countries were Turkey (16.1%), India (15.3%) and Mexico (13.3%; Supplementary Table S1, available at Rheumatology online). Seventy-two per cent had taken at least two doses of COVID-19 vaccine and all had received at least one vaccine dose. Table 1 shows the type and proportion of patients with other AIRDs. The most common vaccine received was Pfizer-BioNTech (BNT162b2) in 39.3%. The most common immunosuppressants or immunomodulatory agents used by the SLE patients at the time of survey completion were antimalarial agents (62.6%), fol- lowed by glucocorticoids (55.6%) and azathioprine (14.4%; Table 1). Other population characteristics are shown in Table 1. Of 10679 survey participants with complete responses, 1227 patients with IIM (11.4%) were excluded (Fig. 1; Table 1). Of 9462 respondents forming the population under study, 583 were patients with SLE (6.2%), 3069 patients with other AIRDs (32.4%), 1079 patients with nrAIDs (11.4%) and 4731 were HC (50.0%). Within the total study population, 7028 (74.3%) respondents were females, the respondents’ mean age was 42.26 14.8years, and the most frequently reported ethnicity was Caucasian (49.3%). The three most represented countries were Turkey (16.1%), India (15.3%) and Mexico (13.3%; Supplementary Table S1, available at Rheumatology online). Within the SLE study population, 328 patients (56.3%) had active disease prior to vaccination. The reported post- vaccination AEs were similar between those with active and inactive disease except for higher fever (OR: 1.6; 95% CI: 1.1, 2.5; P ¼ 0.026), fatigue (OR: 1.5; 95% CI: 1.1, 2.3; P ¼ 0.025) and tachycardia (OR: 8.0; 95% CI: 1.8, 35.0; P ¼ 0.006) in patients with active SLE (Table 2). Hospitalization frequencies were similar between the two groups (Table 2). Data extraction Data were retrieved at the time of survey closure on 30 December 2021. Subjects who had not received at least one dose of any COVID-19 vaccine at the time of the survey com- pletion and subjects who had not completed the survey in full were excluded from the analysis (Fig. 1). Respondents who had received at least one dose of any COVID-19 vaccine and Figure 1. Flow diagram of patients included in the study. An electronic protocol terminated the survey automatically if respondents indicated not having received any COVID-19 vaccine. AIRDs: autoimmune rheumatic diseases (excluding SLE and IIM); HC: healthy controls; IIM: idiopathic inﬂammatory myositis; nrAIDs: non-rheumatic autoimmune diseases; SLE: systemic lupus erythematosus Figure 1. Flow diagram of patients included in the study. An electronic protocol terminated the survey automatically if respondents indicated not having received any COVID-19 vaccine. AIRDs: autoimmune rheumatic diseases (excluding SLE and IIM); HC: healthy controls; IIM: idiopathic inﬂammatory myositis; nrAIDs: non-rheumatic autoimmune diseases; SLE: systemic lupus erythematosus Naveen R. et al. Naveen R. et al. 2456 Characteristics of study participants A greater percentage of patients with self- reported active SLE were on glucocorticoids (64.9% vs 43.9%; P < 0.001) compared with inactive SLE patients; the differences in AEs between patients with active and patients with inactive SLE persisted upon adjustments for glucocorti- coid dose and intake of other immunosuppressive or immuno- modulatory agents, yielding an OR of 1.9 for fever (95% CI: 1.1, 3.2; P ¼ 0.012), an OR of 1.7 for fatigue (95% CI: 1.1, 2.7; P ¼ 0.007) and an OR of 8.7 for tachycardia (95% CI: 1.8, 41.4; P ¼ 0.006). y g p Table 1). Other population characteristics are shown in Table 1. Statistical analysis Statistical analysis was performed using the IBM SPSS version 26 (IBM, Armonk, NY, USA). ( ; ) Patients with SLE were older than HC (median: 39 vs 34 years; P < 0.001), but younger than those with other AIRDs (median: 39 vs 49 years; P < 0.001) and nrAIDs (me- dian: 39 vs 42 years; P < 0.001) and had a higher female rep- resentation compared with all other groups (P < 0.001 for all comparisons; Table 1). Furthermore, differences were noted regarding ethnicity and the proportion of vaccines received by SLE patients (Table 1). Patients with SLE were more fre- quently on glucocorticoids compared with other AIRDs (OR: 3.8; 95% CI: 3.1, 4.5; P < 0.001) as well as compared with nrAIDs (OR: 9.9; 95% CI: 7.7, 12.8; P < 0.001). Also, they were more frequently on mycophenolate mofetil, azathioprine and antimalarial agents compared with patients with other AIRDs (Table 1). Adverse events in SLE and comparisons with other subgroups Overall, AEs within seven days following vaccination were reported by 484 (83.0%) of the SLE patients; all these patients reported minor AEs (Supplementary Table S2, avail- able at Rheumatology online). Major AEs were reported by 15 patients (2.6%) and hospitalization by one (0.2%). Injection site pain was reported by 415 (71.2%) patients with SLE. Among the minor AEs, the most frequently reported was fatigue (n ¼ 162; 27.8%), followed by headache (n ¼ 147; 25.2%) and body ache (n ¼ 120; 20.6%; Supplementary Table S2, available at Rheumatology online). Specific major AEs reported included anaphylaxis (n ¼ 3; 0.5%), throat clo- sure (n ¼ 1; 0.2%), severe rashes (n ¼ 2; 0.3%) and others (n ¼ 11; 1.9%). Among 11 SLE patients with self-reported coexisting antiphospholipid syndrome (APS), none reported thrombosis (stroke, deep vein thrombosis or myocardial in- farction) in the seven-day period post-vaccination, and none reported chest pain, fainting, dizziness or difficulty in breath- ing. Only minor AEs (n ¼ 7; 63.3%) were reported, mainly fa- tigue (n ¼ 6; 54.5%) and headache (n ¼ 3; 27.2%). Population characteristics of SLE patients Among the SLE patients, the mean age was 40.1 6 12.0 years, and the majority (94.5%) were females. The most frequent COVID-19 vaccine safety in lupus 2457 COVID-19 vaccine safety in lupus Table 1. Baseline characteristics of survey respondents Variable Total (n ¼ 9462) SLE (n ¼ 583) HC (n ¼ 4731) Other AIRDs (n ¼ 3069) Other nrAIDs (n ¼ 1079) Median age in years (IQR) 41 (29–53) 39 (32–49) 34 (26–47) 49 (39–60) 42 (32–53) Gender (Male: Female) 2378:7028 (1:2.9) 31:551 (1:17) 1659:3043 (1:1.8) 545:2508 (1:4.6) 143: 926 (1:6.5) No. Population characteristics of SLE patients of vaccine doses, n (%) 1 2617 (27.7) 209 (35.8) 1201 (25.3) 858 (27.9) 349 (32.3) 2 6845 (72.3) 374 (64.2) 3530 (74.7) 2211 (72.1) 730 (67.6) Ethnicity, n (%) Caucasian 4668 (49.3) 221 (37.9) 1984 (41.9) 1823 (59.4) 640 (59.3) African-American/African heritage 66 (0.7) 7 (1.2) 33 (0.7) 21 (0.7) 5 (0.5) Asian 2516 (26.6) 236 (40.5) 1315 (27.7) 750 (24.4) 215 (19.9) Hispanic 1173 (12.4) 73 (12.5) 764 (16.1) 206 (6.7) 130 (12.0) Native American/Indigenous/Paciﬁc Islandic 46 (0.5) 7 (1.2) 24 (0.5) 11 (0.4) 4 (0.4) Do not wish to disclose 559 (5.9) 20 (3.4) 345 (7.3) 152 (4.9) 42 (3.8) Other 434 (4.6) 19 (3.3) 266 (5.6) 106 (3.5) 43 (3.9) Vaccine taken, n (%) Pﬁzer-BioNTech (BNT162b2) 3723 (39.3) 250 (42.9) 1623 (34.3) 1376 (44.8) 474 (43.9) Oxford/Astra Zeneca (ChAdOx1 nCoV-19) 1292 (13.7) 68 (11.7) 466 (9.8) 612 (19.9) 146 (13.5) Johnson & Johnson (J&J) (JNJ-78436735) 89 (0.9) 16 (2.7) 37 (0.8) 18 (0.6) 18 (1.7) Moderna (mRNA-1273) 599 (6.3) 59 (10.1) 160 (3.4) 261 (8.5) 119 (11.0) Novavax (NVX-CoV2373) 10 (0.1) – 2 (0.0) 5 (0.2) 3 (0.3) Covishield (ChAdOx1 nCoV-19) 1149 (12.1) 46 (7.8) 677 (14.3) 323 (10.6) 103 (9.6) Covaxin (BBV152) 222 (2.4) 10 (1.7) 120 (2.6) 75 (2.4) 17 (1.6) Sputnik (Gam-COVID-Vac) 188 (2.0) 7 (1.2) 131 (2.8) 29 (0.9) 21 (1.9) Sinopharm (BBIBP-CorV) 1564 (16.6) 33 (5.7) 1244 (26.3) 163 (5.3) 124 (11.5) Othersa 570 (6.0) 93 (16.0) 238 (5.0) 189 (6.2) 50 (4.6) I am not sure 56 (0.6) 1 (0.2) 33 (0.7) 18 (0.6) 4 (0.4) Diagnosis, n (%) No autoimmune disease 4731 (44.0) — 4731 (44.0) — — Rheumatoid arthritis 1347(14.2) — — 1347 (31) — SLE 583 (5.5) 583 (6.1) — — — Systemic sclerosis 407 (3.8) — — 407 (9.4) — Ankylosing spondylitis or psoriatic arthritis 372 (3.5) 3 (0.5) — 372 (8.6) — Sjo¨gren’s syndrome 218 (2) — — 218 (5.0) — Mixed connective tissue disorder (MCTD) 142 (1.3) — — 142 (3.3) — Vasculitis 114 (1.0) — — 114 (2.6) — Crohn’s disease or ulcerative colitis (IBD) 175 (1.6) 3 (0.5) — — 175 (16) Thyroid (hypothyroid or hyperthyroid) 498 (4.7) 46 (7.8) — — 498 (46) Type 1 Diabetes 63 (0.6) 5 (0.8) — — 63 (5.8) Multiple sclerosis 31 (0.3) — — — 31 (3.0) Myasthenia gravis 30 (0.3) 3 (0.5) — — 30 (2.8) Pernicious anaemia 10 (0.1) 1 (0.1) — — 10 (0.9) Haemolytic anaemia/idiopathic thrombocytopenic purpura (ITP) 18 (0.2) 8 (1.3) — — 18 (1.7) Polymyalgia rheumatica 18 (0.2) 1 (0.1) — 18 (0.4) — Others 705 (6.8) 38 (6.5) — 451 (10.5) 254 (23.5) Treatment, n (%) Methotrexate 989/4731 (21.0) 44 (7.5) — 913 (29.7) 32 (2.9) Mycophenolate mofetil 328 (7.0) 125 (21.4) — 182 (5.9) 20 (1.8) Rituximab 67 (1.4) 7 (1.2) — 56 (1.8) 4 (0.4) Azathioprine 261 (5.6) 84 (14.4) — 120 (3.9) 57 (5.3) Hydroxychloroquine 1007 (21.2) 365 (62.6) — 616 (20.0) 730 (67.6) Sulfasalazine 246 (5.2) 1 (0.2) — 227 (7.4) 18 (1.7) Leﬂunomide 155 (3.2) 8 (1.4) — 144 (4.7) 3 (0.3) Oral tacrolimus 49 (1.0) 18 (3.0) — 24 (0.8) 7 (0.6) Ciclosporin 24 (0.6) 4 (0.7) — 16 (0.5) 4 (0.4) IVIG 45 (1.0) 5 (0.9) — 25 (0.8) 14 (1.3) Cyclophosphamide 13 (0.2) 4 (0.7) — 9 (0.3) 0 (0.0) Glucocorticoids No glucocorticoids 3541 (74.8) 258 (44.2) — 2309 (75.2) 958 (88.8) <10 mg prednisone (or eq.)/day 961 (20.4) 269 (46.1) — 615 (20.0) 75 (6.9) 10–20 mg prednisone (or eq.)/day 184 (3.8) 40 (6.8) — 117 (3.8) 26 (2.4) >20 mg prednisone (or eq.)/day 63 (1.4) 16 (2.7) — 27 (0.9) 20 (1.9) AIDs: autoimmune diseases; AIRDs: autoimmune rheumatic diseases (other than SLE or inﬂammatory myopathies); HC: healthy controls; NA: not applicable. AIDs: autoimmune diseases; AIRDs: autoimmune rheumatic diseases (other than SLE or inﬂammatory myopathies); HC: healthy controls; NA: not applicable. a Others included open-ended responses in different languages. Population characteristics of SLE patients a Others included open-ended responses in different languages. Downloaded from https://academic.oup.com/rheumatology/article/62/7/2453/6839954 by Universita degli Studi di Modena e Reggi user on 31 August 2023 Table Figure 3, Supplementary Tables S2, S6 and S7, all available at Rheumatology online, provide an overview of vaccination- related AEs between patients with SLE and HC, between patients with SLE and patients with other AIRDs, and patients with SLE and patients with nrAIDs, respectively. Compared with HC, patients with SLE had similar overall AEs frequen- cies, frequencies of injection site pain, overall minor AEs, and hospitalization frequencies, except for higher frequencies of rashes (OR: 1.2; 95% CI: 1.0, 1.5; P ¼ 0.038) and anaphylaxis (OR: 8.7; 95% CI: 1.3, 57.0; P ¼ 0.024) in patients with SLE. In terms of absolute numbers, the patients with SLE who reported anaphylaxis were few (n ¼ 3; 0.5%). When compared with other AIRD patients, patients with SLE reported similar frequencies of overall AEs, minor AEs, major AEs and hospital- izations, except for lower frequencies of chills (OR: 0.6; 95% CI: 0.4, 0.8; P ¼ 0.005) in SLE patients. Similarly, when com- pared with nrAID patients, patients with SLE reported similar frequencies of overall AEs, minor AEs, major AEs and hospital- izations, except for chills (OR: 0.5; 95% CI: 0.3, 0.8; P ¼ 0.003) and fatigue (OR: 0.6; 95% CI: 0.4, 0.9; P ¼ 0.020) that were less frequent in the SLE population. Figure 3, Supplementary Tables S2, S6 and S7, all available at Rheumatology online, provide an overview of vaccination- related AEs between patients with SLE and HC, between patients with SLE and patients with other AIRDs, and patients with SLE and patients with nrAIDs, respectively. Compared with HC, patients with SLE had similar overall AEs frequen- cies, frequencies of injection site pain, overall minor AEs, and hospitalization frequencies, except for higher frequencies of rashes (OR: 1.2; 95% CI: 1.0, 1.5; P ¼ 0.038) and anaphylaxis (OR: 8.7; 95% CI: 1.3, 57.0; P ¼ 0.024) in patients with SLE. Major AEs were reported to be similar across recipients of different vaccine types. Hospitalization following vaccination was infrequent, with similar frequencies across recipients of the various vaccine types. Systemic minor AEs such as fever, chills and body ache were more frequently reported by Oxford/AstraZeneca (ChAdOx1 nCoV-19) and Moderna (mRNA-1273) vaccine recipients (OR ranging between 2.5 and 3.0; P < 0.05 for all instances). All other minor AEs were similar across recipients of different vaccine types (Table 3 and Supplementary Table S3, available at Rheumatology online). Downloaded from https://academic.oup.com/rheumatology/article/62/7/2453/6839954 by Universita degli Studi di Modena e Reggi user on 31 August 2023 tivariable binary logistic regression analysis included age, gender, ethnicity, country by human development index. Bold Factors included as covariates in multivariable binary logistic regression analysis included age, gender, ethnicity, country by human development index. Bold text highlights signiﬁcant P-values. AEs: adverse events; OR: odds ratio. antimalarials. The hospitalization frequencies were also similar across the various background treatment groups. Table 3, Supplementary Table S3 (available at Rheumatology online) and Fig. 2 provide an overview of vaccination-related AEs based on the type of vaccine received by patients with SLE. Overall, any AEs were reported more frequently by Pfizer-BioNTech (BNT162b2) vaccine recipients (OR: 2.2; 95% CI: 1.1, 4.2; P ¼ 0.016) compared with the rest. Any minor AEs followed similar trends. Injection site pain was reported more frequently by Pfizer-BioNTech (BNT162b2) vaccine recipients (OR: 2.9; 95% CI: 1.6, 5.0; P < 0.001) and less frequently by Sinopharm (BBIBP-CorV) vaccine recipients (OR: 0.2; 95% CI: 0.1, 0.6; P ¼ 0.003) compared with the rest. Downloaded from https://academic.oup.com/rheumatology/article/62/7/2453/6839954 by Universita degli Studi di Modena e Reggi user on 31 August 2023 Naveen R. et al. 2458 Table 2. Comparison of vaccination-related AEs in patients with active vs inactive SLE Table 2. Comparison of vaccination-related AEs in patients with active vs inactive SLE Active SLE (n ¼ 328) n (%) Inactive SLE (n ¼ 255) n (%) Univariable Multivariable OR (95% CI) P OR (95% CI) P Any AEs 277 (84.5) 207 (81.2) — 0.296 — — Injection site pain 229 (69.8) 186 (73.0) — 0.409 — — Minor AEs Any minor AEs 277 (84.5) 207 (81.2) — 0.296 — — Myalgia 55 (16.8) 36 (14.1) — 0.382 — — Body ache 74 (22.6) 46 (18.0) — 0.180 — — Fever 74 (22.6) 38 (14.9) 1.6 (1.1, 2.5) 0.020 1.6 (1.1, 2.5) 0.026 Chills 37 (11.3) 29 (11.4) — 0.972 — — Nausea and vomiting 29 (8.8) 15 (5.8) — 0.180 — — Headache 84 (25.6) 63 (24.7) — 0.803 — — Rashes 12 (3.7) 3 (1.2) — 0.060 — — Fatigue 102 (31) 60 (23.5) 1.4 (1.0, 2.1) 0.043 1.5 (1.1, 2.3) 0.025 Diarrhoea 9 (2.7) 10 (3.9) — 0.427 — — Abdominal pain 9 (2.7) 4 (1.6) — 0.340 — — Tachycardia 20 (6.0) 2 (0.8) 8.2 (1.9, 35) 0.001 8.0 (1.8, 35) 0.006 Rise in blood pressure 5 (1.5) 3 (1.2) — 0.720 — — Fainting 0 (0.0) 1 (0.4) — 0.256 — — Difﬁculty in breathing 6 (1.8) 1 (0.4) — 0.114 — — Dizziness 26 (7.9) 15 (5.8) — 0.338 — — Chest pain 10 (3.0) 1 (0.4) 7.9 (1.0, 62) 0.019 7.7 (0.9, 62) 0.054 Major AEs Any major AEs 12 (3.7) 3 (1.2) — 0.060 — — Anaphylaxis 2 (0.6) 1 (0.4) — 0.716 — — Marked dyspnoea 1 (0.3) 0 (0.0) — 0.378 — — Throat closure 1 (0.3) 0 (0.0) — 0.378 — — Severe rashes 1 (0.3) 0 (0.4) — 0.858 — — Hospitalization 0 (0.0) 1 (0.4) — 0.256 — — Factors included as covariates in multivariable binary logistic regression analysis included age, gender, ethnicity, country by human development index. Bold text highlights signiﬁcant P-values. AEs: adverse events; OR: odds ratio. Downloaded from https://academic.oup.com/rheumatology/article/62/7/2453/6839954 by Universita degli Studi di Modena e Reggi user on 31 August 2023 Table Supplementary Tables S4 and S5, available at Rheumatology online, provide an overview of vaccination-related AEs strati- fied by the type of background immunosuppressive therapy. The vaccination-related AEs were similar across background treatment categories except for lower chills in SLE patients who were on antimalarial agents (OR: 0.5; 95% CI: 0.3, 0.9; P ¼ 0.042) compared with patients who were not on Supplementary Table S8, available at Rheumatology online, demonstrates that SLE patients with autoimmune comorbid- ities (n ¼ 92; 15.8%) reported similar frequencies of overall AEs, minor AEs, and major AEs compared with SLE patients without autoimmune comorbidities (n ¼ 491; 84.2%), with the exception of fatigue (OR: 2.1; 95% CI: 1.3, 3.4; P ¼ 0.002) and abdominal pain (OR: 3.6; 95% CI: 1.1, 12.1; P ¼ 0.033) COVID-19 vaccine safety in lupus 2459 Table 3. Frequencies of vaccination-related AEs in SLE patients by vaccine type Table 3. Table Frequencies of vaccination-related AEs in SLE patients by vaccine type Pfizer- BioNTech (BNT162b2) (n ¼ 250) Oxford/Astra Zeneca (ChAdOx1 nCoV-19) (n ¼ 68) Johnson & Johnson (J&J) (JNJ-78436735) (n ¼ 16) Moderna (mRNA- 1273) (n ¼ 59) Covishield (Serum Institute India) (ChAdOx1 nCoV- 19) (n ¼ 46) Covaxin (Bharat Biotech) (BBV152) (n ¼ 10) Sputnik (Gam- COVID- Vac) (n ¼ 7) Sinopharm (BBIBP- CorV) (n ¼ 33) Any adverse event 219 (87.6) 60 (88.2) 15 (93.8) 55 (93.2) 31 (67.4) 7 (70.0) 7 (100.0) 28 (84.8) Injection site pain 202 (80.8)*** 53 (77.9) 12 (75.0) 51 (86.4) 19 (41.3) 6 (60.0) 6 (85.7) 17 (51.5)* Minor AEs Any minor AEs 219 (87.6)* 60 (88.2) 15 (93.8) 55 (93.2) 31 (67.4) 7 (70.0) 7 (100.0) 28 (84.8) Myalgia 38 (15.2) 11 (16.2) 3 (18.8) 15 (25.4) 7 (15.2) 0 (0.0) 1 (14.3) 5 (15.2) Body ache 33 (13.2)* 25 (36.8)* 3 (18.8) 19 (32.2)* 8 (17.4) 1 (10.0) 2 (28.6) 7 (21.2) Fever 38 (15.2) 22 (32.4) 4 (25.0) 23 (38.9)* 10 (21.7) 0 (0.0) 0 (0.0) 8 (24.2) Chills 25 (10.0) 14 (20.6)* 2 (12.5) 19 (32.2)* 1 (2.2) 0 (0.0) 0 (0.0) 0 (0.0) Nausea and vomiting 19 (7.6) 7 (10.3) 2 (12.5) 6 (10.2) 0 (0.0) 0 (0.0) 2 (28.6) 2 (6.1) Headache 51 (20.4) 25 (36.8) 8 (50.0) 22 (37.4) 5 (10.9) 2 (20.0) 3 (42.9) 8 (24.2) Rashes 7 (2.8) 1 (1.5) 0 (0.0) 5 (8.5) 1 (2.2) 0 (0.0) 0 (0.0) 0 (0.0) Fatigue 81 (32.4) 21 (30.9) 9 (56.3) 22 (37.3) 5 (10.9) 0 (0.0) 3 (42.9) 5 (15.2) Diarrhoea 8 (3.2) 5 (7.4) 1 (6.3) 2 (3.4) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) Abdominal pain 4 (1.6) 3 (4.4) 1 (6.3) 3 (5.1) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) High pulse rate 10 (4.0) 4 (5.9) 1 (6.3) 5 (8.5) 0 (0.0) 0 (0.0) 0 (0.0) 1 (3.0) Rise in blood pressure 3 (1.2) 1 (1.5) 0 (0.0) 1 (1.7) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) Fainting 0 (0.0) 1 (1.5) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) Difﬁculty in breathing 4 (1.6) 1 (1.5) 0 (0.0) 2 (3.4) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) Dizziness 10 (4.0)* 7 (10.3) 3 (18.8) 5 (8.5) 1 (2.2) 0 (0.0) 1 (14.3) 4 (12.1) Chest pain 5 (2.0) 1 (1.5) 0 (0.0) 3 (5.1) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) Others 31 (12.4) 7 (10.3) 1 (6.3) 2 (3.4) 2 (4.3) 1 (10.0) 0 (0.0) 7 (21.2) Major AEs Any major AEs 9 (3.6) 1 (1.5) 0 (0.0) 3 (5.1) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) Anaphylaxis 2 (0.8) 0 (0.0) 0 (0.0) 1 (1.7) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) Marked dyspnoea 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) Throat closure 1 (0.4) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) Severe rashes 1 (0.4) 1 (1.5) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) Others 6 (2.4) 0 (0.0) 0 (0.0) 2 (3.4) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) Hospitalisation 1 (0.4) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) Results from binary logistic regression analysis. Table Comparisons are made between one drug vs the rest. Bold denotes increased risk (OR) compared with the rest, while bold underlined denotes decreased risk (OR) compared with the rest. * P < 0.05; P < 0.005; * P < 0.001. AEs: adverse events; OR: odds ratio. p p gy y that were more frequently reported by SLE patients with auto- immune comorbidities. seven-day post-vaccination period, with a focus on patients with SLE and comparisons with patient populations with other AIRDs, nrAIDs, as well as healthy individuals. y In analysis stratifying SLE patients into patients with active and inactive disease, overall AE and hospitalization frequen- cies were similar between the groups. However, active SLE in- creased the chance for experiencing fever and fatigue by 1.6 and 1.5 times, respectively, and the chance for experiencing tachycardia by eight times. While the explanation underlying the observed association between active SLE and post- vaccination tachycardia is unclear and difficult to speculate upon, this is to the best of our knowledge the first time it is reported in the literature. The association persisted upon adjustments for glucocorticoid doses and intake of other im- munosuppressive or immunomodulatory medications. Lastly, SLE patients with concurrent APS did not report thrombotic events post-vaccination, which is reassuring and concordant with previous reports on patients with APS [19–22]. Discussion Rapid production of vaccines against COVID-19 was necessi- tated by the pernicious impact of the pandemic on societies. However, the swift pace of vaccine development has resulted in hesitancy in a considerable proportion of the global popu- lation. This hesitancy has been even more prominent in patients with chronic diseases, particularly autoimmune dis- eases, patient groups not only considered more vulnerable to infections but also more prone to drug reactions [8, 9, 18]. Patients with SLE are often on immunosuppressant therapies and/or glucocorticoids, which may impact the biological responses to vaccines. In the present study, we evaluated patient-reported post-vaccination AEs in patients with SLE and found overall reassuring results; while 83.0% of patients reported AEs, these were typically minor and self-resolving. Nevertheless, major AEs were reported by 2.6% of SLE patients and included anaphylaxis, throat closure and severe rashes. To the best of our knowledge, this is the first study of this scale in terms of numbers, ethnic diversity and global reach, to address COVID-19 vaccination-related AEs over a In analysis stratified by vaccine type, AE frequencies reported by SLE patients were overall similar across the different vaccine types, except for Pfizer recipients who reported overall AEs and injection site pain at a 2-fold and 3-fold higher extent com- pared with recipients of other vaccine types, respectively, and Oxford/AstraZeneca and Moderna recipients who reported a 2460 Naveen R. et al. e 2. Selected vaccination-related AEs in patients with SLE. The forest plot illustrates selected results from multivariable logistic regression analysis. s denote odds ratios (ORs) and whiskers denote 95% conﬁdence intervals. AEs: adverse events; LoS: level of signiﬁcance; SLE: systemic lupus ematosus Naveen R. et al. p p gy e 2. Selected vaccination-related AEs in patients with SLE. The forest plot illustrates selected results from multivariable logistic regression analysis. s denote odds ratios (ORs) and whiskers denote 95% conﬁdence intervals. AEs: adverse events; LoS: level of signiﬁcance; SLE: systemic lupus ematosus Downloaded from https://academic.oup.com/rheumatology/article/62/7/2453/68399 Downloaded from https://academic.oup.com/rheumatology/article/62/7/2453/6839954 by Universita degli Studi di Modena e Reggi user on 31 August 2023 Downloaded from https://academic.oup.com/rheumatology/article/62/7/2453/6839954 by Universita degli Studi di Modena e Reggi user on 31 Aug Figure 2. Selected vaccination-related AEs in patients with SLE. The forest plot illustrates selected results from multivariable logistic regression analysis. Circles denote odds ratios (ORs) and whiskers denote 95% conﬁdence intervals. AEs: adverse events; LoS: level of signiﬁcance; SLE: systemic lupus erythematosus Figure 2. Discussion Selected vaccination-related AEs in patients with SLE. The forest plot illustrates selected results from multivariable logistic regression analysis. Circles denote odds ratios (ORs) and whiskers denote 95% conﬁdence intervals. AEs: adverse events; LoS: level of signiﬁcance; SLE: systemic lupus erythematosus Figure 3. Frequencies of vaccination-related adverse events (AEs) across patients with various rheumatic diseases and healthy controls. AEs: adverse events; AIRDs: autoimmune rheumatic diseases (excluding SLE and IIM); HC: healthy controls; nrAIDs: non-rheumatic autoimmune diseases igure 3 Frequencies of vaccination-related adverse events (AEs) across patients with various rheumatic diseases and healthy controls AEs: adverse niversita degli Studi di Modena e Reggi user on 31 August 2023 Figure 3. Frequencies of vaccination-related adverse events (AEs) across patients with various rheumatic diseases and healthy controls. AEs: adverse events; AIRDs: autoimmune rheumatic diseases (excluding SLE and IIM); HC: healthy controls; nrAIDs: non-rheumatic autoimmune diseases 2461 COVID-19 vaccine safety in lupus higher probability of some minor AEs such as body ache, fever and chills. By contrast, Sinopharm recipients less frequently ex- perienced injection site pain. Major AE and hospitalization fre- quencies were similar across recipients of different vaccine types. These findings were in line with those of the VACOLUP study, which reported similar frequencies of AEs across vaccine types, and irrespective of age or gender [13]. addresses concerns and controversies around COVID-19 vac- cination. Most AEs reported were minor, self-resolving, and comparable in nature and frequencies to those reported by healthy controls. Our results will hopefully allow greater con- fidence and better uptake of COVID-19 vaccines in this par- ticularly vulnerable group of individuals, as advocated by European [37] and American [7] guidelines. yp , p g g [ ] Lastly, we performed a subgroup analysis stratifying SLE patients based on background immunosuppressant or immu- nomodulatory therapy. In this analysis, AE frequencies did not differ across patient groups on different medications, with the exception of chills being less frequently reported by patients who were on antimalarial agents compared with patients who were not. Following conflicting initial signals, antimalarial agents were later disproved to be of efficacy in managing patients with COVID-19 infection [23, 24]. Patients with SLE who use antimalarials are recommended to continue the antimalarial treatment during a COVID-19 in- fection without any dose adjustment. There have been specu- lations about cardiotoxicity and electrocardiographic QTc prolongation with the use of antimalarials in COVID-19 in- fection, with a corroborating meta-analysis of several studies [5]. Funding I.P. is supported by grants from the Swedish Rheumatism Association (R-941095), King Gustaf V’s 80-year Foundation (FAI-2020–0741), Professor Nanna Svartz Foundation (2020–00368), Swedish Society of Medicine (SLS-974449), Ulla and Roland Gustafsson Foundation (2021–26), Region Stockholm (FoUI-955483) and Karolinska Institutet. py g g p q y Several limitations need to be acknowledged. Firstly, the study was entirely based on self-reported patient data, with no possibility for verification through medical charts or healthcare professionals. Secondly, online surveys introduce inherent recall bias, as well as selection bias due to an expected higher participation willingness in patients who ex- perienced AEs, a potential lack of internet access for individu- als of lower socioeconomic status, and the likely lower grade of data contribution by patients suffering severe disability. Thirdly, the focus of the study was on short-term safety of COVID-19 vaccination, thus not contributing to the knowl- edge on safety over a longer term. However, the study reaf- firms the safety of COVID-19 vaccination in SLE patients and is in agreement with existing evidence on COVID-19 vaccine safety within SLE populations of different studies [19, 27– 36]. Fourthly, it was beyond the scope of this study to explore humoral responses to vaccines, which may also have impact on the development of AEs. Nevertheless, the large number of study participants, the high frequency of complete survey responses, and the wide geographical spread of survey respondents constituted major strengths of the study. The anonymised and self-reported nature of the questionnaire may also be considered a strength, by facilitating direct and likely unbiased (without external influence) patient and HC representation. Disclosure statement: A.L.T. has received honoraria for advi- sory boards and speaking for Abbvie, Gilead, Janssen, Lilly, Novartis, Pfizer, UCB. E.N. has received speaker honoraria/ participated in advisory boards for Celltrion, Pfizer, Sanofi, Gilead, Galapagos, AbbVie, Lilly and holds research grants from Pfizer and Lilly. H.C. has received grant support from Eli Lilly and UCB; consulting fees from Novartis, Eli Lilly, Orphazyme, Astra Zeneca; speaker for UCB, Biogen. I.P. has received research funding and/or honoraria from Amgen, AstraZeneca, Aurinia Pharmaceuticals, Elli Lilly and Company, Gilead Sciences, GlaxoSmithKline, Janssen Pharmaceuticals, Novartis and F. Hoffmann-La Roche AG. M.K. has received research grants and personal fees from AbbVie, AsahiKasei, Astellas, Boehringer Ingelheim, Chugai, Corbus, GlaxoSmithKline, Horizon, Kissei, MBL, Mitsubishi- Tanabe, Mochida, Nippon Shinyaku, and Ono Pharmaceuticals. J.D. has received research funding from CSL Limited. N.Z. Supplementary data are available at Rheumatology online. Supplementary data are available at Rheumatology online. Data availability The datasets generated during and/or analysed during the cur- rent study are not publicly available but are available from the corresponding author on reasonable request. Discussion However, in many COVID-19 infection treatment proto- cols, patients were given higher doses of antimalarial agents compared with usual doses within rheumatology; for exam- ple, hydroxychloroquine loading with 800 mg [25]. Thus, SLE patients in the present study are anticipated to have re- ceived lower doses than those shown to induce cardiotoxicity; it is worth mentioning that the recommended daily hydroxy- chloroquine dose for patients with SLE is 5 mg/kg [26]. Moreover, many patients in the studies that showed hydroxychloroquine-induced cardiotoxicity also received azithromycin, another drug with potentiality to prolong the QTc [5]. Lastly, no signals were seen for B-cell depleting ther- apy regarding AE or hospitalization frequency. Contribution statement Conceptualisation: N.R., E.N., L.G., I.P. Data curation: All authors. Formal analysis: N.R. Funding acquisition: I.P. Investigation: N.R., E.N., L.G., I.P. Methodology: N.R., E.N., L.G., I.P. Software: L.G. Validation: V.A., R.A., J.B.L., H.C. Visualization: J.L., R.A., V.A., L.G. Writing—original draft: N.R., E.N., I.P. Writing—review and editing: All authors. Disclaimer: No part of this manuscript is copied or pub- lished elsewhere in whole or in part. Acknowledgements COVID-19 Vaccination in Autoimmune Diseases (COVAD) Study Group Author List: Bhupen Barman, Yogesh Preet Singh, Rajiv Ranjan, Avinash Jain, Sapan C Pandya, Rakesh Kumar Pilania, Aman Sharma, Manesh Manoj M, Vikas Gupta, Chengappa G Kavadichanda, Pradeepta Sekhar Patro, Sajal Ajmani, Sanat Phatak, Rudra Prosad Goswami, Abhra Chandra Chowdhury, Ashish Jacob Mathew, Padnamabha Shenoy, Ajay Asranna, Keerthi Talari Bommakanti, Anuj Shukla, Arun Kumar R Pandey, Kunal Chandwar, Sinan Kardes¸, Do¨ndu¨ U¨ sku¨dar Cansu, Minchul Kim, Ashima Makol, Tulika Chatterjee, John D Pauling, Chris Wincup, Lorenzo Cavagna, Nicoletta Del Papa, Gianluca Sambataro, Atzeni Fabiola, Marcello Govoni, Simone Parisi, Elena Bartoloni Bocci, Gian Domenico Sebastiani, Enrico Fusaro, Marco Sebastiani, Luca Quartuccio, Franco Franceschini, Pier Paolo Sainaghi, Giovanni Orsolini, Rossella De Angelis, Maria Giovanna Danielli, Vincenzo Venerito, Marcin Milchert, Lisa S Traboco, Suryo Anggoro Kusumo Wibowo, Erick Adrian Zamora Tehozol, Jorge Rojas Serrano, Ignacio Garcıa-De La Torre, Jesu´s Loarce- Martos, Sergio Prieto-Gonza´lez, Albert Gil-Vila, Raquel Aranega Gonzalez, Masataka Kuwana, Akira Yoshida, Ran Nakashima, Shinji Sato, Naoki Kimura, Yuko Kaneko, Johannes Knitza, Stylianos Tomaras, Margarita Aleksandrovna Gromova, Or Aharonov, Tamer A Gheita, Ihsane Hmamouchi, Leonardo Santos Hoff, Margherita Giannini, Franc¸ois Maurier, Julien Campagne, Alain Meyer, Melinda Nagy-Vincze, Daman Langguth, Vidya Limaye, Merrilee Needham, Nilesh Srivastav, Marie Hudson, Oce´ane Landon-Cardinal, Syahrul Sazliyana Shaharir, Wilmer Gerardo Rojas Zuleta, Jose´ Anto´nio Pereira Silva, Jo~ao Eurico Fonseca, Olena Zimba. 4. 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Vaccine hesitancy in patients with autoimmune diseases: data from the coronavirus disease-2019 vaccination in autoimmune diseases study. Indian J Rheumatol 2022;17:188. 10. Kayesh MEH, Kohara M, Tsukiyama-Kohara K. References 1. Chen L, Cai X, Zhao T et al. Safety of global SARS-CoV-2 vac- cines, a meta-analysis. vaccines. Vaccines 2022;10:596. 2. Grainger R, Kim AHJ, Conway R, Yazdany J, Robinson PC. COVID-19 in people with rheumatic diseases: risks, outcomes, treatment considerations. Nat Rev Rheumatol 2022;18:191–204. 3. Tavares A, de Melo AKG, Cruz VA et al. Guidelines on COVID-19 vaccination in patients with immune-mediated rheumatic diseases: a Brazilian Society of Rheumatology task force. Adv Rheumatol 2022;62:3. Funding has received speaker fees, advisory board fees and research grants from Pfizer, Roche, Abbvie, Eli Lilly, NewBridge, Sanofi-Aventis, Boehringer Ingelheim, Janssen, Pierre Fabre; none is related to this manuscript. O.D. has/had consultancy relationship with and/or has received research funding from or has served as a speaker for the following companies in the area of potential treatments for systemic In conclusion, the findings of the present study provide re- assurance on the safety of COVID-19 vaccination for the SLE patient population, adding to the body of literature that 2462 Naveen R. et al. Naveen R. et al. Naveen R. et al. sclerosis and its complications in the last three years: Abbvie, Acceleron, Alcimed, Amgen, AnaMar, Arxx, Baecon, Blade, Bayer, Boehringer Ingelheim, ChemomAb, Corbus, CSL Behring, Galapagos, Glenmark, GSK, Horizon (Curzion), Inventiva, iQvia, Kymera, Lupin, Medac, Medscape, Mitsubishi Tanabe, Novartis, Roche, Roivant, Sanofi, Serodapharm, Topadur and UCB. Patent issued ‘mir-29 for the treatment of systemic sclerosis’ (US8247389, EP2331143). R.A. has/had a consultancy relationship with and/or has received research funding from the following companies-Bristol Myers-Squibb, Pfizer, Genentech, Octapharma, CSL Behring, Mallinckrodt, AstraZeneca, Corbus, Kezar, and Abbvie, Janssen, Alexion, Argenx, Q32, EMD-Serono, Boehringer Ingelheim, Roivant. The other authors have no conflict of interest relevant to this manuscript. sclerosis and its complications in the last three years: Abbvie, Acceleron, Alcimed, Amgen, AnaMar, Arxx, Baecon, Blade, Bayer, Boehringer Ingelheim, ChemomAb, Corbus, CSL Behring, Galapagos, Glenmark, GSK, Horizon (Curzion), Inventiva, iQvia, Kymera, Lupin, Medac, Medscape, Mitsubishi Tanabe, Novartis, Roche, Roivant, Sanofi, Serodapharm, Topadur and UCB. Patent issued ‘mir-29 for the treatment of systemic sclerosis’ (US8247389, EP2331143). R.A. has/had a consultancy relationship with and/or has received research funding from the following companies-Bristol Myers-Squibb, Pfizer, Genentech, Octapharma, CSL Behring, Mallinckrodt, AstraZeneca, Corbus, Kezar, and Abbvie, Janssen, Alexion, Argenx, Q32, EMD-Serono, Boehringer Ingelheim, Roivant. The other authors have no conflict of interest relevant to this manuscript. dissemination of this survey. Finally, the authors wish to thank all members of the COVAD study group for their invaluable role in data collection. H.C. is supported by the National Institution for Health Research Manchester Biomedical Research Centre Funding Scheme. The views expressed in this publication are those of the authors and not necessarily those of the NHS, the National Institute for Health Research or the Department of Health. Acknowledgements Immunogenicity, safety, and antiphospholipid antibodies after SARS-CoV-2 vaccine in patients with primary antiphospholipid syndrome. Lupus 2022;31:974–84. 30. 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https://openalex.org/W2163825221	https://inserm.hal.science/inserm-00978012/document	English	null	Bone fragility in sarcoidosis and relationships with calcium metabolism disorders: a cross sectional study on 142 patients	Arthritis research & therapy	2,014	cc-by	7,334	Bone fragility in sarcoidosis and relationships with calcium metabolism disorders: a cross sectional study on 142 patients. Bone fragility in sarcoidosis and relationships with calcium metabolism disorders: a cross sectional study on 142 patients. Nathalie Saidenberg-Kermanac’H, Luca Semerano, Hilario Nunes, Danielle Sadoun, Xavier Guillot, Marouane Boubaya, Nicolas Naggara, Dominique Valeyre, Marie-Christophe Boissier Nathalie Saidenberg-Kermanac’H, Luca Semerano, Hilario Nunes, Danielle Sadoun, Xavier Guillot, Marouane Boubaya, Nicolas Naggara, Dominique Valeyre, Marie-Christophe Boissier To cite this version: Nathalie Saidenberg-Kermanac’H, Luca Semerano, Hilario Nunes, Danielle Sadoun, Xavier Guillot, et al.. Bone fragility in sarcoidosis and relationships with calcium metabolism disorders: a cross sectional study on 142 patients.. Arthritis Research and Therapy, 2014, 16 (2), pp.R78. 10.1186/ar4519. inserm-00978012 Abstract Introduction: The prevention of fragility fractures in patients with sarcoidosis is a serious concern and the potential risk of hypercalcemia limits vitamin D and calcium supplementation. The objective of this study was to evaluate the risk factors for low bone mineral density (BMD) and fractures in sarcoidosis. In particular, we aimed to determine the link among bone fragility and calcium and vitamin D metabolism in this population. Methods: We performed a cross-sectional analysis on 142 consecutive patients with histologically proven sarcoidosis. BMD and prevalence of vertebral fractures on X-rays were assessed and the association with potential risk factors was studied by regression analysis. Results: Fragility fractures occurred in 23.5% of patients, despite a normal mean BMD in the study population. In a multivariate analysis, low dietary calcium, fracture, age, gender and menopause were associated with increased risk of low BMD. Low dietary calcium, high current corticosteroid dose and low creatinine clearance were associated with increased risk of fracture. Serum 25(OH)D between 10 and 20 ng/ml was significantly associated with higher BMD. Conversely, values greater than 20 ng/ml were associated with increased risk of fracture. Serum 25(OH)D level was inversely correlated with disease activity. Of note, vitamin D supplements increased serum 25(OH)D in a dose-dependent manner but had no effect on serum calcium level. Conclusions: Sarcoidosis patients have a high risk of fracture despite not having a lowered BMD suggesting that other independent factors are involved. Current corticosteroid dose, low dietary calcium and serum 25(OH)D levels are associated with bone fragility. In sarcoidosis, calcium and vitamin D supplementation might be warranted, but desirable 25(OH)D serum levels might be lower than those advised for the general population. Disorders of calcium and vitamin D metabolism could also interfere with bone mineral density in sarcoidosis. Extra renal synthesis of the active form of vitamin D (1,25 (OH)2D) takes place inside the granulomas under the in- fluence of 1alpha-hydroxylase. In contrast to the renal en- zyme, the 1-alpha-hydroxylase expressed by macrophages is not inhibited by serum 1,25(OH)2D levels; moreover, the stimulation of the 25(OH)D-24-hydroxylase trans- forming the 1,25(OH)2D into inactive 24,25(OH)2D is stimulated only at very high levels of 1,25(OH)2D [3,4]. The resulting high levels of calcitriol could contribute to increased intestinal absorption of calcium, which might partly explain the hypercalcemia sometimes observed in sarcoidosis. Moreover, sarcoidosis patients are more sensi- tive than healthy subjects to vitamin D supplements with © 2014 Saidenberg-Kermanac’h 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. Bone fragility in sarcoidosis and relationships with calcium metabolism disorders: a cross sectional study on 142 patients Nathalie Saidenberg-Kermanac’h1,2,3, Luca Semerano1,2,3, Hilario Nunes4, Danielle Sadoun4, Xavier Guillot1,2,3, Marouane Boubaya5, Nicolas Naggara6, Dominique Valeyre4† and Marie-Christophe Boissier1,2,3† RESEARCH ARTICLE Open Access Open Access HAL Id: inserm-00978012 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. Saidenberg-Kermanac’h et al. Arthritis Research & Therapy 2014, 16:R78 http://arthritis-research.com/content/16/2/R78 Saidenberg-Kermanac’h et al. Arthritis Research & Therapy 2014, 16:R78 http://arthritis-research.com/content/16/2/R78 Clinical parameters These risks factors are nevertheless balanced by the fact that the disease evolves generally in young adults, at lower risk of fracture. Moreover, CS-free remission pe- riods can be very long, and it was shown that in sarcoid- osis the effect of CS on bone might be, at least partially, reversible [6,7]. Few studies with contradictory results are available in the literature, probably due to limited sample size and heterogeneous clinical presentation of included patients. Previous studies using quantitative computed tomography (QCT) showed a reduction of bone mineral content (BMC) even in patients not treated with CS [7,8]. Thereafter, similar results were found only in lumbar spine BMD of post-menopausal women [9] or in CS-treated subjects [10-12]. In contrast, in a four-year longitudinal study, no bone loss was ob- served even in CS-treated patients despite a high rate of fracture (38%) observed by vertebral fracture assessment (VFA) [13]. This suggests that other determinants apart from BMD are probably involved in fracture risk. So far, the link between serum 25(OH)D level and BMD has not been studied in sarcoidosis. For each patient, a questionnaire assessed the following risk factors for osteoporosis: age, sex, menopausal status, tobacco and alcohol consumption, body mass index (BMI), personal or family history of fracture of low energy (defined as resulting from a fall from standing height or lower; skull, metacarpal and metatarsal fractures were excluded). Anti-osteoporotic treatments, current and cumulated CS dose and vitamin D supplements in the six months preceding the study were recorded. Dietary calcium intake was evaluated by the Fardellone auto-questionnaire [15]. The data related to sarcoidosis were also collected: dis- ease duration, localization (graded from 0 to 6 according to the degree of severity of each organ involvement), current flare (at least one active localization or new localization in the three months preceding the study), number of relapses, stage of pulmonary involvement (stage 0: normal chest radiography; stage I: bilateral hilar lymphadenopathy with- out pulmonary infiltrates; stage II: bilateral hilar lymph- adenopathy with pulmonary infiltrates; stage III: pulmonary infiltrates without bilateral hilar lymphadenopathy; stage IV: end-stage fibrosis, bullae, honeycombing and cavity), stage of dyspnoea from I to IV according to New York Heart Association (NYHA) classification. Introduction Sarcoidosis is a multifaceted granulomatous disease ran- ging from regressive localized forms to chronic systemic involvement. The risk of fracture in sarcoidosis has not been clearly evaluated although most patients may present risk factors of osteoporosis. Like in other chronic diseases, as rheumatoid arthritis (RA) [1] or spondyloarthritis [2], both corticosteroid (CS) use and systemic inflammation could promote bone loss in sarcoidosis. Correspondence: nathalie.saidenberg@avc.aphp.fr †Equal contributors 1INSERM UMR1125, Bobigny, France 2Sorbonne Paris Cité-Université Paris 13, Bobigny, France Full list of author information is available at the end of the article * Correspondence: nathalie.saidenberg@avc.aphp.fr †Equal contributors 1INSERM UMR1125, Bobigny, France 2Sorbonne Paris Cité-Université Paris 13, Bobigny, France Full list of author information is available at the end of the article Page 2 of 9 Saidenberg-Kermanac’h et al. Arthritis Research & Therapy 2014, 16:R78 http://arthritis-research.com/content/16/2/R78 Saidenberg-Kermanac’h et al. Arthritis Research & Therapy 2014, 16:R78 http://arthritis-research.com/content/16/2/R78 10 (N° ID RCB 2011-A00202-39). All patients gave their informed consent prior to their inclusion in the study. a higher increase in serum calcium after intake [5]. For these reasons, many experts advise patients to avoid sun exposure and vitamin D and calcium supplements, with the potential risk of chronic vitamin D deficiency. Study design l d d We included 142 consecutive patients with sarcoidosis ac- cording to the criteria retained in the Consensus Confer- ence ATS/ERS [14], that is, combining clinical, biological and radiological presentation compatible with diagnosis and excluding other granulomatous diseases. Among 222 patients attending pneumonology consultation or day- hospital during the inclusion period, 74 were excluded be- cause they did not meet inclusion criteria and 6 refused to participate in the study. All 142 remaining included pa- tients presented documented histological lesions of granu- loma without caseous necrosis, in at least one site of biopsy. Patients with other chronic progressive diseases, chronic respiratory or renal insufficiency stage IV and V whose origin was not related to sarcoidosis and those on diuretics able to interfere with calcium metabolism were excluded from the study. At inclusion, all patients under- went clinical examination evaluating the risk factors for osteoporosis and calcium intake. All patients had bio- chemical, radiological and BMD assessment. Clinical parameters Our objective was to determine the risk factors for bone fragility evaluated by BMD and fracture prevalence in sarcoidosis patients and in particular to evaluate the relationship with vitamin D and calcium metabolism in a pilot cross-sectional study. Biochemical parameters p The serum levels of calcium (corrected with albumin), phosphate, 25(OH)D (Diasorin radioimmunoassay, Stillwater, US), 1,25(OH)2D (IDS radioimmunoassay, Frankfurt, Germany), creatinine, parathyroid hormone (PTH) (immunometry LIA, Immulite 2000, Puteau, France), thyroid stimulating hormone (TSH), bone markers: bone alkaline phosphatases (BALP) (ELISA, reactif microrevue), C-terminal telopeptide of type I collagen (CTX) (ECLIA/ Cobas-Roche, Switzerland), osteocalcin (immunometry TRACES, Kryptor), 24-hour urinary calcium, phosphate and creatinine, were gathered. Other parameters: erythro- cyte sedimentation rate (ESR), C-reactive protein (CRP), serum angiotensin-converting enzyme (ACE) level, blood count and serum albumin level were also gathered. Imaging parameters Radiographs of thoracic and lumbar spine (face and profile) were carried out. Assessment of vertebral fractures was in- dependently performed by both a radiologist (NN) and a rheumatologist (NSK) according to the semi-quantitative method of Genant [16] and their respective results were blinded during assessment. Inter-rater reliability was good K = 0.78, 95% CI (0.63 to 0.93). Vertebral fracture was de- fined by reduced height loss above 20% of the mean, pos- terior or anterior wall. This study complies with the Declaration of Helsinki and was approved by the Ethical Committee of France Page 3 of 9 Saidenberg-Kermanac’h et al. Arthritis Research & Therapy 2014, 16:R78 http://arthritis-research.com/content/16/2/R78 Table 1 Patients characteristics and biochemical parameters Women/men (number of patients) -80/62 Menopausal women (number of patients) 51/80 Age (years) 51.6 ± 11.6 Mean disease duration (years) 9.5 ± 7.1 BMI (mean ± SD) 27.5 ± 5.4 Radiological Stage I/ II/ III/IV/ (number of patients) 15/69/14/41 Dyspnoea Stage NYHA I and II/ NYHA III and IV (number of patients) 118/45 History of low energy fracture (number of patients) 21 Current CS intake (mean dose): 88 patients 12.4 ± 11.8 mg Mean cumulative CS dose per patient 27.6 ± 19.9 g Vitamin D supplements (mean dose) in the six months before the study: 31 patients 181,161 ± 137,430 U BP treatment (mean treatment duration) before study: 46 patients 25.3 ± 26.7 months Mean serum 25(OH)D level (N >30 ng/ml) 14.5 ± 7.61 Mean serum 1,25(OH)2D level (66 < N <167 pmol/l) 137.4 ± 50.3 Mean serum PTH level (10 < N <70 pmol/l) 45.36 ± 31.4 Mean creatinine clearance (ml/min) 110.7 ± 35.9 Mean serum calcium level (2.2 < N <2.6 mmol/l) 2.35 ± 0.9 Mean ESR (mm/h) 17.6 ± 14.5 Mean CRP (mg/L) 7.0 ± 9.8 Mean ACE (19 < N <41) 51.7 ACE, angiotensin-converting enzyme; BMI, Body mass index; BP, bisphosphonates; CRP, C-reactive protein; CS, Corticosteroids; ESR, erythrocyte sedimentation rate; NYHA, New York Heart Association; PTH, parathyroid hormone. Table 1 Patients characteristics and biochemical parameters Table 1 Patients characteristics and biochemical BMD was measured by dual X-ray absorptiometry (DXA Lunar Prodigy, GE Healthcare) at the lumbar spine and the total hip. Osteopenia and osteoporosis were de- fined by a-1SD < T-score < -2.5 SD and T-score ≤-2.5 SD, respectively. Low BMD was defined by a T-score < -1 SD (osteopenia or osteoporosis). Statistical analysis Data are summarized as the mean and standard devi- ation for continuous data and frequency for categorical data. A logistic regression model was used to identify factors associated with low BMD (defined as a T-Score lower than -1 SD) and risk of fracture. 25(OH)D was grouped in three categories as retained by the Institute of Medecine [17]: ≤10 ng/ml (deficiency), 10 to 20 ng/ml (insufficiency) and ≥20 ng/ml (desirable). All factors with P <0.20 at univariate analysis were included in a multiple logistic regression model with backward selection. Age, gender and menopause were grouped together to avoid a colinearity problem in multivariate analysis. Serum 25 (OH)D and calcium levels according to different criteria were compared with the Mann-Whitney U-test, with a Bonferroni correction for multiple tests. The associa- tions between the continuous factors were determined with Spearman’s correlation coefficients. All tests were two-sided at a 0.05 significance level. Analyses were car- ried out using R statistical software version 2.14.1. ACE, angiotensin-converting enzyme; BMI, Body mass index; BP, bisphosphonates; CRP, C-reactive protein; CS, Corticosteroids; ESR, erythrocyte sedimentation rate; NYHA, New York Heart Association; PTH, parathyroid hormone. ACE, angiotensin-converting enzyme; BMI, Body mass index; BP, bisphosphonates; CRP, C-reactive protein; CS, Corticosteroids; ESR, erythrocyte sedimentation rate; NYHA, New York Heart Association; PTH, parathyroid hormone. Clinical characteristics of the patients One hundred forty two patients, 80 women (51 meno- paused) and 62 men, were included. Eighty-five patients were Caucasians, 54 Caribbeans and 3 Indians. Mean age was 51.6 ± 11.6 years and BMI 27.5 ± 5.4. Mean disease duration was 9.5 ± 7.1 years and 104 patients had pre- sented more than one relapse; 21/137 patients experi- enced a low energy fracture before inclusion. Eighty-eight patients were receiving CS treatment at the time of the study (mean dose 12.4 ± 11.8 mg/d of prednisone equiva- lent), 28 patients had never received CS, 45 interrupted CS treatment for at least six months. The mean cumulated CS dose was 27.6 ± 19.9 g of prednisone equivalent. Thirty-one patients had received vitamin D supplements in the six months preceding the study (mean dose 181,161 ± 137,430 U) and 15 were on supplementation at inclusion. Forty-six patients had received a specific bone treatment before the study (bisphosphonates in the major- ity) on average for 25.3 ± 26.7 months; 24 were still treated during the study. The mean daily dietary calcium was 717.2 ± 360 mg (Table 1). difference between summer (June to August) and winter (December to February), respectively, with a mean level of 2.36 ± 0.06 mmol/l vs 2.32 ± 0.1 mmol/l ( P = 0.16). Only one patient had high serum calcium (>2.6 mmol/L), (without hypercalciuria) related to a systemic form with documented bone sarcoidosis lesions. Nine out of 94 patients presented hypercalciuria (24-hour urinary cal- cium >0.1 mmol/kg). Serum 25(OH)D levels were low (Figure 1) with a mean of 14.5 ± 7.61 ng/ml but with normal mean PTH serum level. The 31 patients who had received vitamin D supple- ments had significantly higher 25(OH)D but not higher serum calcium levels vs. those not supplemented and no significant change in 1,25(OH)2D serum level (Figure 2). CS-treated or untreated patients did not differ in 25 (OH)D and 1,25(OH)2D serum levels nor in CRP or ESR (trend toward a lower ESR on CS). There was no association between bone markers and BMD value or fracture. However, as expected, CS-treated patients had significantly lower bone marker serum levels: (CTX : 372.1 pg/ml with CS vs. 480.6 pg/ml without CS, P = 0.01; BALP : 30.0 ± 13.5 UI/L vs 33.5 ± 10.8 UI/L, Biochemical parameters of bone metabolism Biochemical parameters are shown in Table 1. Serum calcium was within the normal range with no significant Saidenberg-Kermanac’h et al. Arthritis Research & Therapy 2014, 16:R78 http://arthritis-research.com/content/16/2/R78 Saidenberg-Kermanac’h et al. Arthritis Research & Therapy 2014, 16:R78 http://arthritis-research.com/content/16/2/R78 Page 4 of 9 Figure 1 Distribution of 25(OH)D (A) and 1,25(OH)2D (B) serum level in the study population. igure 1 Distribution of 25(OH)D (A) and 1,25(OH)2D (B) serum level in the study population. tribution of 25(OH)D (A) and 1,25(OH)2D (B) serum level in the study population. P = 0.37) that was found by D Kavathia et al. [18] or the number of relapses. P <0.05; osteocalcin: 15.5 ± 10.1 ng/ml vs 25.2 ± 13.2 ng/ml, P <0.001). Vitamin D supplementation was positively correlated with serum 25(OH)D (r = 0.38, P <0.001) but not with serum calcium level. Risk factors for bone fragility in sarcoidosis Table 2 shows the odds ratio of low BMD and fractures at univariate analysis. Low BMD (T-score < -1 SD) is as- sociated with age, menopause, prevalent fracture, low dietary calcium intake, cumulative CS dose, long disease duration, advanced-stage dyspnoea (III or IV), lympho- penia, high ESR and low creatinine clearance. The pa- tients with serum levels of 25(OH)D between 10 and 20 ng/ml have the lowest odds of low BMD, whereas the odds increase when this threshold is exceeded. Fractures were significantly associated with age, low dietary cal- cium intake, cumulative and current CS dose, advanced- stage dyspnoea (III or IV), low creatinine clearance and low BMD. Of note, 25(OH)D levels exceeding 20 ng/ml are associated with significantly higher odds of fracture (Table 2). BMI, ethnicity, type of sarcoidosis involve- ment, CS-free period duration, smoking, 1,25(OH)2D serum level, CRP, bone remodelling markers showed no association with BMD or fracture. The relationship between 25(OH)D serum level and risk of low BMD in our population, using a generalized addi- tive model followed a U-shaped curve: patients with serum 25(OH)D levels between 10 and 20 ng/ml are at lower risk of low BMD. The relationship between 25(OH) D and fracture is more linear, but with a steeper slope for 25(OH)D values above 20 ng/ml (Figure 3A, B). We then evaluated whether history of vitamin D sup- plementation or BP treatment modified the relationship between 25(OH)D serum level, low BMD and fracture. In our sample, vitamin D-supplemented or BP-treated patients had higher prevalence of bone fragility fracture (P <0.01) and lower BMD at both the lumbar spine (P <0.01) and hip (P <0.05) (Table 4). The relationship between 25(OH)D serum level and BMD is unmodified if those patients are excluded from the analysis (interaction between 25(OH)D serum level and vitamin D supplements or BP treatment, P = 0.71), (Figure 3C). Conversely, as far as fracture is concerned, We then assessed factors associated with low BMD using multivariate analysis (Table 3). Osteoporosis prevalence in sarcoidosis The mean BMD was normal: Tscore: -0.5 SD at the lum- bar spine and -0.09 SD at the total hip. However, 34.8% presented low BMD at the lumbar spine (37 patients had osteopenia and 11 patients had osteoporosis, re- spectively) and 24% had osteopenia or osteoporosis at the hip (24 patients and 9 patients, respectively). On the whole, 40.15% (55/137) were osteopenic at one skeletal site at least, and 14.6% (20/137) were osteoporotic. Serum levels of 25(OH)D show significant but weak in- verse correlation with disease flares (r = -0.18, P <0.05), the severity of the pulmonary involvement (r = -0.18, P <0.05), ACE (r = -0.19, P <0.05) and ESR (r = -0.19, P <0.05). 1,25 (OH)2D serum levels are positively correlated with serum 25(OH)D (r = 0.35, P <0.001) and with no other parameter. In particular, we did not find correlation between 1,25 (OH)2D serum level and chronicity of the disease (r = -0.08, Figure 2 Changes in serum 25(OH)D, calcium and 1,25 (OH) 2D level after vitamin D supplementation. Changes in serum 25(OH)D (A) calcium (B) and 1,25 (OH) 2D (C) level according to the dose of vitamin D supplementation in the six months that preceded the study. There is no significant change in calcium and 1,25 (OH) 2D serum level depending on the total amount of vitamin D supplementation. Figure 2 Changes in serum 25(OH)D, calcium and 1,25 (OH) 2D level after vitamin D supplementation. Changes in serum 25(OH)D (A) calcium (B) and 1,25 (OH) 2D (C) level according to the dose of vitamin D supplementation in the six months that preceded the study. There is no significant change in calcium and 1,25 (OH) 2D serum level depending on the total amount of vitamin D supplementation. Page 5 of 9 Page 5 of 9 Saidenberg-Kermanac’h et al. Arthritis Research & Therapy 2014, 16:R78 http://arthritis-research.com/content/16/2/R78 A total of 13.6% of the patients (19/139) had at least one vertebral fracture and 10 patients presented with two or more vertebral fractures. Overall, 23.5% of patients (32/ 136) had at least one vertebral or peripheral fracture. lymphopenia and vitamin D supplementation are associ- ated with higher odds of low BMD. Again, the patients with 25(OH)D serum levels between 10 and 20 ng/ml are at lower risk of low BMD vs. those with levels <10 ng/ml (reference class), while levels exceeding 20 ng/ml are associated with a higher risk of fracture. Osteoporosis prevalence in sarcoidosis Low dietary calcium and high CS doses are also associated with a higher risk of fracture. Risk factors for bone fragility in sarcoidosis Age, prevalent frac- ture, female gender, menopause, low dietary calcium, Table 2 Factors associated with low BMD and fracture at univariate analysis BMD < −1SD Fracture OR [95% CI] P OR [95% CI] P Menopause 5.29 [1.83; 15.27] 0.002 2.1 [0.68; 6.53] 0.2 Age, years 1.06 [1.03; 1.1] <0.001 1.09 [1.05; 1.14] <0.001 Calcium intake <500 mg/d 4.28 [1.89; 9.72] <0.001 2.2 [0.93; 5.18] 0.072 Stage NHYA 3 or 4 3.34 [1.3; 8.54] 0.012 3.18 [1.23; 8.2] 0.017 Disease duration 1.05 [1; 1.1] 0.047 1 [0.95; 1.06] 0.9 Cumulative CS dose 1.15 [0.98; 1.36] 0.088 1.2 [0.99; 1.44] 0.059 Current CS treatment 1.68 [0.83; 3.4] 0.15 2.63 [1.08; 6.37] 0.033 Vitamin D supplements 3.82 [1.66; 8.8] 0.002 2.07 [0.87; 4.95] 0.1 BP treatment 3.03 [1.44; 6.36] 0.003 1.83 [0.8; 4.15] 0.15 ESR >10 mm/h 2.49 [1.1; 5.62] 0.028 1.38 [0.55; 3.46 0.5 Lymphocytes >1,000/mm3 0.45 [0.21; 0.94] 0.033 1.19 [0.5; 2.85] 0.7 Creatinine clearance, ml/mn 0.99 [0.98; 1] 0.034 0.97 [0.95; 0.99] <0.001 25(OH)D serum level 25(OH)D ≤10 ng/ml 1 - 1 - 10 < 25(OH)D <20 ng/ml 0.48 [0.21; 1.09] 0.079 1.24 [0.45; 8.23] 0.67 25(OH)D >20 ng/ml 2.00 [0.8; 4.97] 0.13 2.94 [1.05; 8.23] 0.04 Serum PTH level, pmol/l Fracture/low BMD 4.39 [1.86; 10.37] <0.001 4.39 [1.86; 10.37] <0.001 BMD, bone mineral density; BP, bisphosphonates; CS, Corticosteroids; ESR, erythrocyte sedimentation rate; PTH, parathyroid hormone; NYHA, New York Heart Association. Table 2 Factors associated with low BMD and fracture at univariate analysis Saidenberg-Kermanac’h et al. Arthritis Research & Therapy 2014, 16:R78 http://arthritis-research.com/content/16/2/R78 Saidenberg-Kermanac’h et al. Arthritis Research & Therapy 2014, 16:R78 http://arthritis-research.com/content/16/2/R78 Saidenberg-Kermanac’h et al. Arthritis Research & Therapy 2014, 16:R78 http://arthritis-research.com/content/16/2/R78 Saidenberg-Kermanac’h et al. Risk factors for bone fragility in sarcoidosis Arthritis Research & Therapy 2014, 16:R78 http://arthritis-research.com/content/16/2/R78 Page 6 of 9 Table 3 Factors associated with low BMD and fracture at multivariate analysis BMD < −-SD Fracture OR [95% CI] P OR [95% CI] P Menopausal female 13.84 [2.28; 84.11] 0.004 Male ≥50 years 12.2 [1.42; 104.71] 0.023 Calcium intake <500 mg/d 3.98 [1.19; 13.25] 0.025 3.5 [1.09; 11.27] 0.036 Vitamin D supplements 12.86 [2.98; 55.53] 0.001 BP treatment 4.6 [1.47; 14.3 ] 0.009 Fracture 3.88 [0.99 ;15.23] 0.052 25(OH)D ≤10 ng/ml 1 - 1 - 10 < 25(OH)D ≤20 ng/ml 0.29 [0.08; 1.06] 0.062 2.05 [0.57; 7.45] 0.274 25(OH)D >20 ng/ml 0.96 [0.25; 3.67] 0.95 3.93 [1.02; 15.17] 0.047 Current CS treatment 3.73 [1.06; 13.16] 0.04 Creatinine clearance 0,97 [0.94; 0.99] 0.002 BMD, Bone mineral density; BP, Bisphosphonate; CS, Corticosteroids. Empty cells correspond to variables not included in the multivariate analysis because they were not significant at univariate analysis or to variables included in the multivariate analysis but not retained in the final model because they were no longer significant (for example, age). Table 3 Factors associated with low BMD and fracture at multivariate analysis BMD, Bone mineral density; BP, Bisphosphonate; CS, Corticosteroids. Empty cells correspond to variables not included in the multivariate analysis because they were not significant at univariate analysis or to variables included in the multivariate analysis but not retained in the final model because they were no longer significant (for example, age). Figure 3 Relation between 25(OH)D and low BMD and fracture. Relation between 25(OH)D (ng/ml) and the odds of low BMD (A, C) and fracture (B, D) with the use of a generalized additive model (GAM) for the totality of patients (A, B) and after exclusion of Vitamin D or BP-treated patients (C, D) respectively. The relationship between vitamin D levels and the odds of low BMD follows a U-shaped curve (A, C) while the relationship with fracture risk follows a linear relationship. Shaded areas represent the 95% confidence intervals and the tick marks show the distribution (with median and interquartile range) of 25(OH)D values. BMD, bone mineral density; BP, bisphosphonates. Figure 3 Relation between 25(OH)D and low BMD and fracture. Discussion This study is the first that assesses the link between the metabolism of calcium and vitamin D and the risk of osteoporosis in a population of patients with sarcoidosis. We observe that 25(OH)D levels are associated with low BMD and fracture and might be a risk factor for both. In addition, we found a high prevalence of fracture con- trasting with a normal mean BMD in this population. The results of this study allow us to highlight three sig- nificant points concerning vitamin D in sarcoidosis. Even if the biological mechanism of the toxicity of high vitamin D levels remains speculative [23-25], extra- renal synthesis of 1.25(OH)2D in sarcoid granuloma resulting in excessive 1.25(OH)2D levels, could be in- volved [3]. In our sample, there was a positive correl- ation between 25(OH)D and 1,25(OH)2D serum levels. While physiological levels of 1.25(OH)2D inhibit PTH- dependent bone resorption, higher levels are conversely known to induce bone resorption. Hamada et al. [26] found that in female (but not male) CS-free sarcoidosis patients, 1.25(OH)2D levels were negatively correlated with lumbar Z-score and serum calcium and positively correlated with osteocalcin. In our patients, whether treated with CS or not, we did not find any correlation between serum 1,25(OH)2D and bone markers apart from a negative correlation with bone alkaline phosphat- ase. However, dosing of 1.25(OH)2D is subject to fluctu- ations [27], and our population was not homogeneous: the patients had variable forms of the disease and most of them were on CS. All these factors might have inter- fered with the results. Nevertheless, consensus exists that the level of 25(OH)D (precursor of 1,25(OH)2D) is more stable and more relevant to evaluate vitamin D status [28]. Hence, 25(OH)D might be better suited to assess the relationship between vitamin D and both os- seous and extra-osseous involvement of the disease. First, we found a significant association among 25(OH) D serum level, BMD and risk of fracture. Levels ranging between 10 and 20 ng/ml are associated with higher BMD while this association is lost for higher values, which are conversely associated with higher risk of fracture. These associations do not seem to be due to vitamin D supple- ment in patients with lower BMD or higher risk of frac- ture. In fact, the associations persisted at multivariable analysis after correction for vitamin D supplementation in the last six months. Risk factors for bone fragility in sarcoidosis Relation between 25(OH)D (ng/ml) and the odds of low BMD (A, C) and fracture (B, D) with the use of a generalized additive model (GAM) for the totality of patients (A, B) and after exclusion of Vitamin D or BP-treated patients (C, D) respectively. The relationship between vitamin D levels and the odds of low BMD follows a U-shaped curve (A, C) while the relationship with fracture risk follows a linear relationship. Shaded areas represent the 95% confidence intervals and the tick marks show the distribution (with median and interquartile range) of 25(OH)D values. BMD, bone mineral density; BP, bisphosphonates. Page 7 of 9 Saidenberg-Kermanac’h et al. Arthritis Research & Therapy 2014, 16:R78 http://arthritis-research.com/content/16/2/R78 Page 7 of 9 Table 4 Prevalence of patients having low BMD (≤1 SD) or at least one prevalent fracture according to 25(OH)D serum levels BMD Fracture All patients No BP or Vitamin D All patients No BP or Vitamin D 25OHD <10 20/46 (43%) 8/29 (27%) 8/46 (17%) 2/27 (7%) 10 < 25OHD <20 15/56 (27%) 5/31 (16%) 11/53 (21%) 8/28 (28%) 25OHD >20 20/33 (61%) 4/7 (57%) 13/34 (38%) 2/7 (28%) BMD, bone mineral density; BP, bisphosphonates. The results are reported for the whole sample and for those patients that had not received BP treatment or vitamin D supplementation. aving low BMD (≤1 SD) or at least one prevalent fracture according to 25(OH)D serum there is a significant interaction between 25(OH)D and vitamin D and BP treatment (P <0.05), explained by the low prevalence of fracture in patients with serum levels of 25(OH)D <10 ng/ml if vitamin D and BP-treated pa- tients are excluded (Table 4). Nevertheless, even after exclusion of these patients, the relationship between 25 (OH)D levels and fracture remains linear (Figure 3D). mean maximum serum level 74 ng/ml) is associated with lower BMD and decrease of bone remodeling in osteope- nic post-menopausal women. Finally, Ensrud et al. [21] found that association between 25(OH)D and frailty status (a risk factor of fall and fracture) may have a U-shaped pattern with increasing odds of frailty at the lower (<20 ng/ml) and higher (≥30 ng/ml) 25(OH)D levels. In sarcoidosis patients, known to be more sensitive to vitamin D [5], the optimal range of 25(OH)D levels might be lower than that desirable for the general population as it has been described in idiopathic infantile hypercalcemia. Risk factors for bone fragility in sarcoidosis In this rare disease, the presence of CYP24A1 mutation causes inactivation of 24-hydroxylase and explains the in- creased sensitivity to vitamin D [22]. In this disease, 25 (OH)D serum must be maintained at low levels to avoid hypercalcemia. Received: 6 August 2013 Accepted: 4 March 2014 Published: 24 March 2014 Received: 6 August 2013 Accepted: 4 March 2014 Published: 24 March 2014 Discussion Moreover, the exclusion of patients having received vitamin D or BP supplements to prevent corticosteroid-induced osteoporosis did not affect the re- lationship between 25(OH)D and BMD or fracture at gen- eralized additive model analysis. These data suggest that excessive vitamin D supplement could be deleterious in these patients. This notion might be supported by the results of recent studies on the general population: Vital D Study [19], a double-blind, randomized, controlled trial involving 2,317 community-dwelling women (mean age 70 years) ran- domly assigned to receive either a single oral dose of cho- lecalciferol 500,000 IU or placebo yearly for three to five years, found a higher risk of fracture and fall in supple- mented women in whom baseline 25(OH)D serum level increased from 19.6 to 48.07 ng/ml. Grimnes et al. [20] found that excessive vitamin D supplementation (inducing The second point concerning vitamin D is the possible correlation between low 25(OH)D serum level (and not Page 8 of 9 Saidenberg-Kermanac’h et al. Arthritis Research & Therapy 2014, 16:R78 http://arthritis-research.com/content/16/2/R78 Saidenberg-Kermanac’h et al. Arthritis Research & Therapy 2014, 16:R78 http://arthritis-research.com/content/16/2/R78 Saidenberg-Kermanac’h et al. Arthritis Research & Therapy 2014, 16:R78 http://arthritis-research.com/content/16/2/R78 1.25(OH)2D) and the parameters of disease activity. This was suggested in other inflammatory diseases, such as RA, where low 25(OH)D levels were associated with dis- ease flares [29,30]. 1.25(OH)2D) and the parameters of disease activity. This was suggested in other inflammatory diseases, such as RA, where low 25(OH)D levels were associated with dis- ease flares [29,30]. Conclusion This is the first study that establishes a link between vitamin D levels and bone mineral density in patients with sarcoidosis and suggests an optimal threshold of 25 (OH)D in this population. Furthermore, these data sug- gest that particular risk factors for osteoporosis should be taken into account for sarcoidosis patients, whose fracture risk is high and poorly related to BMD. The third important point is that patients supple- mented with vitamin D have significantly higher serum 25(OH)D but not higher serum calcium vs. non- supplemented patients. Accordingly, Adler et al. [10] did not find any impact of calcium and vitamin D supple- mentation on serum or urinary calcium in sarcoidosis patients. Authors’ contribution k d h d Nevertheless, the risk of hypercalcemia after vitamin D supplementation reported in other series should lead to caution [31]. Further studies are needed to better identify what patient can be safely supplemented in cal- cium and Vitamin D and at what dose. NSK worked on the study conception and design, study conduct, data collection and analysis, data interpretation, drafting of the manuscript and revision of the manuscript content, and takes responsibility for the integrity of the data analysis, and final approval of the manuscript. LS was responsible for data analysis and interpretation, drafting and revising the manuscript content, and final approval of the manuscript. HN, DS, XG, MB, NN and MCB contributed to data collection and analysis, revision and final approval of the manuscript. DV was responsible for data interpretation, revising manuscript content and final approval of the manuscript. All authors read and approved the final manuscript. We observed a higher prevalence of fracture com- pared to epidemiological data on healthy adults of the same age [32]. This prevalence is also close to that found in young, adult CS-treated patients with other diseases [33] and to that found by Heijckmann [34] in a cross-sectional study in sarcoidosis patients. Even if low BMD was correlated with the risk of fracture, mean BMD was normal in our study patients, contrasting with the high prevalence of fracture. This suggests that other factors than BMD are involved, and must be taken into account, in the evaluation of global fracture risk in these patients. Among them, cumulative CS dose, age, respiratory insufficiency and altered renal function were all associated with increased risk of fracture at univari- able analysis. Acknowledgements h d ll This study was partially supported by the GRIO “Groupe de recherche et d’information sur les ostéoporoses” (France). The authors wish to thank Evelyne Avice for her outstanding assistance with data collection. Competing interest ll h h Competing interest All authors state that they have no conflicts of interest. Abbreviations ACE: Angiotensin-converting enzyme; BALP: Bone alkaline phosphatases; BMD: Bone mineral density; BMI: Body mass index; BP: Bisphosphonate; CRP: C-reactive protein; CS: Corticosteroids; CTX: C-terminal telopeptide of type I collagen; ESR: Erythrocyte sedimentation rate; NYHA: New York Heart Association; PTH: Parathyroid hormone; QCT: Quantitative computed tomography; RA: Rheumatoid arthritis; TSH: Thyroid stimulating hormone; VFA: Vertebral fracture assessment. Overall, our findings suggest that vitamin D supple- mentation should be considered in sarcoidosis patients but should probably target a threshold that might be lower than that advised for the general population. In addition, low dietary calcium correlated with low BMD and high risk of fracture support the need for adequate calcium intake in these patients. Author details 1 1INSERM UMR1125, Bobigny, France. 2Sorbonne Paris Cité-Université Paris 13, Bobigny, France. 3Assistance Publique-Hôpitaux de Paris, Groupe Hospitalier Avicenne-Jean Verdier-René Muret, Department of Rheumatology, Bobigny, France. 4Assistance Publique-Hôpitaux de Paris, Groupe Hospitalier Avicenne-Jean Verdier-René Muret, Department of Respiratory Diseases, Bobigny, France. 5Assistance Publique-Hôpitaux de Paris, Groupe Hospitalier Avicenne-Jean Verdier-René Muret, Clinical Research Unit, Bobigny, France. 6Assistance Publique-Hôpitaux de Paris, Groupe Hospitalier Avicenne-Jean Verdier-René Muret, Department of Radiology, Bobigny, France. We did not find any correlation between BMD or frac- tures and parameters of inflammation but in this study the average ESR and CRP were low. However, lympho- cyte count was inversely correlated with BMD at both univariable and multivariable analysis. References ld Bouvard B, Annweiler C, Sallé A, Beauchet O, Chappard D, Audran M, Legrand E: Extraskeletal effects of vitamin D: facts, uncertainties, and controversies. Joint Bone Spine 2011, 78:10–16. 9. Sipah S, Tuzun S, Ozaras R, Calis HT, Ozaras N, Tuzun F, Karayel T: Bone mineral density in women with sarcoidosis. J Bone Miner Metab 2004, 22:48–52. 31. Baughman RP, Janovcik J, Ray M, Sweiss N, Lower EE: Calcium and vitamin D metabolism in sarcoidosis. Sarcoidosis Vasc Diffuse Lung Dis 2013, 1:113–120. 10. Adler RA, Funkhouser HL, Petkov VI, Berger MM: Glucocorticoid-induced osteoporosis in patient with sarcoidosis. Am J Med Sci 2003, 325:1–6. 32. van der Klift M, de Laet CE, McCloskey EV, Hofman A, Pols HA: The incidence of vertebral fractures in men and women: the Rotterdam Study. J Bone Miner Res 2002, 17:1051–1056. 11. Montemurro L, Fraioli P, Riboldi A, Delpiano S, Zanni D, Rizzato G: Bone loss i d i d id i f ll A I l M d I 11. Montemurro L, Fraioli P, Riboldi A, Delpiano S, Zanni D, Rizzato G: Bone loss in prednisone treated sarcoidosis: a two-year follow-up. Ann Ital Med Int 1990, 5:164–168. in prednisone treated sarcoidosis: a two-year follow-up. Ann Ital Med Int 1990, 5:164–168. y 33. Siffledeen JS, Siminoski K, Jen H, Fedorak RN: Vertebral fractures and role of low bone mineral density in Crohn’s disease. Clin Gastroenterol Hepatol 2007, 5:721–728. 12. Rizzato G, Tosi G, Mella C, Zanni D, Sisti S, Loglisci T: Researching osteoporosis in prednisone treated sarcoid patients. Sarcoidosis 1987, 4:45–48. 34. Heijckmann AC, Huijberts MS, De Vries J, Menheere PP, Van Der Veer E, Kruseman AC, Wolffenbuttel BH, Geusens P, Drent M: Bone turnover and hip bone mineral density in patient with sarcoidosis. Sarcoidosis Vasc Diffuse Lung Dis 2007, 24:51–58. 13. Heijckmann AC, Drent M, Dumitrescu B, De Vries J, Nieuwenhuijzen Kruseman AC, Wolffenbuttel BH, Geusens P, Huijberts MS: Progressive vertebral deformities despite unchanged bone mineral density in patients with sarcoidosis: a 4-year follow-up study. Osteopors Int 2008, 19:839–847. doi:10.1186/ar4519 Cite this article as: Saidenberg-Kermanac’h et al.: Bone fragility in sarcoidosis and relationships with calcium metabolism disorders: a cross sectional study on 142 patients. Arthritis Research & Therapy 2014 16:R78. 14. Statement on sarcoidosis. References ld 1. Goldring SR, Gravallese EM: Mechanisms of bone loss in inflammatory arthritis: diagnosis and therapeutic implications. Arthritis Res 2000, 2:33–37. 1. Goldring SR, Gravallese EM: Mechanisms of bone loss in inflammatory arthritis: diagnosis and therapeutic implications. Arthritis Res 2000, 2:33–37. The main study limitations lie in the cross-sectional and monocentric design, in the lack of a control popula- tion and the heterogeneity of the disease profile in the study population. This last point is difficult to prevent to get a sufficient sample size of patients but the multivari- ate analysis allows us to identify the main risk factors of osteoporosis. The main strength is the large sample size for this disease. These preliminary data need to be con- firmed in longitudinal studies in particular to verify the association between the different levels of 25(OH)D and the risk of fracture or low BMD. 2. Confavreux CB, Chapurlat RD: Systemic bone effects of biologic therapies in rheumatoid arthritis and ankylosing spondylitis. Osteoporos Int 2011, 22:1023–1036. 3. Reichel H, Koeffer H, Barbers R, Norman AW: Regulation of 1,25-dihydroxyvitamin D production by cultured alveolar macrophages from normal human donors and from patient with pulmonary sarcoidosis. J Clin Endocrinol Metab 1987, 65:1201–1209. 4. Rizzato G: Clinical impact of bone and calcium metabolism changes in sarcoidosis. Thorax 1998, 53:425–429. 5. Stern PH, De Olazabal J, Bell NH: Evidence for abnormal regulation of circulating 1 alpha,25-dihydroxyvitamin D in patients with sarcoidosis and normal calcium metabolism. J Clin Invest 1980, 66:852–855. 6. Rizzato G, Montemurro L: Reversibility of exogenous corticosteroid-induced bone loss. Eur Respir J 1993, 6:116–119. Page 9 of 9 Page 9 of 9 Saidenberg-Kermanac’h et al. Arthritis Research & Therapy 2014, 16:R78 http://arthritis-research.com/content/16/2/R78 7. Rizzato G, Fraioli P: Natural and corticosteroid-induced osteoporosis in sarcoidosis: prevention, treatment, follow up and reversibility. Sarcoidosis 1990, 7:89–92. 29. Welsh P, Peters MJ, McInnes IB, Lems WF, Lips PT, McKellar G, Knox S, Michael Wallace A, Dijkmans BA, Nurmohamed MT, Sattar N: Vitamin D deficiency is common in patients with RA and linked to disease activity, but circulating levels are unaffected by TNFα blockade: results from a prospective cohort study. Ann Rheum Dis 2011, 70:1165–1167. 8. Rizzato G, Tosi G, Mella C, Montemurro L, Zanni D, Sisti S: Prednisone-induced bone loss in sarcoidosis: a risk especially frequent in postmenopausal women. Sarcoidosis 1988, 5:93–98. 30. References ld Joint Statement of the American Thoracic Society (ATS), the European Respiratory Society (ERS) and the World Association of Sarcoidosis and Other Granulomatous Disorders (WASOG) adopted by the ATS Board of Directors and by the ERS Executive Committee, February 1999. Am J Respir Crit Care Med 1999, 160:736–755. 15. Fardellone P, Sebert JL, Bouraya M, Bonidan O, Leclercq G, Doutrellot C, Bellony R, Dubreuil A: Evaluation of the calcium content of diet by frequential self-questionnaire. Rev Rhum Mal Osteoartic 1991, 58:99–103. 16. Genant HK, Wu CY, van Kuijk C, Nevitt MC: Vertebral fracture assessment using a semiquantitative technique. J Bone Miner Res 1993, 8:1137–1148. 17. Ross AC, Manson JE, Abrams SA, Aloia JF, Brannon PM, Clinton SK, Durazo-Arvizu RA, Gallagher JC, Gallo RL, Jones G, Kovacs CS, Mayne ST, Rosen CJ, Shapses SA: The 2011 report on dietary reference intakes for calcium and vitamin D from the Institute of Medicine: what clinicians need to know. J Clin Endocrinol Metab 2011, 96:53–58. 18. Kavathia D, Buckley JD, Rao D, Rybicki B, Burke R: Elevated 1, 25-dihydroxyvitamin D levels are associated with protracted treatment in sarcoidosis. Respir Med 2010, 104:564–570. 19. Sanders KM, Stuart AL, Williamson EJ, Simpson JA, Kotowicz MA, Young D, Nicholson GC: Annual high-dose oral vitamin D and falls and fractures in older women: a randomized controlled trial. JAMA 2010, 303:815–822. 20. Grimnes G, Joakimsen R, Figenschau Y, Torjesen PA, Almås B, Jorde R: The effect of high-dose vitamin D on bone mineral density and bone turnover markers in postmenopausal. Osteoporos Int 2012, 23:201–211. 21. Ensrud KE, Ewing SK, Fredman L, Hochberg MC, Cauley JA, Hillier TA, Cummings SR, Yaffe K, Cawthon PM, Study of Osteoporotic Fractures Research Group: Circulating 25-hydroxyvitamin D levels and frailty status in older women. J Clin Endocrinol Metab 2010, 95:5266–5273. 22. Schlingmann KP, Kaufmann M, Weber S, Irwin A, Goos C, John U, Misselwitz J, Klaus G, Kuwertz-Bröking E, Fehrenbach H, Wingen AM, Güran T, Hoenderop JG, Bindels RJ, Prosser DE, Jones G, Konrad M: Mutations in CYP24A1 and idiopathic infantile hypercalcemia. N Engl J Med 2011, 365:410–421. 23. Dawson-Hughes B, Harris SS: High-dose vitamin D supplementation: too much of a good thing? JAMA 2010, 303:1861–1862. 24. Ueno Y, Shinki T, Nagai Y, Murayama H, Fujii K, Suda T: In vivo administration of 1,25-dihydroxyvitamin D3 suppresses the expression of RANKL mRNA in bone of thyroparathyroidectomized rats constantly infused with PTH. J Cell Biochem 2003, 90:267–277. Saidenberg-Kermanac’h et al. Arthritis Research & Therapy 2014, 16:R78 http://arthritis-research.com/content/16/2/R78 References ld Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: 25. Anderson PH, Iida S, Tyson JH, Turner AG, Morris HA: Bone CYP27B1 gene expression is increased with high dietary calcium and in mineralising osteoblasts. J Steroid Biochem Mol Biol 2010, 121:71–75. • Convenient online submission 26. Hamada K, Nagai S, Tsutsumi T, Izumi T: Bone mineral density and vitamin D in patients with sarcoidosis. Sarcoidosis Vasc Diffuse Lung Dis 1999, 16:219–223. 27. Lips P: Vitamin D deficiency and secondary hyperparathyroidism in the elderly: consequences for bone loss and fractures and therapeutic implications. Endocr Rev 2001, 22:477–501. 28. Lips P: The relative value of 25(OH)D and 1,25(OH)2D measurements. J Bone Miner Res 2007, 22:1668–1671. 28. Lips P: The relative value of 25(OH)D and 1,25(OH)2D measurements. J Bone Miner Res 2007, 22:1668–1671.
https://openalex.org/W2331404222	https://publications.goettingen-research-online.de/bitstream/2/41186/2/cddis201676a.pdf	English	null	Cathepsin B pulls the emergency brake on cellular necrosis	Cell death and disease	2,016	cc-by	1,842	Cathepsin B pulls the emergency brake on cellular necrosis FS Wouters,1,2 and G Bunt1,3 FS Wouters,1,2 and G Bunt1,3 FS Wouters,1,2 and G Bunt1,3 Cell Death and Disease (2016) 7, e2170; doi:10.1038/cddis.2016.76; published online 31 March 2016 Cell death can take many shapes. Programmed ‘apoptotic’ and uncontrolled ‘necrotic’ cell death mark the extremes of the spectrum of possibilities.1 Whereas the induction of apoptosis follows distinct signaling steps and cleanly removes a cell from its tissue, necrosis represents the cell’s wholesale disintegra- tion and is in general harmful to the organism. Massive cellular insults lead to necrosis. It appears, however, that cells have some choice in the mode of their demise depending on the severity of the insult. Hypoxic cores in solid tumors2 or perfusion-impaired brain tissue in stroke,3 for instance are typically necrotic, but possess an apoptotic shell. occurs rapidly and is completed within minutes. Among the observed bcl-2 members, anti-apoptotic bcl-xl is degraded before pro-apoptotic bid and bax. Cells thus appear to accumulate pro-apoptotic reactants in the early stages of lysosomal rupture. The pro-apoptotic bid showed a conspicuous behavior. Before the overall sigmoidal degradation, a pronounced and very rapid cleavage was observed in the first minutes of lysosomal lysis. Fast proteolytic processing of bid is used in apoptosis and might serve the same role in lysosomal lysis. In apoptosis, the caspase 8 protease activates bid by its truncation, directly connecting the extrinsic and intrinsic apoptotic signaling pathways. This mechanism is called the ‘bid shunt’ and processing of bid by cathepsin B has been suggested to operate similarly.11 Our work places the bid shunt very early in lysosomal cell death signaling and this is the first time this event is caught ‘on film’. The same can be seen for lysosomal damage.4–6 Lysosomes can rupture during ischemic or traumatic cell injury,7 giving rise to both apoptotic and necrotic outcomes. Lysosomes are cellular organelles with a proteolytic function and possess an impressive set of protease enzymes8 dedicated to the degradation of both intracellular material, such as damaged or old organelles, and extracellular components such as matrix proteins. The release of the lysosomal proteases into the cytosol sentences the cell to death by autodigestion. In order to gain a better understanding of the regulation of bcl-2 protein levels by cathepsins and the involvement in programmatic steps in necrosis and apoptosis, we investi- gated the interplay of both systems in more detail. 1Laboratory for Molecular and Cellular Systems, Institute of Neuropathology, University Medical Center Göttingen, Göttingen, Germany; 2Centre for Nanoscale Molecular Physiology of the Brain, Göttingen, Germany and 3Clinical Optical Microscopy, Institute of Neuropathology, University Medical Center Göttingen, Göttingen, Germany Corresponding author: FS Wouters, Laboratory for Molecular and Cellular Systems, Institute of Neuropathology, University Medical Center Göttingen, Waldweg 33, D-37073 Göttingen, Germany. Tel: +49 551 3912368; Fax: +49 551 396031; E-mail: fred.wouters@gwdg.de Citation: Cell Death and Disease (2016) 7, e2170; doi:10.1038/cddis.2016.76 & 2016 Macmillan Publishers Limited All rights reserved 2041-4889/16 Citation: Cell Death and Disease (2016) 7, e2170; doi:10.1038/cddis.2016.76 & 2016 Macmillan Publishers Limited All rights reserved 2041-4889/16 OPEN www.nature.com/cddis News and Commentary Conflict of Interest The authors declare no conflict of interest. general inhibition of the thiol-cathepsin classes resulted in the complete inhibition of the proteolysis of these same sensors, we concluded that their accelerated degradation upon cathepsin B inhibition is most likely caused by another, unidentified, thiol-cathepsin. This proteolytic cascade pre- vents the degradation of apoptosis-supporting proteins, shifting the balance toward apotosis. The identity of this cathepsin requires further research. The authors declare no conflict of interest. Acknowledgements. This work was supported by a grant from the Federal Ministry of Science and Education, BMBF (13N9243) and the Open Access Publication Fund of the Göttingen University. 1. Yuan J et al. Genes Dev 2010; 24: 2592–2602. 2. Riva C et al. Anticancer Res 1998; 18: 4729–4736. 3. Memezawa H et al. Stroke 1992; 23: 552–559. 4. Kilinc M et al. Neurobiol Dis 2010; 40: 293–302. 5. Qin AP et al. Neurosci Bull 2008; 24: 117–123. 6. Yamashima T et al. Prog Neurobiol 2009; 89: 343–358. 7. Boya P et al. Oncogene 2008; 27: 6434–6451. 8. Müller S et al. Biochim Biophys Acta 2012; 1824: 34–43. 9. de Castro MAG et al. Cell Death Discov 2016; 2: e16012. 10. Cirman T et al. J Biol Chem 2004; 279: 3578–3587. 11. Guicciardi ME et al. J Clin Invest 2000; 106: 1127–11378. p q Cells thus appear to be able to pull an ‘emergency brake’ upon lethal injury with damaging of lysosomes. Starting with cathepsin B, an early programmatic protease cascade is initiated that appears to avoid necrosis in favor of apoptosis. In this apoptotic exit effort, cathepsin B preferentially degrades anti-apoptotic bcl-xl, activates bid by its rapid and selective truncation, and degrades a thiol-cathepsin to avoid the removal of apoptotic bid and bax (Figure 1, right). Up to a certain level of lysosomal damage, this three-pronged rescue program steers dying cells away from necrosis. When massive damage overwhelms the cell with thiol-cathepsins that eat away at essential cellular proteins, necrosis becomes unavoidable. Our research contributes to the understanding of cell death on a molecular mechanistic level by uncovering programmatic steps at the interface between necrosis and apoptosis. Knowledge on cell death decision signaling is therapeutically relevant, for example, in the formulation of more effective chemotherapy and in combatting toxic drug side effects. 11. Guicciardi ME et al. J Clin Invest 2000; 106: 1127–11378 Cell Death and Disease is an open-access journal published by Nature Publishing Group. Cathepsin B pulls the emergency brake on cellular necrosis g y y Given the relatively low amount of lysosomes that were disrupted in the early stages after lysosomal disruption, we selected the neutral-active cathepsin B as likely candidate for most of the early proteolytic actions. Hence, we repeated the experiments in the presence of a selective cathepsin B inhibitor and an inhibitor for the thiol-cathepsin class, to which cathepsin B belongs. Inhibition of the thiol-cathepsins con- firmed their predominant role in the degradation of bcl-2 family proteins, as the proteolysis of all three bcl-2 constructs was inhibited. Selective cathepsin B inhibition helped uncover its role in steering cell death. We found that early bid truncation was abolished, confirming cathepsin B as the responsible protease. Furthermore, when we limited lysosomal protease action to the time window of the early bid truncation, we detected late-apoptotic caspase 3/7 activation, showing that cells had chosen an apoptotic exit. In a recent Cell Death Discovery article,9 we looked at the fate of the major regulators of apoptosis, the bcl-2 protein family, in order to understand how cell fate upon lysosomal rupture is directed toward necrosis or apoptosis. This protein family contains both pro- and anti-apoptotic members and has been shown to be part of the proteolytic spectrum of cathepsin lysosomal proteases.10 We reasoned that a regulated degradation of these proteins, by affecting the equilibrium of pro- and anti-apoptotic signaling, might offer a means for switching between cell death forms. To this end, we imaged the proteolytic degradation of the major bcl-2 family representatives bcl-xl, bid and bax upon induced lysosomal disruption in real time using förster resonance energy transfer (FRET) microscopy of single cells. We created proteolytic FRET sensors by sandwiching full- length bcl-2 proteins between cyan and yellow fluorescent proteins. A strong FRET signal is detected in the intact sensor, which is lost upon cleavage (Figure 1, left). Unexpectedly and paradoxically, bax and also bid exhibited a new accelerated proteolytic behavior upon selective inhibi- tion of cathepsin B. The delay phase before the onset of cleavage was completely abolished, in particular for bax. As a The bcl-2 sensors show a common sigmoidal ‘delay-snap’ cleavage behavior: after a delay of tens of minutes, cleavage 2 News and Commentary 2 Figure 1 Left: upon lysosomal damage, cathepsin proteases are released in the cytosol, finally resulting in cell death. The apoptosis-regulating bcl-2 family proteins bcl-xl, bid and bax are cleaved by cathepsins. Cathepsin B pulls the emergency brake on cellular necrosis Their degradation was followed by FRET microscopy on single cells in real time. Cleavage of FRET sensors consisting of full-length bcl-2 proteins sandwiched between cyan and yellow fluorescent protein was imaged. The intact sensors show FRET, the cleaved sensors do not. Right: cathepsin B initiates an apoptotic exit program by (i) proteolytic removal and consequent inactivation of anti-apoptotic bcl-xl, (ii) the rapid and controlled proteolytic activation (t-bid) of pro-apoptotic bid, and (iii) the proteolytic inactivation of an unknown thiol-cathepsin that would otherwise have removed pro-apoptotic bax and bid. Even in the background of a strong necrotic stimulus as lysosomal lysis, cathepsin B thus appears to launch an early apoptotic exit program. FRET, förster resonance energy transfer; t-bid, truncated bid. Figure 1 Left: upon lysosomal damage, cathepsin proteases are released in the cytosol, finally resulting in cell death. The apoptosis-regulating bcl-2 family proteins bcl-xl, bid and bax are cleaved by cathepsins. Their degradation was followed by FRET microscopy on single cells in real time. Cleavage of FRET sensors consisting of full-length bcl-2 proteins sandwiched between cyan and yellow fluorescent protein was imaged. The intact sensors show FRET, the cleaved sensors do not. Right: cathepsin B initiates an apoptotic exit program by (i) proteolytic removal and consequent inactivation of anti-apoptotic bcl-xl, (ii) the rapid and controlled proteolytic activation (t-bid) of pro-apoptotic bid, and (iii) the proteolytic inactivation of an unknown thiol-cathepsin that would otherwise have removed pro-apoptotic bax and bid. Even in the background of a strong necrotic stimulus as lysosomal lysis, cathepsin B thus appears to launch an early apoptotic exit program. FRET, förster resonance energy transfer; t-bid, truncated bid. Figure 1 Left: upon lysosomal damage, cathepsin proteases are released in the cytosol, finally resulting in cell death. The apoptosis-regulating bcl-2 family proteins bcl-xl, bid and bax are cleaved by cathepsins. Their degradation was followed by FRET microscopy on single cells in real time. Cleavage of FRET sensors consisting of full-length bcl-2 proteins sandwiched between cyan and yellow fluorescent protein was imaged. The intact sensors show FRET, the cleaved sensors do not. Right: cathepsin B initiates an apoptotic exit program by (i) proteolytic removal and consequent inactivation of anti-apoptotic bcl-xl, (ii) the rapid and controlled proteolytic activation (t-bid) of pro-apoptotic bid, and (iii) the proteolytic inactivation of an unknown thiol-cathepsin that would otherwise have removed pro-apoptotic bax and bid. Cathepsin B pulls the emergency brake on cellular necrosis Even in the background of a strong necrotic stimulus as lysosomal lysis, cathepsin B thus appears to launch an early apoptotic exit program. FRET, förster resonance energy transfer; t-bid, truncated bid. Conflict of Interest The authors declare no conflict of interest. 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/ Cell Death and Disease Cell Death and Disease
https://openalex.org/W4221107561	https://www.nature.com/articles/s41598-022-09448-7.pdf	English	null	Surgical outcomes of acute type A aortic dissection in dialysis patients: lessons learned from a single-center’s experience	Scientific reports	2,022	cc-by	5,799	www.nature.com/scientificreports www.nature.com/scientificreports Surgical outcomes of acute type A aortic dissection in dialysis patients: lessons learned from a single‑center’s experience PEN The hemodynamic and elec-t It has been well studied that patients with end-stage renal disease (ESRD) associate with decreased life expec- tancy and are more vulnerable to develop cardiovascular events compared to healthy individuals1–3. As a well- established treatment method, increasing ESRD patients are receiving regular dialysis treatment. In 2010, the incidence and prevalence of patients who require hemodialysis were 147.3/million and 509/million in Beijing4. Acute type A aortic dissection (ATAAD) is a critical disease that often associates with lethal outcomes. It often progresses rapidly and develops life-threatening complications, such as aortic rupture and cardiac tamponade5. Although outcomes of ATAAD have been improving in recent years due to the advance of techniques6, it is still a dangerous condition, especially in patients with other comorbidities like ESRD. The hemodynamic and elec- trolytes homeostasis is often disturbed under dialysis which posts additional challenges for the surgical repair of ATAAD7. However, the outcomes of such patients had not been well described. In this study we described both the short- and long-term outcomes of dialysis patients who received ATAAD repair surgery. p p q y j g Acute type A aortic dissection (ATAAD) is a critical disease that often associates with lethal outcomes. It often progresses rapidly and develops life-threatening complications, such as aortic rupture and cardiac tamponade5. Although outcomes of ATAAD have been improving in recent years due to the advance of techniques6, it is still a dangerous condition, especially in patients with other comorbidities like ESRD. The hemodynamic and elec- trolytes homeostasis is often disturbed under dialysis which posts additional challenges for the surgical repair of ATAAD7. However, the outcomes of such patients had not been well described. In this study we described both the short- and long-term outcomes of dialysis patients who received ATAAD repair surgery. Surgical outcomes of acute type A aortic dissection in dialysis patients: lessons learned from a single‑center’s experience PEN Zhigang Wang1,4, Pingping Ge2,4, Lichong Lu1,4, Min Ge1, Cheng Chen1, Lifang Zhang3 & Dongjin Wang1* There is a paucity of data describing the safety and efficacy of acute type A aortic dissection (ATAAD) repair surgeries in dialysis patients. Our study aimed to investigated the influence of dialysis on early and late outcomes in end-stage renal disease (ESRD) patients who received repair surgery for ATAAD. A total of 882 ATAAD patients who received emergency aortic dissection repair at our center from January 2015 to December 2019 were retrospectively screened in this study and divided into the dialysis group (n = 16) and the non-dialysis group (n = 866), depending on whether they required dialysis for preoperative ESRD. No significant difference of age, preoperative hemodynamics, organ ischemia conditions, operative variables as well as the 30-Day mortality and in-hospital complications was discovered between two groups. However, the survival rates and the proportion of late aortic event (sudden death and reoperation) free population at 1 and 3 years after surgery were significantly decreased in dialysis patients compared to non-dialysis patients. Our study indicated that the short-term surgical outcomes of ATAAD in dialysis patients were comparable to non-dialysis patient. However, the dialysis patients were associated with a worse long-term prognosis. It has been well studied that patients with end-stage renal disease (ESRD) associate with decreased life expec- tancy and are more vulnerable to develop cardiovascular events compared to healthy individuals1–3. As a well- established treatment method, increasing ESRD patients are receiving regular dialysis treatment. In 2010, the incidence and prevalence of patients who require hemodialysis were 147.3/million and 509/million in Beijing4. d ( ) l d h ft h l h l ft It has been well studied that patients with end-stage renal disease (ESRD) associate with decreased life expec- tancy and are more vulnerable to develop cardiovascular events compared to healthy individuals1–3. As a well- established treatment method, increasing ESRD patients are receiving regular dialysis treatment. In 2010, the incidence and prevalence of patients who require hemodialysis were 147.3/million and 509/million in Beijing4. Acute type A aortic dissection (ATAAD) is a critical disease that often associates with lethal outcomes. It often progresses rapidly and develops life-threatening complications, such as aortic rupture and cardiac tamponade5. Although outcomes of ATAAD have been improving in recent years due to the advance of techniques6, it is still a dangerous condition, especially in patients with other comorbidities like ESRD. www.nature.com/scientificreports/ (No. BL2014004) and waived the requirement for informed consent because of the retrospective nature of the study. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013).h (No. BL2014004) and waived the requirement for informed consent because of the retrospective nature of the study. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013).h h The Follow-up was accomplished by telephone interview with the patient, family members, or the patient’s referring physicians from December 2015 to December 2020. Late aortic events were defined as residual aneu- rysm or anastomotic pseudoaneurysm, that requires another surgical repair, fatal aortic rupture, sudden death, and expansion of more than 6 cm in diameter of a residual aneurysm8. Surgical procedure and postoperative treatment. The ATAAD repair surgery were carried out as described previously9. Briefly, surgical procedures including routine median sternotomy, cardiopulmonary bypass, and intermittent cardioplegic arrest with hypothermic circulatory arrest were conducted similarly between two groups. Appropriate distal surgical method was chosen depending on the location of the intimal tear and the extent of dissection. For the proximal segment, a root reinforcement reconstruction was routinely performed. The aortic valve replacement or Bentall procedure was performed when the dissection involved the coronary ostia or aortic valve, or was in the presence of an aortic root aneurysm.t y p y After the operation, all patients were transferred to the intensive care unit (ICU). Continuous renal replace- ment therapy was started for dialysis patients 6 h after the operation. Statistical analyses. Continuous variables were expressed as mean ± standard deviations or median with interquartile and were analyzed by Student’s t-test or the Mann–Whitney U test. Categorical variables were pre- sented as n (%) and analyzed with the chi-square or Fisher’s exact test.i A systematic literature review was conducted and identified potential predictors such as age, sex, cause, medi- cal history, and operative procedures as predictors for prognosis in ATAAD. To reduce the influence of these confounding baseline parameters, a one-to-one propensity score matching method was applied to analyze the short-term outcomes (calipers of width 0.02 standard deviations of the logit of the propensity score), baseline characteristics and variables of interest that associated with outcomes (variables excepting for laboratory data listed in Table 1 and intro-operative variables listed in Table 3) were included in the analysis. Results ’ Patients’ preoperative parameters and anatomical characteristics of the ATAAD lesions were shown in Table 1. Our data showed that the average age of patients in the dialysis group was similar to those in the non-dialysis group (47.1 ± 11.2 years vs. 53.1 ± 13.2 years, p > 0.05). However, significantly more patients in the dialysis group had hypertension histories (p = 0.009). Interestingly, no significant difference was identified in preoperative hemodynamic measurements and organ malperfusion conditions between the two groups. On the other hand, the levels of leukocyte count, haemoglobin, creatinine, and blood urea nitrogen were significantly different between the two groups. In addition, we found out a clear trend that dialysis patients were more likely to have primary entry tear in the aortic arch, even though the difference was not statistically significant. fi As shown in Table 2, the leading primary cause for ESRD were hypertension (n = 10) followed by chronic glomerulonephritis (n = 5). The mean duration of dialysis history before the onset of ATAAD was 4.5 ± 3.5 years, and 87.5% of these patients were receiving hemodialysis (rather than peritoneal dialysis). As shown in Table 3, operative variables like arterial cannulation sites, aortic arch surgery methods, distal surgical techniques, and cardiopulmonary bypass duration were similar between the two groups. However, dialysis patients were more likely to receive root construction, compared to patients in the non-dialysis group. y p y p p y g p Next, we examined the early prognosis of ATAAD repair surgery in both groups (Table 4). Before propensity score matching, the 30-Day mortality rate of patients in the dialysis group was similar to the non-dialysis group (12.5% vs. 11.4%, p > 0.05). In the dialysis group, the causes for 30-Day in-hospital death were intracranial hem- orrhage (n = 1) and multi-organ failure (n = 1). Interestingly, the ICU and hospital stay were similar between the two groups as well as operation associated complications. Meanwhile, the drainage volume 24 h after sur- gery, mechanical ventilation duration, and re-intubation rate were significantly increased in the dialysis group (p < 0.05). After propensity score matching, the 30-Day mortality remained similar between the two groups. In addition, the differences of other postoperative parameters were no longer identifiable between the two groups after propensity score matching.h t 105 patients (11.9%) died during the hospitalization period. The median follow-up was 29 months. www.nature.com/scientificreports/ Cumulative survival and late aortic event free rate were calculated by the Kaplan–Meier method which was performed using STATA, version 15.0 (Stata Corporation, College Station, TX), and the difference was determined by the long-rank test. The rest of statistical analyses were carried out using IBM SPSS Version 25.0 (SPSS Science, Chicago, IL, USA). A two-sided p-value < 0.05 was considered statistically significant. Ethics declarations. All procedures performed in studies involving human participants were in accord- ance with the ethical standards of Nanjing Drum Tower Hospital of Medicine Ethics Committee for Clinical studies at which the studies were conducted (Approval number: No. BL2014004). Written informed consent was waived due to the nature of the study. Methods and materials A total of 882 consecutive patients who received emergent ATAAD surgery at Nanjing Drum Tower Hospital between January 2015 and December 2019 were retrospective screened for this study. The diagnosis of ATAAD was made on the basis of enhanced computed tomography and the acute ATAAD was characterized as patients within 14 days of symptom onset. Among all 882 patients, 16 patients were receiving hemodialysis or peritoneal dialysis therapy for ESRD before the onset of aortic dissection. No patients received renal transplantation before the onset of the ATAAD.h The 882 patients were divided into two groups according to whether they were receiving dialysis therapy before the surgery (dialysis group, n = 16; non-dialysis group, n = 866). Patients’ medical records and imaging results were reviewed. The institutional review board of the Nanjing Drum Tower Hospital approved this study 1Department of Cardio‑Thoracic Surgery, Affiliated Drum Tower Hospital, Medical School of Nanjing University, Zhongshan Road 321, Nanjing 210008, China. 2Department of General Practice, Nanjing First Hospital, Nanjing Medical University, Nanjing, China. 3Department of Psychiatry, The First Affiliated Hospital, Zhengzhou University, Zhengzhou, China. 4These authors contributed equally: Zhigang Wang, Pingping Ge and Lichong Lu. *email: glyywdj@163.com \| https://doi.org/10.1038/s41598-022-09448-7 Scientific Reports \| (2022) 12:5372 www.nature.com/scientificreports/ Results ’ 46 patients (5.9%) who were lost to follow-up and 1 patient who committed suicide 6 months after hospital discharge were identified as censored data. A total of 43 patients in the non-dialysis group and 5 patients in the dialysis group died during the follow-up period (Fig. 1). The 1-year and 3-year survival rates was significantly decreased in dialysis patients compared to non-dialysis patients (59.3 ± 14.3% vs. 96.8 ± 0.7% and 29.7 ± 16.5% vs. 90.1 ± 1.7%, respectively p < 0.001, log rank).f p y p g Late aortic events including sudden death (n = 3) and reoperation at a different site of the aorta (n = 1) were identified in the dialysis group. Results ’ On the other hand, fatal aortic rupture (n = 9), sudden death (n = 12) and https://doi.org/10.1038/s41598-022-09448-7 Scientific Reports \| (2022) 12:5372 \| www.nature.com/scientificreports/ Variables Total (n = 882) Overall cohort PSM cohort Non-dialysis (n = 866) Dialysis (n = 16) P Value Non-dialysis (n = 16) Dialysis (n = 16) P Value DeBakey type I (%) 727 (82.4) 715 (82.6) 12 (75.0) 0.503 13 (81.3) 12 (75.0) 1.000 Demographic data Age (year) 53.0 ± 13.2 53.1 ± 13.2 47.1 ± 11.2 0.069 54.2 ± 10.5 47.1 ± 11.2 0.073 Male (%) 646 (73.2) 638 (73.7) 8 (50.0) 0.045 8 (50.0) 8 (50.0) 1.000 Obesity (BMI > 30 kg/m2) (%) 99 (14.1) 99 (14.4) 0 (0) 0.147 2 (18.2) 0 (0) 0.157 Medical history Hypertension (%) 639 (72.4) 623 (71.9) 16 (100) 0.009 16 (100) 16 (100) – Diabetes mellitus (%) 20 (2.3) 20 (2.3) 0 (0) 1.000 0 (0) 0 (0) – Previous cardiovascular disease (%) 28 (3.2) 28 (3.2) 0 (0) 1.000 1 (6.3) 0 (0) 1.000 Cerebrovascular disease (%) 33 (3.8) 32 (3.7) 1 (9.1) 0.346 1 (6.3) 1 (9.1) 1.000 Marfan syndrome (%) 24 (2.7) 24 (2.8) 0 (0) 1.000 2 (12.5) 0 (0) 0.484 Previous cardiac surgery (%) PCI (%) 9 (1.0) 9 (1.0) 0 (0) 1.000 1 (6.3) 0 (0) 1.000 TEVAR (%) 18 (2.1) 18 (2.1) 0 (0) 1.000 2 (12.5) 0 (0) 0.499 CABG (%) 1 (0.1) 1 (0.1) 0 (0) 1.000 0 (0) 0 (0) – AVR (%) 14 (1.6) 14 (1.6) 0 (0) 1.000 1 (6.3) 0 (0) 1.000 Limb ischemia (%) 107 (12.1) 107 (12.4) 0 (0) 0.242 3 (18.8) 0 (0) 0.226 Mesenteric ischemia (%) 34 (3.9) 34 (3.9) 0 (0) 1.000 1 (6.3) 0 (0) 1.000 Cerebral ischemia (%) 78 (8.8) 77 (8.9) 1 (6.3) 1.000 4 (25.0) 1 (6.3) 0.333 Coronary ischemia (%) 46 (5.2) 46 (5.3) 0 (0) 1.000 3 (18.8) 0 (0) 0.226 Location of the entry tear Ascending aorta (%) 556 (63.0) 549 (63.4) 7 (43.8) 0.107 8 (50.0) 7 (43.8) 0.723 Aortic arch (%) 117 (13.3) 113 (13.0) 4 (25.0) 0.252 3 (18.8) 4 (25.0) 1.000 Descending aorta or unknown (%) 209 (23.7) 204 (23.6) 5 (31.3) 0.552 4 (25.0) 5 (31.3) 1.000 Hypotension (%) 26 (2.9) 24 (2.8) 2 (12.5) 0.078 3 (18.8) 2 (12.5) 1.000 Pericardial tamponade (%) 151 (17.1) 147 (17.0) 4 (25.0) 0.498 0 (0) 4 (25.0) 0.101 Preoperative laboratory data WBC (109/L) 11.0 (8.3, 14.1) 11.1 (8.4, 14.1) 7.7 (6.3, 9.7) 0.014 14.0 ± 6.3 8.6 ± 3.0 0.008 Haemoglobin (g/L) 123.5 ± 29.2 124.1 ± 29.0 88.3 ± 14.7 < 0.001 111.6 ± 26.3 88.3 ± 14.7 0.012 PLT (109/L) 144.0 (108.0, 184.0) 144.0 (108.0, 184.0) 141.5 (123.8, 177.3) 0.899 113.1 ± 56.3 149.6 ± 43.0 0.067 Fibrinogen (g/L) 2.5 ± 1.4 2.5 ± 1.4 2.9 ± 1.1 0.140 2.2 ± 0.9 2.9 ± 1.1 0.077 Triglyceride (mmol/L) 1.0 (0.7, 1.5) 1.0 (0.7, 1.5) 1.4 (0.6, 1.6) 0.639 0.8 (0.5, 1.3) 1.4 (0.6, 1.6) 0.417 CRP (mg/dl) 19.2 (4.6, 75.7) 19.2 (4.6, 76.4) 28.2 (4.9, 54.7) 0.902 29.9 (7.4, 71.0) 28.2 (4.9, 54.7) 0.461 D-dimer (ng/mL) 4.7 (2.3, 9.4) 4.6 (2.3, 9.4) 5.9 (4.5, 10.4) 0.076 7.1 (2.8, 22.8) 5.9 (4.5, 10.4) 0.661 Albumin (g/L) 37.3 (33.8, 40.1) 37.3 (33.8, 40.1) 34.1 (31.0, 38.0) 0.134 34.6 ± 5.3 34.8 ± 4.7 0.934 TnT (ng/ml) 0.02 (0.01, 0.14) 0.02 (0.01, 0.14) 0.06 (0.03, 0.12) 0.046 0.08 (0.04, 0.21) 0.06 (0.03, 0.12) 0.568 ALT (U/L) 25.6 (15.7, 46.9) 25.6 (15.8, 47.0) 15.2 (9.9, 53.5) 0.185 49.3 (18.4, 194.5) 15.2 (9.9, 53.5) 0.062 Bun (mmol/L) 7.2 (5.6, 9.5) 7.1 (5.5, 9.4) 18.3 (13.5, 28.4) < 0.001 10.7 ± 2.8 20.8 ± 9.5 < 0.001 sCr (mg/dl) 112.7 ± 129.3 99.2 ± 70.3 841.8 ± 345.1 < 0.001 160.6 (115.5, 203.1) 786.9 (585.8, 988.9) < 0.001 eGFR (ml/min) 85.6 ± 43.0 87.8 ± 41.6 9.4 ± 5.6 < 0.001 80.3 ± 20.5 9.4 ± 5.6 < 0.001 Total bilirubin (mg/dl) 15.3 (10.8, 22.6) 15.3 (10.9, 22.6) 7.0 (4.9, 16.2) 0.002 18.5 (11.9, 29.7) 7.0 (4.9, 16.2) 0.004 INR 1.1 (1.0, 1.2) 1.1 (1.0, 1.2) 1.3 (1.1, 1.6) 0.011 1.2 (1.1, 1.3) 1.3 (1.1, 1.6) 0.415 APTT (s) 28.9 (26.2, 35.2) 28.9 (26.2, 35.1) 29.3 (25.4, 42.3) 0.568 30.1 (27.0, 38.6) 29.3 (25.4, 42.3) 0.968 Table 1. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Table 2. Characteristics of the renal disease. Values for categorial variables are given as count (percentage); values for continuous variables are given as mean ± standard deviation. Total (n = 16) Primary cause of end-stage renal disease Hypertension 10 (62.5%) Chronic glomerulonephritis 5 (31.3%) Unknown 1 (6.3%) Type of dialysis Hemodialysis 14 (87.5%) Peritoneal 2 (12.5%) Type of blood access Upper limb hemodialysis shunt 13 (81.3%) Superficialization of the brachial artery 1 (6.3%) Duration of dialysis (years) 4.5 ± 3.5 Table 2. Characteristics of the renal disease. Values for categorial variables are given as count (percentage); values for continuous variables are given as mean ± standard deviation. Table 2. Characteristics of the renal disease. Values for categorial variables are given as count (percentage) values for continuous variables are given as mean ± standard deviation. Table 2. Characteristics of the renal disease. Values for categorial variables are given as count (percentage); values for continuous variables are given as mean ± standard deviation. Table 3. Comparison of operative variables. Values for categorial variables are given as count (percentage); values for continuous variables are given as median (interquartile range) or mean ± standard deviation. MVR mitral valve replacement, MVP mitral valvuloplasty, TVP tricuspid valvuloplasty, CABG coronary artery bypass graft, CPB cardiopulmonary bypass, DHCA deep hypothermic circulatory arrest, PSM propensity score matching. www.nature.com/scientificreports/ Variables Total (n = 882) Overall cohort PSM Cohort Non-dialysis (n = 866) Dialysis (n = 16) P Value Non-dialysis (n = 16) Dialysis (n = 16) P Value Intro-operative variables CABG (%) 51 (5.8) 50 (5.8) 1 (6.3) 1.000 2 (12.5) 1 (6.3) 1.000 CPB time (min) 232.3 ± 67.9 232.3 ± 67.6 235.8 ± 85.1 0.984 272.3 ± 95.3 235.8 ± 85.1 0.291 Aortic cross- clamp time (min) 154.0 (124.0, 194.0) 154.0 (124.0, 194.0) 138.0 (106.8, 194.0) 0.374 171.5 ± 60.6 156.0 ± 63.9 0.366 DHCA time (min) 29.5 ± 12.5 29.5 ± 12.5 28.2 ± 11.9 0.710 24.3 ± 12.2 28.2 ± 11.9 0.335 Cannulation Axillary artery (%) 171 (19.4) 166 (19.2) 5 (31.3) 0.213 6 (37.5) 5 (31.3) 0.710 Femoral artery (%) 230 (26.1) 225 (26.0) 5 (31.3) 0.578 5 (31.3) 5 (31.3) 1.000 Axillary + femoral artery (%) 442 (50.1) 436 (50.3) 6 (37.5) 0.309 5 (31.3) 6 (37.5) 1.000 Root procedure Bentall (%) 202 (22.9) 201 (23.2) 1 (6.3) 0.139 6 (37.5) 1 (6.3) 0.083 Root reconstruc- tion (%) 641 (72.7) 626 (72.3) 15 (93.8) 0.085 10 (62.5) 15 (93.8) 0.083 Valve sparing root replacement (%) 35 (4.0) 35 (4.0) 0 (0) 1.000 3 (18.8) 0 (0) 0.248 Distal surgical technique Hemi-arch replacement (%) 179 (20.3) 175 (20.2) 4 (25.0) 0.546 7 (43.8) 4 (25.0) 0.264 Total arch + frozen elephant trunk (%) 422 (47.8) 415 (47.9) 7 (43.8) 0.741 10 (62.5) 7 (43.8) 0.288 Arch fenestrated stent graft (%) 268 (30.4) 263 (30.4) 5 (31.3) 1.000 10 (62.5) 5 (31.3) 0.288 Table 3. Comparison of operative variables. Values for categorial variables are given as count (percentage); values for continuous variables are given as median (interquartile range) or mean ± standard deviation. MVR mitral valve replacement, MVP mitral valvuloplasty, TVP tricuspid valvuloplasty, CABG coronary artery bypass graft, CPB cardiopulmonary bypass, DHCA deep hypothermic circulatory arrest, PSM propensity score matching. Results ’ Comparison of preoperative variables. Values for categorial variables are given as count (percentage); values for continuous variables are given as median (interquartile range) or mean ± standard deviation. BMI Table 1. Comparison of preoperative variables. Values for categorial variables are given as count (percentage); values for continuous variables are given as median (interquartile range) or mean ± standard deviation. BMI body mass index, WBC white blood cell, Bun blood urea nitrogen, sCr serum creatinine, PLT platelet, ALB albumin, CRP c-reactive protein, eGFR estimated glomerular filtration rate, INR international normalized ratio, PSM propensity score matching. reoperation at a different site of the aorta (n = 21) were identified in the non-dialysis group. As shown in the Fig. 2, the late aortic event free survival was significantly decreased in dialysis patients compared to non-dialysis patients at both 1 and 3 years after the operation (72.5 ± 14.1% vs. 97.7 ± 0.6% and 60.4 ± 14.1% vs. 90.6 ± 1.8%, respectively p < 0.001). Scientific Reports \| (2022) 12:5372 \| https://doi.org/10.1038/s41598-022-09448-7 Table 3. Comparison of operative variables. Values for categorial variables are given as count (percentage); values for continuous variables are given as median (interquartile range) or mean±standard deviation MV Discussion h d In this study, our data indicated that the preoperative parameters, including hemodynamics and organ malperfu- sion conditions, were similar between patients without or without dialysis. Furthermore, no significant differ- ence of postoperative parameters as well as other short-term prognosis measurements was identified in dialysis patients after propensity score matching. However, the long-term mortality and incidence of late aortic events was significantly increased in dialysis patients compared to non-dialysis patients. gi y y p p y p Our study indicated that only 1.8% (16/882) of all ATAAD patients were receiving dialysis treatment due to ESRD, which was similar to the 1–3% prevalence identified in other previous studies7,10,11. However, the treatment https://doi.org/10.1038/s41598-022-09448-7 Scientific Reports \| (2022) 12:5372 \| www.nature.com/scientificreports/ Table 4. Comparison of postoperative variables. Values for categorial variables are given as count (percentage); values for continuous variables are given as median (interquartile range) or mean ± standard deviation. ICU intensive care unit, PSM propensity score matching. Discussion h d Variables Total (n = 882) Overall cohort PSM Cohort Non-dialysis (n = 866) Dialysis (n = 16) P Value Non-dialysis (n = 16) Dialysis (n = 16) P Value Postoperative complications (%) Re-exploration for bleeding (%) 33 (3.7) 33 (3.8) 0 (0) 1.000 4 (25.0) 0 (0) 0.101 Dialysis (%) 148 (16.8) 132 (15.2) 16 (100.0) < 0.001 8 (50.0) 16 (100.0) 0.002 Stroke (%) 69 (7.8) 68 (7.9) 1 (6.3) 1.000 0 (0) 1 (6.3) 1.000 Paraplegia (%) 29 (3.3) 28 (3.2) 1 (6.3) 0.417 0 (0) 1 (6.3) 1.000 Re-intubation (%) 37 (4.2) 34 (3.9) 3 (18.8) 0.026 2 (12.5) 3 (18.8) 1.000 Tracheostomy (%) 36 (4.1) 35 (4.0) 1 (6.3) 0.490 1 (6.3) 1 (6.3) 1.000 Deep sternal wound infection (%) 13 (1.5) 12 (1.4) 1 (6.3) 0.213 1 (6.3) 1 (6.3) 1.000 Sepsis (%) 8 (0.9) 8 (0.9) 0 (0) 1.000 1 (6.3) 0 (0) 1.000 Intracranial hemorrhage (%) 6 (0.7) 5 (0.6) 1 (6.3) 0.104 1 (6.3) 1 (6.3) 1.000 Gastrointestinal bleeding (%) 4 (0.5) 4 (0.5) 0 (0) 1.000 1 (6.3) 0 (0) 1.000 Drainage volume 24 h after surgery (ml) 520.0 (300.0, 869.5) 510.0 (300.0, 864.5) 680.0 (602.5, 1042.5) 0.033 520.0 (345.0, 835.0) 680.0 (602.5, 1042.5) 0.051 Ventilation time (hour) 17.0 (11.0, 43.0) 17.0 (11.0, 43.0) 33.0 (14.6, 60.6) 0.046 61.5 (16.8, 146.8) 33.0 (14.6, 60.6) 0.269 ICU Stay time (day) 4.0 (3.0, 7.0) 4.0 (3.0, 7.0) 6.5 (4.3, 9.0) 0.083 8.0 (6.0, 12.0) 6.5 (4.3, 9.0) 0.196 Hospital stay time (day) 20.9 ± 12.1 21.0 ± 12.2 18.4 ± 11.5 0.279 27.5 (10.8, 35.8) 17.5 (9.3, 21.3) 0.086 30-Day mortality (%) 101 (11.5) 99 (11.4) 2 (12.5) 0.704 2 (12.5) 2 (12.5) 1.000 Table 4. Comparison of postoperative variables. Values for categorial variables are given as count (percentage); values for continuous variables are given as median (interquartile range) or mean ± standard deviation. ICU intensive care unit, PSM propensity score matching. Table 4. Comparison of postoperative variables. Values for categorial variables are given as count (percentage); values for continuous variables are given as median (interquartile range) or mean ± standard deviation. ICU intensive care unit, PSM propensity score matching. Figure 1. Kaplan–Meier curves for overall cumulative survival of dialysis and non-dialysis patients suffering from acute type A aortic dissection. Figure 1. Kaplan–Meier curves for overall cumulative survival of dialysis and non-dialysis patients suffering from acute type A aortic dissection. Table 4. Comparison of postoperative variables. Values for categorial variables are given as count ( t ) l f ti i bl i di (i t til ) ± t d Discussion h d for this subgroup of patients is difficult and often associates with higher morbidity, such as cerebrovascular diseases, hypertension, and diabetes12. A multi-center registry study conducted in Germany reported that the incidence of primary entry tear in aortic arch was 14.5%13, which was similar to the 13% (113/866) we identified in our study among non-dialysis patients. In contrast, 25% (4/16) of the dialysis patients developed a primary entry tear in aortic arch. This dif- ference might due to the increased calcification in the aortic arch area during dialysis treatment7, which might explain why intimal tears are more likely to occur in the atherosclerotic aortic arch as well.l Conflicting studies had been published about the safety of surgical repair in dialysis patients. Some previous studies identified ESRD as a major risk factor for postoperative morbidity and mortality. Liu and colleagues14 reported that the adjusted mortality rate in dialysis-dependent patients was 3-times higher compared to those with normal renal function. In addition, Okada et al. identified that the severe renal dysfunction was an inde- pendent risk factor for in-hospital death in non-dialysis patients15. On the contrary, another retrospective study which included 960 patients suggested that although the in-hospital mortality rate was increased in dialysis patients (16% vs. 6%), no statistically difference was achieved7. Similarly, no significant difference of 30-Day mortality rate was identified in our cohort between dialysis patients and non-dialysis patients. Furthermore, our results also seemed contradicting to some previous studies which suggested that the ESRD was associated Scientific Reports \| (2022) 12:5372 \| https://doi.org/10.1038/s41598-022-09448-7 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 2. Kaplan–Meier curves for freedom from late aortic events of dialysis and non-dialysis patients suffering from acute type A aortic dissection. Figure 2. Kaplan–Meier curves for freedom from late aortic events of dialysis and non-dialysis patients suffering from acute type A aortic dissection. with increasing postoperative complications16,17. We hypothesized the relatively improved short-term prognosis we observed in this study was due to the improvement of surgical as well as critical care techniques and more careful matching of baseline characteristics. A dilated aorta is believed to be associated with worse long-term prognosis of ATAAD. However, our clini- cal experience suggests that extended aortic replacement can be especially dangerous for dialysis patients due to the increased operative invasiveness and prolonged operation time. Discussion h d Therefore, we suggest that the extended aortic replacement in dialysis patients should be avoided unless the clear identification of expanded lesions on imaging results.i g g It has been well known in the field that the control of hypertension is critical to manage patients with residual aneurysms18. All dialysis patients in our study had hypertension, compared to the 70% identified among non- dialysis patients. Previous studies have shown the efficacy of beta-blocking agents on prevention of aortic dis- section and dilatation in Marfan syndrome patients19. Similarly, our previous study also suggested that regular beta-blockers treatment after discharge was associated with decreased long-term mortality in ATAAD patients who received aortic dissection repair surgery20. Considering the fact that the renin-angiotensin system was more activated in ESRD due to the hemodynamic changes, beta-blockers seemed to be more beneficial in such group of patients. For dialysis patients with hypertension, strong consideration should be given to the prescription of beta-blockers after aortic dissection repair surgery.i t p g y In addition, one of our previous studies showed that the concomitant hypertension identified upon hospital administration was an independent risk factor for long-term mortality in ATAAD patients20. These observa- tions indicated that strict medication adherence as well as blood pressure control after discharge is critical in the management of patients who received aortic dissection surgery repair, especially for dialysis patients. g p g y p p y y p In summary, these results indicated that the ATAAD repair surgery was relatively safe in dialysis patients but closer follow-up should be planned. Limitationsh This study had some limitations. Firstly, this was a retrospective study conducted in a single center with limited dialysis patients. A multi-center study with a larger cohort is needed to validate our findings in future. Secondly, our surgical technique had evolved over the study period which might influence the results. Finally, the result of this study should be interpreted with caution due to limited follow-up period and incomplete demographic data from some patients. Conclusion Our study indicated that the short-term outcomes for dialysis patients who received conventional ATAAD repair surgery were acceptable. However, these patients were associated with a worse long-term prognosis. These results reemphasized the need for close follow-up examination and precautions should be made for late aortic events in dialysis patients who received ATAAD repair surgery. Further prospective multicenter studies aim to identify approaches to reduce late complications are required. Received: 24 September 2021; Accepted: 23 March 2022 References 1. Rahmanian, P. B., Adams, D. H., Castillo, J. G., Vassalotti, J. & Filsoufi, F. Early and late outcome of cardiac surgery in dialysis- dependent patients: Single-center experience with 245 consecutive patients. J. Thorac. Cardiovasc. Surg. 135, 915–922 (2008). Acknowledgementsh g There are no acknowledgements to declare. g There are no acknowledgements to declare. Author contributions D.W. and Z.W. conceived and designed the experiments; P.G., L.L., M.G., and C.C. collected the samples; Z.W. and L.Z. analyzed the data; Z.W., P.G., and L.L. wrote the paper. All authors reviewed the manuscript before submission. www.nature.com/scientificreports/ www.nature.com/scientificreports/ 2. Dewey, T. M. et al. Does coronary artery bypass graft surgery improve survival among patients with end-stage renal disease?. Ann. Thorac. Surg. 81, 591–598 (2006) (discussion 598).t h g 3. Liang, N. L. et al. High mortality rates after both open surgical and endovascular thoracic aortic interventions in patients with end-stage renal disease. J. Vasc. Surg. 66, 991–996 (2017). t end-stage renal disease. J. Vasc. Surg. 66, 991–996 (2017). g J g ( ) 4. Zuo, L., Wang, M., Control, F. B. B. P. Q., Improvement, C. Current status of maintenance hemodialysis in Beijing China (2011). Kidney Int. Suppl. 3, 167–169 (2013). y pp 5. Nienaber, C. A. & Eagle, K. A. Aortic dissection: New frontiers in diagnosis and management: Part I: From etiology to diagnostic strategies. Circulation 108, 628–635 (2003).h g 6. Chemtob, R. A. et al. Stroke in acute type A aortic dissection: The nordic consortium for acute type A aortic dissection (NOR- CAAD). Eur. J. Cardiothorac. Surg. 58, 1027–1034 (2020).h g 7. Akiyoshi, K. et al. Surgical outcomes of acute type A aortic dissection in dialysis patients. Gen. Thorac. Cardiovasc. Surg. 67, 501–509 (2019).h 8. Morishita, K. et al. Midterm results of surgical treatment of thoracic aortic disease in dialysis patients. Ann. Thorac. Surg. 80 96–100 (2005). 9. Wang, Z. et al. Acute kidney injury in patients operated on for type A acute aortic dissection: Incidence, risk factors and short-term outcomes. Interact. Cardiovasc. Thorac. Surg. 31, 697–703 (2020).hth outcomes. Interact. Cardiovasc. Thorac. Surg. 31, 697–703 (2020 h g 0. Lawton, J. S. et al. The impact of surgical strategy on survival after repair of type A aortic dissection. J. Thorac. Cardiovasc. Surg 150, 294-301 e291 (2015).th 1. Etz, C. D. et al. Impact of perfusion strategy on outcome after repair for acute type A aortic dissection. Ann. Thorac. Surg. 97, 78–85 (2014). 12. Wang, X. et al. Predictors and in-hospital outcomes of preoperative acute kidney injury in patients with type A acute aortic dis- section. J. Geriatr. Cardiol. 13, 679–684 (2016). 13. Conzelmann, L. O. et al. Analysis of risk factors for neurological dysfunction in patients with acute aortic dissection type A: from the German registr for acute aortic dissection t pe A (GERAADA) Eur J Cardiothorac Surg 42 557 565 (2012) 13. Conzelmann, L. O. et al. Competing interests h p g The authors declare no competing interests. www.nature.com/scientificreports/ Analysis of risk factors for neurological dysfunction in patients with acute aortic dissection type A: Data from the German registry for acute aortic dissection type A (GERAADA). Eur. J. Cardiothorac. Surg. 42, 557–565 (2012). g y yp g 4. Liu, J. Y. et al. Risks of morbidity and mortality in dialysis patients undergoing coronary artery bypass surgery. Northern new england cardiovascular disease study group. Circulation 102, 2973–2977 (2000).fi 15. Okada, K. et al. Outcome of elective total aortic arch replacement in patients with non-dialysis-dependent renal insufficiency stratified by estimated glomerular filtration rate. J. Thorac. Cardiovasc. Surg. 147, 966-972 e962 (2014). iih 16. Horst, M., Mehlhorn, U., Hoerstrup, S. P., Suedkamp, M. & de Vivie, E. R. Cardiac surgery in patients with end-stage renal disease: 10-year experience. Ann. Thorac. Surg. 69, 96–101 (2000). y ph g 7. Charytan, D. M. & Kuntz, R. E. Risks of coronary artery bypass surgery in dialysis-dependent patients–analysis of the 2001 nationa inpatient sample. Nephrol. Dial. Transplant. 22, 1665–1671 (2007).hl 18. Okamoto, R. J., Xu, H., Kouchoukos, N. T., Moon, M. R. & Sundt, T. M. 3rd. The influence of mechanical properties on wall stress and distensibility of the dilated ascending aorta. J. Thorac. Cardiovasc. Surg. 126, 842–850 (2003).f h 19. Salim, M. A., Alpert, B. S., Ward, J. C. & Pyeritz, R. E. Effect of beta-adrenergic blockade on aortic root rate of dilation in the Marfan syndrome. Am. J. Cardiol. 74, 629–633 (1994). y 0. Wang, Z. et al. Impact of hypertension on short- and long-term survival of patients who underwent emergency surgery for type A acute aortic dissection. J. Thorac. Dis. 12, 6618–6628 (2020). References 1. Rahmanian, P. B., Adams, D. H., Castillo, J. G., Vassalotti, J. & Filsoufi, F. Early and late outcome of cardiac surgery in dialysis- dependent patients: Single-center experience with 245 consecutive patients. J. Thorac. Cardiovasc. Surg. 135, 915–922 (2008). https://doi.org/10.1038/s41598-022-09448-7 Scientific Reports \| (2022) 12:5372 \| Additional information Correspondence and requests for materials should be addressed to D.W. Correspondence and requests for materials should be addressed to D.W. 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. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2022 https://doi.org/10.1038/s41598-022-09448-7 Scientific Reports \| (2022) 12:5372 \|
https://openalex.org/W3008927905	https://europepmc.org/articles/pmc7028738?pdf=render	English	null	Publisher Correction: Data-driven acceleration of photonic simulations	Scientific reports	2,020	cc-by	1,252	www.nature.com/scientificreports www.nature.com/scientificreports Publisher Correction: Data- driven acceleration of photonic simulations Rahul Trivedi, Logan Su, Jesse Lu, Martin F. Schubert & Jelena Vuckovic Correction to: Scientific Reports https://doi.org/10.1038/s41598-019-56212-5, published online 23 December 2019 This Article contains an error in the order of the Figures. Figure 1 was incorrectly published as Figure 3. Figure 2 was incorrectly published as Figure 1. Figure 3 was incorrectly published as Figure 2. The correct Figures are reproduced below with their corresponding legends. Figure 1. (a) Schematic of the grating splitter device that comprises the dataset. All the gratings in the dataset are 3 μm long and are designed for a 220 nm silicon-on-insulator (SOI) platform with oxide cladding. We use a uniform spatial discretization of 20 nm while representing Eq. 1 as a system of linear equations. The resulting system of linear equations has 229 × 90 = 20,610 unknown complex numbers. (b) Visualizing samples from the dataset — shown are permittivity distribution, simulated electric fields and effective index fields for 4 randomly chosen samples. All fields are shown at a wavelength of 1.4 μm. Publisher Correction: Data- driven acceleration of photonic simulations OPEN (b) Performance of data-driven GMRES on the evaluation dataset when supplied with different number of principal components (~200 samples from the training set were used for computing the principal components) — the dotted line shows the mean residual, and the solid colored background indicates the region within one standard deviation around the mean residual. (c) Histogram of the residual after 100 data-driven GMRES iterations for different h Figure 2. (a) First five principal components of the electric fields in the grating splitter dataset. (b) Performance of data-driven GMRES on the evaluation dataset when supplied with different number of principal components (~200 samples from the training set were used for computing the principal components) — the dotted line shows the mean residual, and the solid colored background indicates the region within one standard deviation around the mean residual. (c) Histogram of the residual after 100 data-driven GMRES iterations for different N computed over 100 randomly chosen samples from the evaluation dataset. The black vertical dashed line indicates the mean residual after 100 iterations of GMRES over the evaluation dataset. Figure 3. (a) Schematic of the CNN based data-driven GMRES — a convolutional neural network takes as input the permittivity and effective index field and produces as an output the vectors v1, v2 … vN. These vectors are then supplied to the data-driven GMRES algorithm, which produces the full simulated field. (b) Histogram of the residual after 1 and 100 data-driven GMRES iterations evaluated over the evaluation dataset. We consider neural networks trained with both the projection loss function lproj and residual loss function lres. The vertical dashed lines indicate the mean residual after 1 and 100 iterations of GMRES over the evaluation dataset. (c) Performance of the data-driven GMRES on the evaluation dataset when supplied with the vectors at the output of the convolutional neural networks trained with the projection loss function lproj and the residual loss function lres. The dotted line shows the mean residual, and the solid colored background indicates the region within ± standard deviation around the mean residual. igure 3. (a) Schematic of the CNN based data-driven GMRES — a convolutional neural network takes as h i i i d ff i i d fi ld d d h 1 2 N Th Figure 3. Publisher Correction: Data- driven acceleration of photonic simulations OPEN Published: xx xx xxxx Figure 1 was incorrectly published as Figure 3. g y p g Figure 2 was incorrectly published as Figure 1. Figure 1. (a) Schematic of the grating splitter device that comprises the dataset. All the gratings in the dataset are 3 μm long and are designed for a 220 nm silicon-on-insulator (SOI) platform with oxide cladding. We use a uniform spatial discretization of 20 nm while representing Eq. 1 as a system of linear equations. The resulting system of linear equations has 229 × 90 = 20,610 unknown complex numbers. (b) Visualizing samples from the dataset — shown are permittivity distribution, simulated electric fields and effective index fields for 4 randomly chosen samples. All fields are shown at a wavelength of 1.4 μm. Figure 1. (a) Schematic of the grating splitter device that comprises the dataset. All the gratings in the dataset are 3 μm long and are designed for a 220 nm silicon-on-insulator (SOI) platform with oxide cladding. We use a uniform spatial discretization of 20 nm while representing Eq. 1 as a system of linear equations. The resulting system of linear equations has 229 × 90 = 20,610 unknown complex numbers. (b) Visualizing samples from the dataset — shown are permittivity distribution, simulated electric fields and effective index fields for 4 randomly chosen samples. All fields are shown at a wavelength of 1.4 μm. Scientific Reports \| (2020) 10:3330 \| https://doi.org/10.1038/s41598-020-59308-5 www.nature.com/scientificreports/ Figure 2. (a) First five principal components of the electric fields in the grating splitter dataset. (b) Performance of data-driven GMRES on the evaluation dataset when supplied with different number of principal components (~200 samples from the training set were used for computing the principal components) — the dotted line shows the mean residual, and the solid colored background indicates the region within one standard deviation around the mean residual. (c) Histogram of the residual after 100 data-driven GMRES iterations for different N computed over 100 randomly chosen samples from the evaluation dataset. The black vertical dashed line indicates the mean residual after 100 iterations of GMRES over the evaluation dataset. Figure 2. (a) First five principal components of the electric fields in the grating splitter dataset. Publisher Correction: Data- driven acceleration of photonic simulations OPEN (a) Schematic of the CNN based data-driven GMRES — a convolutional neural network takes as input the permittivity and effective index field and produces as an output the vectors v1, v2 … vN. These vectors are then supplied to the data-driven GMRES algorithm, which produces the full simulated field. (b) Histogram of the residual after 1 and 100 data-driven GMRES iterations evaluated over the evaluation dataset. We consider neural networks trained with both the projection loss function lproj and residual loss function lres. The vertical dashed lines indicate the mean residual after 1 and 100 iterations of GMRES over the evaluation dataset. (c) Performance of the data-driven GMRES on the evaluation dataset when supplied with the vectors at the output of the convolutional neural networks trained with the projection loss function lproj and the residual loss function lres. The dotted line shows the mean residual, and the solid colored background indicates the region within ± standard deviation around the mean residual. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not 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) 2020 Scientific Reports \| (2020) 10:3330 \| https://doi.org/10.1038/s41598-020-59308-5
https://openalex.org/W1581794476	https://europepmc.org/articles/pmc4503692?pdf=render	English	null	Lifestyle-Related Factors Contributing to Decline in Knee Extension Strength among Elderly Women: A Cross-Sectional and Longitudinal Cohort Study	PloS one	2,015	cc-by	7,840	RESEARCH ARTICLE Narumi Kojima1, Miji Kim1, Kyoko Saito2, Hideyo Yoshida1, Yuko Yoshida1, Hirohiko Hirano1, Shuichi Obuchi1, Hiroyuki Shimada3, Takao Suzuki3, Hunkyung Kim1 1 Tokyo Metropolitan Institute of Gerontology, Itabashi-ku, Tokyo, Japan, 2 Yokohama City University Graduate School of Medicine, Kanazawa-ku, Yokohama, Japan, 3 Research Institute, National Center for Geriatrics and Gerontology, 35 Gengo, Morioka-machi, Obu-shi, Aichi, Japan nkojima@tmig.or.jp Data Availability Statement: All relevant data are within the Supporting Information files. Data Availability Statement: All relevant data are within the Supporting Information files. Data Availability Statement: All relevant data are within the Supporting Information files. Funding: The authors have no support or funding to report. Abstract This cross-sectional and 4-year longitudinal cohort study aimed to clarify how various life- style-related variables affect knee extension strength in elderly Japanese women. The par- ticipants were community-dwelling women (n = 575) living in the Itabashi Ward of Tokyo, Japan aged 75–85 years at baseline (in 2008) who returned for a follow-up examination 4 years later (in 2012). Maximum isometric knee extension strength in the dominant leg was measured during comprehensive medical check-ups at baseline and follow-up. Interviews with participants included questions on their history of 11 diseases and lifestyle-related fac- tors such as physical activity as well as dietary, smoking, and drinking habits. Cross-sec- tional and longitudinal analyses yielded inconsistent results regarding the associations between lifestyle-related factors and knee extension strength. While going out more fre- quently and regular physical exercise positively affected baseline knee extension strength, they did not affect knee extension strength in the longitudinal analysis. The longitudinal analysis revealed that more frequent intake of soy products or green and yellow vegetables at baseline decreased age-related knee extension strength decline. The inconsistent results from the cross-sectional and longitudinal analyses indicate that conducting both types of analyses is crucial for researching this type of subject. The present study demonstrates that the age-related decline in muscle strength is lower in those who frequently eat soy products or green and yellow vegetables. Thus, recommending higher intake of soy products, and green and yellow vegetables for the elderly might help maintain their muscle health. OPEN ACCESS Citation: Kojima N, Kim M, Saito K, Yoshida H, Yoshida Y, Hirano H, et al. (2015) Lifestyle-Related Factors Contributing to Decline in Knee Extension Strength among Elderly Women: A Cross-Sectional and Longitudinal Cohort Study. PLoS ONE 10(7): e0132523. doi:10.1371/journal.pone.0132523 Editor: Andrea Macaluso, University of Rome Foro Italico, ITALY Received: January 8, 2015 Accepted: June 15, 2015 Published: July 15, 2015 Copyright: © 2015 Kojima 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. Lifestyle-Related Factors Contributing to Decline in Knee Extension Strength among Elderly Women: A Cross-Sectional and Longitudinal Cohort Study Narumi Kojima1*, Miji Kim1, Kyoko Saito2, Hideyo Yoshida1, Yuko Yoshida1, Hirohiko Hirano1, Shuichi Obuchi1, Hiroyuki Shimada3, Takao Suzuki3, Hunkyung Kim1 Introduction As the average life expectancy in Japan is increasing, finding ways to maintain basic levels of activities of daily living (ADL) and instrumental ADL (IADL) among the elderly is important Competing Interests: The authors have declared that no competing interests exist. PLOS ONE \| DOI:10.1371/journal.pone.0132523 July 15, 2015 1 / 13 Factors Associated with Knee Extension Strength for them to lead independent lives. Muscle strength is well known to directly affect ADL and IADL [1–3]. The prospective study of Rantanen et al. shows that the strength of multiple mus- cle groups predicts ADL dependence among persons aged 75 years; at the 5-year follow-up, those who were in the lowest tertile of muscle strength had a 2- to 3-fold greater risk of becom- ing ADL-dependent than those in the highest tertile [2]. Furthermore, Kojima et al. report that older women with greater knee extension strength (KES) have a lower prevalence of IADL dis- ability [1]. Several studies use KES as an index of lower-limb function [1], [4–6]. KES declines with aging [7], particularly in the later stage. In a 10-year follow-up study of 120 participants aged 46–78 years at baseline, older participants demonstrated a greater rate of decline in the strength of knee and elbow extensors and flexors [8]. Therefore, minimizing the age-related decline in KES is important for maintaining independence. Few longitudinal cohort studies have investigated the relationships between lifestyle-related factors and changes in muscle strength. In a 27-year follow-up study of 3,741 men aged 45–68 years at baseline, age as well as a history of stroke, diabetes mellitus, arthritis, coronary heart disease, and chronic obstructive pulmonary disease were factors related to a steeper decline in grip strength [9]. However, the authors did not report any data regarding the influence of mod- ifiable lifestyle-related factors such as eating and exercise habits on muscle strength. A follow- up study of 5,214 women aged 65–91 years at baseline suggests that age, difficulty accomplish- ing functional tasks, and lower physical activity at baseline are related to a steeper decline in handgrip strength during follow-up [10]. Strenuous work, overweight, smoking, cardiovascular diseases, hypertension, diabetes mellitus, and asthma predicted a greater decline in handgrip strength after 22 years of follow-up of 963 persons aged 30–73 years at baseline in a longitudi- nal cohort study [11]. Nevertheless, the effects of modifiable lifestyle-related factors such as dietary and exercise habits on age-related changes in KES remain unclear. Ethics statement The scientific purpose of the study was clearly explained to the participants at enrolment and follow-up. Only data for participants who provided written informed consent were used in this study. This research complies with the ethical rules for human experimentation stated in the Declaration of Helsinki [17]. The study was approved by the Ethics Committee of the Tokyo Metropolitan Institute of Gerontology. No monetary reward was given to the participants, but each participant later received a letter of feedback describing her health status with easily understandable graphs and explanations. Measurement of knee extension strength Isometric KES (in N) was measured in the dominant leg using a hand-held dynamometer (μTas F-1, ANIMA, Chofu, Japan) incorporated into a custom-made frame. Each participant first sat on the horizontal surface of the custom-made frame with her lower legs dangling and knees flexed at 90 degrees; then, she practiced pressing against the tester’s hand until she understood how to execute maximum isometric contraction of the quadriceps. The sensor of the dynamometer was placed 5 cm above the top of the lateral malleolus. The participant was then instructed to exert maximal knee extension force, receiving verbal encouragement as rein- forcement. We conducted 2 trials with an interval of approximately 30 seconds. The greater value of the 2 trials was used for analysis. Participants who had been diagnosed with a serious medical problem (e.g., systolic blood pressure >180 mmHg, diastolic blood pressure >110 mmHg, or a history of heart attack or cerebral stroke within the past 6 months) were excluded from the KES test. Study Population The study population consisted of women who participated in a comprehensive health check- up conducted by the Tokyo Metropolitan Institute of Gerontology in 2008 and who returned for a follow-up in October 2012. During the initial check-up in October or November 2008, 1,288 community-dwelling women from the Itabashi Ward of Tokyo were randomly selected and recruited. Their age ranged between 74 and 85 years (mean ± standard deviation: 78.51 ± 2.69 years). Invitation letters were sent to the 1,288 participants of the 2008 check-up (the local government approved using a list of addresses for the residents), and 575 participants (44.6%) returned for follow-up in 2012. The remaining 713 (55.4%) people did not participate because of death (n = 39); refusal to participate, schedule conflict, or inability to attend (n = 606); or unknown reasons (n = 68). Introduction Factors that could negatively affect muscle strength in the long term include low levels of physical activity, poor eating habits, and smoking. To our knowledge, no epidemiological study indicates that higher consumption of meat or eggs is beneficial for muscle strength; however, because they are major sources of animal protein, which is an essential material in human mus- cles, they are likely beneficial. A survey of 2,983 people in the UK indicates that fatty fish con- sumption positively affects grip strength in both men and women aged 59–73 years [12]. Milk, a good source of quality protein, calcium, and vitamin D, is also expected to have a beneficial effect on muscle health. For example, whey, one of the main proteins in milk, reportedly increases muscle volume when taken regularly as a supplement [13]. Soy products such as tofu, miso, and natto, which are routinely consumed by Japanese people, are considered good sources of plant-based proteins. In addition, soy isoflavone and vitamin K, which are found in natto, promote bone formation in Japanese women [14–16]. Furthermore, the consumption of green and yellow vegetables and/or fruits is expected to help minimize the age-related decline in muscle strength. A cross-sectional study revealed that daily dietary intake of vitamin C and β-carotene is significantly associated with KES [6]. Alcohol consumption and smoking might also affect muscle strength; for example, a follow-up survey in Finland reports former or cur- rent smokers had a greater decline in handgrip strength over 22 years than nonsmokers [11]. Therefore, dietary intake of these food groups, alcohol intake, and smoking habit are factors that potentially affect KES. Developing a strategy to minimize muscle strength decline among the elderly should be a research priority. In order to develop measures to prevent excessive decline in muscle strength during old age, it is crucial to study how various modifiable lifestyle-related factors affect age- related changes in muscle strength. Therefore, this retrospective cohort study investigated 2 / 13 PLOS ONE \| DOI:10.1371/journal.pone.0132523 July 15, 2015 Factors Associated with Knee Extension Strength which modifiable lifestyle-related factors have beneficial or detrimental effects on the age- related decline in KES among Japanese community-dwelling women aged 75 or older. which modifiable lifestyle-related factors have beneficial or detrimental effects on the age- related decline in KES among Japanese community-dwelling women aged 75 or older. Interviews and variables During the baseline check-up, the participants were asked closed-ended questions about life- style and health status. The interview included items on going out, walking, light exercise, regu- lar exercise and sports, alcohol intake, smoking, intake frequencies of 10 food groups (i.e., seafood, meat, eggs, milk, soy products, green or yellow vegetables, seaweed, potatoes, fruits, and oils and fats), and cohabitation status as well as history of hypertension, stroke, heart dis- ease, diabetes mellitus, hyperlipidemia, osteoporosis, anemia, asthma, chronic obstructive pul- monary disease, hip osteoarthritis, and gonarthrosis. 3 / 13 PLOS ONE \| DOI:10.1371/journal.pone.0132523 July 15, 2015 Factors Associated with Knee Extension Strength To evaluate each participant’s habitual dietary patterns, the 10 food groups mentioned above were selected from the 15 original food groups, excluding the 5 food groups that include traditionally eaten staples (e.g., rice, miso soup, pickled vegetables, bread, and noodles) [18]. The questionnaire included examples of specific foods for each category to assist with appro- priate selection of the food groups by the interviewers and participants. Examples of seafood included raw fish and all kinds of fish or clam products. The meat category included meat and all meat products. Eggs included chicken or quail eggs but not fish spawn. Milk included only pure milk, excluding milk flavored with coffee or fruit. Soy products were foods made from soybeans, such as tofu and natto, which is a traditional Japanese food made of fermented soy- beans. Green and yellow vegetables were explained as vegetables that have dark colors, includ- ing carrot, spinach, pumpkin, and tomato. The seaweed category included both raw and dried products. Because it was determined that the potato category obviously consisted of white potato, sweet potato, and taro—all of which are consumed by Japanese people—a detailed explanation was not provided. Fruits included all fruits, both fresh and canned. Tomatoes were specified as vegetables, because some Japanese people consider them fruits. Oils and fats included butter, margarine, and all other kinds of oils used for cooking. While some of the questions were multiple choice (5 response options at most), the responses for all variables were dichotomized for the analysis to ensure that the numbers of positive and negative responders were as similar as possible based on the baseline data. The thresholds based on the baseline data between positive and negative responses were also applied to the follow-up data. Interviews and variables “Frequency of going out” had 4 response options (at least once/ day, once/2–3 days, once/week, and very rarely), and the participants were classified as “once/ 2–3 days or less” or “at least once/day” for analysis. Responses to the questions about “strolling or light exercise” and “frequency of participating in such activities/week” were classified as “at least 2–4 days/week” or “once/week or less” for strolling and “at least 5–6 days/week” or “2–4 days/week or less” for light exercise. Regarding the question about “regular exercise and sports,” participants were classified as “yes” or “no.” Participants had 4 response options (almost every day, once/2 days, once or twice/week, or almost never) for the questions on the intake frequency of each food group and were ultimately classified as “once or twice/week or less” or “at least once/2 days” for meat and eggs and “once/2 days or less” or “almost every day” for all other food groups. The Dietary Variety Score (DVS), which is an index of dietary variety introduced by Kumagai et al. [19], was calculated by summing the number of times each partic- ipant answered “almost every day” for the intake of each of the 10 food groups [18–20]. The total score ranges from 0 (consuming once/2 days or less for all of the food groups) to 10 (con- suming almost every day for all of the food groups). The women were then classified as “DVS 5” or “DVS 6.” For “alcohol consumption” and “smoking habit,” the participants were clas- sified as “current drinker/smoker” or “non-drinker (never or ex-drinker)/non-smoker (never or ex-smoker).” The response options for the history of the previously listed diseases were “no history” or “with history.” Participants who had a history of a disease were also asked about the current disease status; participants were ultimately classified as “currently negative” or “cur- rently positive.” Participant characteristics at baseline and follow-up The age of the 575 participants ranged between 75 and 85 years (78.07 ± 2.56) in 2008 and between 78 and 89 years (82.07 ± 2.55) in 2012 (Table 1). At baseline, the participants who did not return for the 2012 check-up were significantly older (78.86 ± 2.74 versus 78.07 ± 2.56 years, respectively) and had lower KES (192.36 ± 48.60 versus 205.95 ± 53.36 N, respectively) than those who returned. Both average the height and weight of the 575 final study participants decreased significantly over the 4 years of the study (P < 0.001, Table 1). No changes in body mass index or %body fat were observed. KES decreased significantly by approximately 10% over the course of 4 years. The data for physical activities and other lifestyle factors showed that the participants gener- ally became less active 4 years after baseline (Table 1). The proportion of women who went out at least once/day or participated in regular exercise and sports decreased significantly (P < 0.001, P = 0.007, respectively). The proportions of women who drank alcohol or smoked decreased by 4.2% and 1.2%, respectively (Table 1). Regarding intakes of the different food groups, the proportion of women who consumed some of the food groups (i.e., meats, and oils and fats) more frequently (once per 2 days for meat and eggs, almost every day for other food groups) increased significantly over the course of 4 years (Table 1). The proportion of participants who ate meat at least once/2 days increased significantly by 5.7% (from 59.3% at baseline to 65.0% at follow-up; P = 0.011). The proportion of those who ate oils and fats almost every day increased significantly by 9.4% (50.6–60.0%; P = 0.039). In contrast, the proportions of women who ate soy products, green or yellow vege- tables, seaweed, or fruits almost every day decreased significantly (all P < 0.05; Table 1). The proportions of women who had hypertension, osteoporosis, anemia, or gonarthrosis increased significantly over the 4 years (all P < 0.05; Table 1). Statistical analysis Somatometric parameters and KES are presented as population means ± standard deviations. Changes of these parametric variables over 4 years were examined using paired t-tests. The population status of each lifestyle-related variable is presented as the number of positive/nega- tive responses. The statistical significance of changes in the ratios of these non-parametric vari- ables was examined using the McNemar test. 4 / 13 PLOS ONE \| DOI:10.1371/journal.pone.0132523 July 15, 2015 Factors Associated with Knee Extension Strength A cross-sectional analysis was conducted to examine the relationships between the lifestyle- related variables and KES at baseline in the 575 women who also participated in the follow-up survey. For each lifestyle-related variable, we compared KES between those with positive and negative responses at baseline by using ANCOVA adjusted for baseline age and current status of all diseases, with each disease included as a separate variable. We also conducted a longitudinal analysis to examine the associations between baseline life- style-related variables and changes in KES over 4 years. For each variable, we compared the changes in KES between those with positive and negative responses at baseline by using ANCOVA adjusted for baseline age, KES, and the status of all diseases, with each disease included as a separate variable. All statistical analyses were conducted using PASW Statistics 18 (IBM Corp., Armonk, NY, USA). The results were regarded statistically significant when the P-value was <0.05. Longitudinal analysis of the effects of lifestyle-related factors on knee extension strength Longitudinal analysis showed that except for 3 food groups, no lifestyle-related variables at baseline were related to changes in KES over 4 years (Table 3). Those who ate seafood almost baseline were related to changes in KES over 4 years (Table 3). Those who ate seafood almost Table 1. Subjects' characteristics at baseline and follow-up at 4 years (n = 575). Factors Associated with Knee Extension Strength Cross-sectional analysis of knee extension strength at baseline Cross-sectional analysis of the baseline data showed that some factors related to physical activ- ity were significantly associated with KES at baseline (Table 2). The mean KES was higher in those who went out once/day or more or participated in regular exercise and sports than those who did not. We found no significant relationship between KES and other lifestyle-related fac- tors including the frequency of walking, frequency of food intake of the studied food groups, DVS, alcohol intake, and smoking (Table 2). 5 / 13 PLOS ONE \| DOI:10.1371/journal.pone.0132523 July 15, 2015 SD = standard deviation, COPD = chronic obstructive pulmonary disease, P-Values were outcomes of paired t-tests for continuous variables, and of McNemar tests for binary variables. Longitudinal analysis of the effects of lifestyle-related factors on knee extension strength Variables Baseline Follow-Up P-Value Mean SD Mean SD Measured values 　　 Age 78.07 ±2.56 82.07 ±2.55 Height, cm 148.42 ±5.29 147.32 ±5.50 P<0.001 Weight, kg 50.20 ±7.89 49.33 ±8.18 P<0.001 Body mass index, kg/m2 22.78 ±3.34 22.72 ±3.53 0.286 %Body fat 31.94 ±4.71 32.07 ±7.74 0.528 Knee extension strength, N 205.95 ±53.36 186.01 ±54.60 P<0.001 n (%) n (%) Physical activities 　　 Going out (at least once per day) 472 (82.1) 419 (72.9) P<0.001 Going for a stroll (at least 2–4days per week) 343 (59.7) 371 (64.5) 0.054 Light exercises (at least 5–6days per week) 287 (49.9) 314 (54.6) 0.060 Regular exercises and sports (Yes) 234 (40.7) 201 (35.0) 0.007 Foods & discretionary items 　　　　 Seafood (almost every day) 260 (45.2) 238 (41.4) 0.132 Meat (at least once per 2 days) 341 (59.3) 374 (65.0) 0.011 Egg (at least once per 2 days) 369 (64.2) 393 (68.3) 0.071 Milk (almost every day) 357 (62.1) 368 (64.0) 0.375 Soy products (almost every day) 395 (68.7) 358 (62.3) 0.006 Green and yellow vegetables (almost every day) 507 (88.3) 483 (84.0) 0.021 Seaweeds (almost every day) 314 (54.6) 252 (43.8) P<0.001 Potatoes (almost every day) 248 (43.1) 211 (36.7) 0.326 Fruits (almost every day) 500 (87.0) 487 (84.7) 0.006 Oils and fats (almost every day) 291 (50.6) 345 (60.0) 0.039 Dietary variety score (6 points) 290 (50.5) 267 (46.4) 0.073 Drinking (Current drinker) 149 (25.9) 125 (21.7) 0.006 Smoking (Smoker) 22 (3.8) 15 (2.6) 0.039 Diseases 　　　　 Hypertension (positive) 306 (53.2) 365 (63.5) P<0.001 Stroke (positive) 27 (4.7) 36 (6.3) 0.124 Heart disease (positive) 106 (18.4) 123 (21.4) 0.075 Diabetes mellitus (positive) 46 (8.0) 48 (8.3) 0.791 Hyperlipidemia (positive) 225 (39.1) 246 (42.9) 0.083 Osteoporosis (positive) 160 (27.8) 215 (37.5) P<0.001 Anemia (positive) 10 (1.7) 21 (3.7) 0.043 Asthma (positive) 18 (3.1) 23 (4.0) 0.267 COPD (positive) 1 (0.2) 3 (0.5) 0.625 Osteoarthritis of hip (positive) 11 (1.9) 18 (3.1) 0.143 Gonarthritis (positive) 126 (22.0) 156 (27.1) 0.005 SD = standard deviation, COPD = chronic obstructive pulmonary disease, P-Values were outcomes of paired t-tests for continuous variables, and of McNemar tests for binary variables. Table 1. Subjects' characteristics at baseline and follow-up at 4 years (n = 575). PLOS ONE \| DOI:10.1371/journal.pone.0132523 July 15, 2015 6 / 13 Factors Associated with Knee Extension Strength Table 2. Result of the cross-sectional analysis showing the average knee extension strength accord- ing to binarized baseline lifestyle factors. Table 2. PLOS ONE \| DOI:10.1371/journal.pone.0132523 July 15, 2015 Longitudinal analysis of the effects of lifestyle-related factors on knee extension strength Result of the cross-sectional analysis showing the average knee extension strength accord- ing to binarized baseline lifestyle factors. Mean SE P-Value Physical activities Going out Once per 2–3days or less (N = 98) 191.09 (5.24) At least once per day (N = 448) 209.62 (2.44) .001 Going for a stroll 1day per week or less (N = 214) 205.52 (3.56) At least 2–4days per week (N = 332) 206.77 (2.86) .784 Light exercises 2–4days per week or less (N = 272) 201.54 (3.15) At least 5–6days per week (N = 274) 211.02 (3.15) .035 Regular exercises and sports No (N = 322) 199.99 (2.89) Yes (N = 224) 215.26 (3.47) .001 Foods & discretionary items Seafood Once per 2 days or less (N = 299) 204.98 (3.01) Almost every day (N = 247) 207.87 (3.32) .521 Meat 1–2 times per week or less (N = 228) 203.31 (3.46) At least once per 2 days (N = 318) 208.41 (2.92) .263 Egg 1–2 times per week or less (N = 197) 200.51 (3.73) At least once per 2 days (N = 349) 209.55 (2.79) .055 Milk Once per 2 days or less (N = 204) 203.45 (3.67) Almost every day (N = 342) 207.96 (2.82) .333 Soy products Once per 2 days or less (N = 173) 201.72 (3.98) Almost every day (N = 373) 208.39 (2.69) .168 Green and yellow vegetables Once per 2 days or less (N = 66) 203.52 (6.43) Almost every day (N = 479) 206.64 (2.38) .650 Seaweeds Once per 2 days or less (N = 248) (3.31) Almost every day (N = 298) 209.31 (3.02) .139 Potatoes Once per 2 days or less (N = 311) (2.97) Almost every day (N = 235) 208.22 (3.43) .457 Fruits Once per 2 days or less (N = 71) (6.22) Almost every day (N = 475) 207.24 (2.38) .270 Oils and fats Once per 2 days or less (N = 271) (3.18) Almost every day (N = 275) 205.53 (3.15) .736 Dietary variety score DVS 5 points (N = 269) 198.11 (2.07) (Continued) No (N = 322) Yes (N = 224) Foods & discretionary items (Continued) PLOS ONE \| DOI:10.1371/journal.pone.0132523 July 15, 2015 7 / 13 Factors Associated with Knee Extension Strength Table 2. Longitudinal analysis of the effects of lifestyle-related factors on knee extension strength (Continued) Mean SE P-Value DVS 6 points (N = 276) 199.17 (2.00) .714 Drinking Non-drinker (N = 404) 204.08 (2.58) Current drinker (N = 142) 212.50 (4.37) .099 Smoking Non-smoker (N = 525) (2.26) Smoker (N = 21) 220.99 (11.41) .189 SE; Standard Error, Analyses of covariance were applied incorporating baseline age, and baseline status of all the diseases (hypertension, stroke, heart disease, diabetes mellitus, hyperlipidemia, osteoporosis, anemia, asthma, chronic obstructive pulmonary disease, hip osteoarthritis, gonarthrosis) as covariates. doi:10.1371/journal.pone.0132523.t002 Table 2. (Continued) SE; Standard Error, Analyses of covariance were applied incorporating baseline age, and baseline status of all the diseases (hypertension, stroke, heart disease, diabetes mellitus, hyperlipidemia, osteoporosis, anemia, asthma, chronic obstructive pulmonary disease, hip osteoarthritis, gonarthrosis) as covariates. every day had a significantly greater decrease (1.5 times) in KES (24.68 N) than those who ate seafood once/2 days or less (16.88 N) (P = 0.022). In contrast, the intake of soy products and green or yellow vegetables had a beneficial effect on KES. The decrease of KES in participants who ate soy products almost every day (17.87 N) was approximately 69% of that in those who ate soy products once/2 days or less (26.06 N) (P = 0.026), and the decrease of KES in partici- pants who ate green or yellow vegetables almost every day (18.82 N) was approximately 60% of that in those who ate these vegetables once/2 days or less (31.46 N; P = 0.015; Table 3). PLOS ONE \| DOI:10.1371/journal.pone.0132523 July 15, 2015 Discussion We conducted both cross-sectional and longitudinal analyses of the factors associated with KES on a cohort of elderly women in Japan. However, the associations between lifestyle-related factors and KES were inconsistent between analyses. Our cross-sectional analysis showed that women who go out or exercise regularly had a higher KES than those who did not. However, these variables were not significantly associated with changes in KES in the longitudinal analy- sis. Therefore, the significant association between physical activity-related lifestyle variables and KES at baseline might merely reflect the fact that women with strong legs were able to per- form physical activities with fewer limitations. The intake frequencies of seafood, soy products, and green or yellow vegetables, which were not associated with KES at baseline, were associated with changes in KES over 4 years. The rou- tine intake of seafood negatively affected KES, while those of soy products and green and yel- low vegetables had beneficial effects. The apparent discrepancy between the results of the 2 analyses indicates that conducting a cross-sectional analysis alone is insufficient for investigat- ing the influences of lifestyle-related factors on muscle strength. We did not expect that variables found to be associated with KES in the cross-sectional anal- ysis (i.e., going out, and regular exercise and sports) would not show associations in the longi- tudinal analysis. Several intervention studies have established that exercise is effective for maintaining or even increasing muscle strength in the elderly [21–23]. One possible reason why our data do not seem to support the effect of physical activities on muscle strength is that some participants did not maintain their baseline physical activity level. However, an addi- tional comparison between those who reported consistently going out and those who consis- tently reported not going out yielded no significant association with a change in KES (P = 0.307; data not shown). Therefore, the consistency of physical activity does not seem to be critical for maintaining muscle strength among elderly women. Although our study failed to 8 / 13 PLOS ONE \| DOI:10.1371/journal.pone.0132523 July 15, 2015 Factors Associated with Knee Extension Strength Table 3. Result of the longitudinal analysis showing the effect of baseline lifestyle factors on decline in muscle strength. PLOS ONE \| DOI:10.1371/journal.pone.0132523 July 15, 2015 Discussion Mean SE P-Value Physical activities Going out Once per 2–3days or less (N = 95) -19.87 (4.05) At least once per day (N = 439) -20.51 (1.86) .886 Going for a stroll 1day per week or less (N = 207) -22.53 (2.70) At least 2–4days per week (N = 327) -19.04 (2.15) .314 Light exercises 2–4days per week (N = 266) -19.94 (2.39) At least 5–6days per week (N = 268) -20.86 (2.39) .787 Regular exercises and sports No (N = 314) -21.61 (2.22) Yes (N = 220) -18.67 (2.66) .404 Foods & discretionary items Seafood Once per 2 days or less (N = 292) -16.88 (2.26) Almost every day (N = 242) -24.68 (2.50) .022 Meat 1–2 times per week or less (N = 223) -18.88 (2.62) At least once per 2 days (N = 311) -21.48 (2.21) .451 Egg 1–2 times per week or less (N = 192) -19.37 (2.84) At least once per 2 days (N = 342) -20.98 (2.12) .654 Milk Once per 2 days or less (N = 199) -22.31 (2.78) Almost every day (N = 335) -19.26 (2.13) .388 Soy products Once per 2 days or less (N = 165) -26.06 (3.04) Almost every day (N = 369) -17.87 (2.02) .026 Green and yellow vegetables Once per 2 days or less (N = 64) -31.46 (4.85) Almost every day (N = 469) -18.82 (1.78) .015 Seaweeds Once per 2 days or less (N = 240) (2.52) Almost every day (N = 294) -18.10 (2.27) .135 Potatoes Once per 2 days or less (N = 302) (2.25) Almost every day (N = 232) -18.08 (2.57) .237 Fruits Once per 2 days or less (N = 70) (4.68) Almost every day (N = 464) -19.18 (1.80) .068 Oils and fats Once per 2 days or less (N = 264) (2.41) Almost every day (N = 270) -22.30 (2.37) .256 Dietary variety score DVS 5 points (N = 261) -22.73 (2.42) (Continued) Table 3. Result of the longitudinal analysis showing the effect of baseline lifestyle factors on decline in muscle strength. Result of the longitudinal analysis showing the effect of baseline lifestyle factors on decline strength No (N = 314) Yes (N = 220) Foods & discretionary items (Continued) PLOS ONE \| DOI:10.1371/journal.pone.0132523 July 15, 2015 9 / 13 Factors Associated with Knee Extension Strength Table 3. Discussion (Continued) Mean SE P-Value DVS 6 points (N = 272) -18.06 (2.37) .171 Drinking Non-drinker (N = 395) -20.06 (1.96) Current drinker (N = 139) -21.33 (3.31) .742 Smoking Non-smoker (N = 514) -20.25 (1.71) Smoker (N = 20) -24.14 (8.77) .664 SE; Standard Error, Analyses of covariance were applied incorporating baseline age, baseline knee extensor strength, and baseline status of all the diseases (hypertension, stroke, heart disease, diabetes mellitus, hyperlipidemia, osteoporosis, anemia, asthma, chronic obstructive pulmonary disease, hip osteoarthritis, and gonarthrosis) as covariates. Table 3. (Continued) SE; Standard Error, Analyses of covariance were applied incorporating baseline age, baseline knee extensor strength, and baseline status of all the diseases (hypertension, stroke, heart disease, diabetes mellitus, hyperlipidemia, osteoporosis, anemia, asthma, chronic obstructive pulmonary disease, hip osteoarthritis, and gonarthrosis) as covariates. y pp p g g extensor strength, and baseline status of all the diseases (hypertension, stroke, heart disease, diabetes mellitus, hyperlipidemia, osteoporosis, anemia, asthma, chronic obstructive pulmonary disease, hip osteoarthritis, and gonarthrosis) as covariates. doi:10.1371/journal.pone.0132523.t003 find an association of regular physical activity with KES, the beneficial effect of exercise on muscle strength cannot be denied. The decline in KES was greater in women who ate seafood almost every day at baseline. However, this result may not be very robust, because the KES at baseline was slightly higher in women who ate seafood almost every day. Several studies indicate that regular seafood intake contributes to maintaining muscle strength [12,24,25]. For example, a cross-sectional study in the UK shows that fatty fish consumption positively influences grip strength in older people [12]. Rats fed Alaska pollock protein exhibit greater fast-twitch muscle hypertrophy than rats fed casein [24]. In a study conducted in older people, leg extension power was positively associ- ated with the serum concentration of an active vitamin D derivative, which is abundant in sea- food [25]. However, methyl mercury, a compound found in seafood, might negatively affect muscle strength. Accordingly, frequent intake of seafood with high levels of mercury in coastal villages is associated with a risk of toxicity, including weakened muscles [26,27]. However, because our study was conducted in Tokyo where no such cases have been reported, seafood intake does not seem to have affected muscle strength. Therefore, a study with a larger cohort seems necessary to clarify the effect of daily seafood intake on muscle strength. PLOS ONE \| DOI:10.1371/journal.pone.0132523 July 15, 2015 Discussion The 4-year decline in KES was smaller in those who ate soy products almost every day than those who ate soy products once/2 days or less. Although we could not find direct evidence supporting the beneficial effect of soy products on muscle health in the literature, several stud- ies suggest that some ingredients found in soy products, such as vitamin K2 and isoflavone, improve bone metabolism [16–14,28]. In a study of 4 groups of participants eating different amounts of natto (rich in vitamin K2) for 1 year, the risk of a reduction in bone formation markers in the group that ate natto most frequently was 0.07 times of that in the group that ate no natto [14]. Another prospective study of middle-aged Japanese women shows that 24 weeks of soy isoflavone supplementation increases bone mineral density [15]. A placebo-controlled study shows that menopausal women treated with isoflavone tablets exhibited a significant decrease in urinary excretion of urinary deoxypyridinoline, a specific biomarker of bone resorption [16]. An analysis of the association of regional differences in natto intake and hip fracture incidence revealed that fractures are less prevalent in regions where natto consump- tion is prevalent due to local culture [28]. The daily intake of soy products in our study may have contributed to the maintenance of muscle strength through a reduction of the incidence of fractures and resulting inactivity. 10 / 13 PLOS ONE \| DOI:10.1371/journal.pone.0132523 July 15, 2015 Factors Associated with Knee Extension Strength However, the questionnaire items about food intake used in the present study were not detailed enough to establish concrete data about the dietary habits of elderly people. Because our study was based on a retrospective analysis of existing data, we were unable to use very detailed questions. Therefore, studies using more detailed items about food intake aiming to understand how dietary habits affect elderly people’s muscle strength seem necessary to make suggestions regarding the diet of elderly people for maintaining muscle strength. The smaller decline of KES in women who ate green or yellow vegetables almost every day may be explained by the antioxidant effects of carotenoids and vitamin C. Recent epidemiolog- ical studies in community-dwelling elderly show that low serum concentrations of carotenoids or vitamin C are associated with low muscle strength [4,6,29–31]. A cross-sectional study revealed that daily dietary intake of vitamin C and β-carotene is significantly associated with KES [6]. Discussion A longitudinal observational study of people >64 years old further shows that those in the lowest quartile of total plasma carotenoid level have a significantly higher risk of muscle weakening 6 years later than those in the highest quartile [5]. Concordant with these studies, the present study corroborates the beneficial effect of the daily intake of green or yellow vegeta- bles on age-related decline in muscle strength. Contrary to our expectation, the intake frequency of meat at baseline did not influence changes in muscular strength. In a study of 1,844 Japanese senior citizens, those with the lowest initial density of serum albumin, which is processed from other proteins within the human body, had the highest incidence of ADL disability 12 years later [32]. In contrast, a longitudinal study in healthy aged men suggests that a low albumin concentration does not predict a decline in muscle strength [33]. Furthermore, a low baseline concentration of serum albumin failed to predict a decline in grip strength over 2 years in a study of frail elderly participants [34]. Contrary to our expectation, the intake frequency of meat at baseline did not influence changes in muscular strength. In a study of 1,844 Japanese senior citizens, those with the lowest initial density of serum albumin, which is processed from other proteins within the human body, had the highest incidence of ADL disability 12 years later [32]. In contrast, a longitudinal study in healthy aged men suggests that a low albumin concentration does not predict a decline in muscle strength [33]. Furthermore, a low baseline concentration of serum albumin failed to predict a decline in grip strength over 2 years in a study of frail elderly participants [34]. Another longitudinal study in Japanese elderly shows that a higher intake frequency of animal protein is associated with a lower risk of decline in functional capacity only in men [35]. As the association between protein intake and muscle health remains controversial, more cohort stud- Another longitudinal study in Japanese elderly shows that a higher intake frequency of animal protein is associated with a lower risk of decline in functional capacity only in men [35]. As the association between protein intake and muscle health remains controversial, more cohort stud- ies are needed to elucidate how meat intake affects muscle strength in the elderly. This study has several limitations. Discussion First, we could not determine the amounts of each food consumed at the time of the baseline survey; instead, the questionnaire collected information about the number of days in a week that a given food was eaten. Nevertheless, we expect that the results of the questionnaire are at least generally related with the actual amounts of food eaten. Second, the data regarding lifestyle factors were only based on the answers to questions at a single time point. Therefore, it is uncertain whether a particular subject kept eating soy products, for example, at the same frequency for 4 years. Third, the data about physical activity levels were collected by interview. Although an objective measurement of physical activity is more appropriate for evaluating lifestyle, the retrospective design forced us to use the data obtained through the interviews. In conclusion, the cross-sectional and longitudinal analyses yielded inconsistent results regarding the associations of lifestyle-related factors with KES. This inconsistency suggests that conducting both types of analyses is crucial. Because our study demonstrated that the age- related decline in muscle strength was lower in those who frequently ate soy products or green and yellow vegetables, recommending higher intakes of these foods might be a useful measure for protecting the muscle health of the elderly. S1 Dataset. Dataset containing all relevant data of the participants in 2008 and 2012. (XLSX) References 1. Kojima N, Kim H, Saito K, Yoshida H, Yoshida Y, Hirano H, et al. Association of knee-extension strength with instrumental activities of daily living in community-dwelling older adults. Geriatr Gerontol Int. 2014; 14: 674–680. doi: 10.1111/ggi.12158 PMID: 24215603 2. Rantanen T, Avlund K, Suominen H, Schroll M, Frändin K, Pertti E. 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Katsuyama H, Ideguchi S, Fukunaga M, Fukunaga T, Saijoh K, Sunami S. Promotion of bone formation by fermented soybean (Natto) intake in premenopausal women. J Nutr Sci Vitaminol (Tokyo). 2004; 50: 114–120. 15. Mori M, Aizawa T, Tokoro M, Miki T, Yamori Y. Soy isoflavone tablets reduce osteoporosis risk factors and obesity in middle-aged Japanese women. Clin Exp Pharmacol Physiol. 2004; 31: S39–S41. PMID: 15649285 16. Mori M, Sagara M, Ikeda K, Miki T, Yamori Y. Soy isoflavones improve bone metabolism in postmeno- pausal Japanese women. Clin Exp Pharmacol Physiol. 2004; 31: S44–S46. PMID: 15649287 17. No authors. World Medical Association declaration of Helsinki. Recommendations guiding physicians in biomedical research involving human subjects. JAMA. 1997; 277: 925–926. PMID: 9062334 PLOS ONE \| DOI:10.1371/journal.pone.0132523 July 15, 2015 12 / 13 Factors Associated with Knee Extension Strength 18. Kwon J, Suzuki T, Kumagai S, Shinkai S, Yukawa H. Risk factors for dietary variety decline among Jap- anese elderly in a rural community: a 8-year follow-up study from TMIG-LISA. Eur J Clin Nutr. 2006; 60: 305–311. PMID: 16234831 19. Kumagai S, Watanabe S, Shibata H, Amano H, Fujiwara Y, Shinkai S, et al. [Effects of dietary variety on declines in high-level functional capacity in elderly people living in a community]. Nihon Koshu Eisei Zasshi. 2003; 50: 1117–1124. PMID: 14750363 20. Kimura M, Moriyasu A, Kumagai S, Furuna T, Akita S, Kimura S, et al. Community-based intervention to improve dietary habits and promote physical activity among older adults: a cluster randomized trial. BMC Geriatr. 2013; 13: 8. doi: 10.1186/1471-2318-13-8 PMID: 23343312 21. Fiatarone MA, O'Neill EF, Ryan ND, Clements KM, Solares GR, Nelson ME, et al. Exercise training and nutritional supplementation for physical frailty in very elderly people. N Engl J Med. 1994; 330: 1769– 1775. PMID: 8190152 22. Kim H, Suzuki T, Saito K, Yoshida H, Kojima N, Kim M, et al. PLOS ONE \| DOI:10.1371/journal.pone.0132523 July 15, 2015 References Lower levels of serum albumin and total cholesterol associated with decline in activities of daily living and excess mortality in a 12-year cohort study of elderly Japanese. J Am Geriatr Soc. 2008; 56: 529–535. doi: 10.1111/j.1532- 5415.2007.01549.x PMID: 18179493 33. Snyder CK, Lapidus JA, Cawthon PM, Dam TT, Sakai LY, Marshall LM, et al. Serum albumin in relation to change in muscle mass, muscle strength, and muscle power in older men. J Am Geriatr Soc. 2012; 60: 1663–1672. doi: 10.1111/j.1532-5415.2012.04115.x PMID: 22905696 34. Kitamura K, Nakamura K, Nishiwaki T, Ueno K, Nakazawa A, Hasegawa M. Determination of whether the association between serum albumin and activities of daily living in frail elderly people is causal. Environ Health Prev Med. 2012; 17: 164–168. doi: 10.1007/s12199-011-0233-y PMID: 21861116 35. Imai E, Tsubota-Utsugi M, Kikuya M, Satoh M, Inoue R, Hosaka M, et al. Animal protein intake is asso- ciated with higher-level functional capacity in elderly adults: the Ohasama study. J Am Geriatr Soc. 2014; 62: 426–434. doi: 10.1111/jgs.12690 PMID: 24576149 13 / 13 PLOS ONE \| DOI:10.1371/journal.pone.0132523 July 15, 2015
https://openalex.org/W2068010802	https://zenodo.org/records/2158259/files/article.pdf	English	null	Section of Geology and Mineralogy of the New York Academy of Sciences	Science	1,908	public-domain	822	SECTION OF GEOLOGY AND MINERALOGY OF THE NEW YORE ACADEMY OF SCIENCES CJontinental Formations of the North Amerioan Paleozoic: Professor A. WV. GRABAU. CJontinental Formations of the North Amerioan Paleozoic: Professor A. WV. GRABAU. A REGuLAR monthly meeting of the section was held October 5 in the academy rooms at the American Museum of Natural History. Four papers were presented, as follows: The change of opinion in regard to conditions under which many of the well-known sedimentary formations are originally deposited was outlined. A tabulated list of those formations of the Ap- palachian region whose characters seem to indi- cate continental origin was exhibited and the evidence was briefly discussed. This article is to be published in full in SCIENCE. Outline of the Geology of Long Island, N. Y.: Professor W. 0. CROSBY. Outline of the Geology of Long Island, N. Y.: Professor W. 0. CROSBY. Professor Crosby is of the opinion that the Pleistocene history of Long Island is relatively simple, and the known facts are accounted for by a single ice-invasion. The recent reference of the underlying lignitic and pyritic Chesapeake (Mio- cene) clays and the Lafayette (Pliocene) yellow gravel to the Pleistocene glacial series is believed to be a mistake. From the early Pleistocene up- lift dates the cuesta of Long Island, to which Long Island Sound holds the relation of an inner lowland. This lowland is still floored by Creta- ceous clays and sands. The transverse valleys and deep bays of the north shore of Long Island are essentially preglacial, though greatly modified by glacial erosion and deposition. CHARLES P. BERxEY, Secretary of Section CHARLES P. BERxEY, , Secretary of Section [N. S. VOL. XXVIII. No. 730 [N. S. VOL. XXVIII. No. 730 SCIENCE [N. S. VOL. XXVIII. No. 730 936 the wild ducks that frequent this brook through the summer season. W. A. MuRRILL, Secretary pro tern. steam shovels, in huge open cuts. They range in copper from less than two to two and a half per cent. The operation and processes of the mills and smelters were briefly outlined. The paper was based upon visits made the past summer. THE club met at the American Museum of Natural History on November 10, 1908, and was called to order by Vice-president Burgess at 8:15 P.M. About 95 persons were present. Limnestones Interbedded with the Fordhamr Gneiss in New York City: Dr. CHARLES P. BERKEY. y The discovery of beds of limestone at three points in such relation as to indicate interbed- ding with the banded gneisses was announced. This is an additional feature of similarity between the gneisses of the Highlands and the Fordham at its type locality. The largest bed is about 27 feet thick and is exposed in the east wall of the new Jerome Park Reservoir at 205th Street. In all cases these limestones are very impure and coarsely crystalline, carrying many unusual min- erals arising chiefly from recrystallization. Chon- drodite and actinolite are abundant. Sphalerite and galenite are also found. After the reading of the minutes of the meeting of October 29, Dr. N. L. Britton delivered the lec- ture of the evening on " Trees of the Vicinity of New York." The lecture was illustrated by lan- tern slides from the Van Brunt collection and was of a popular nature. The trees were taken up in a biological order, beginning with the gymno- sperms, and the photographs exhibited illustrated both the general habit of the trees discussed and the details of their flowers and fruit. MARSHALL A. HOWE, Secretary pro tem. WASHINGTON SECTION OF THE AMERICAN CHEMICAL SOCIETY THE 185th meeting was called to order by President Walker on Thursday evening, November 12. The attendance was 90, this large number being due to the fact that many visiting member of the A. 0. A. C. were present. The followin papers were read: p p " Color of Lead Chromate," E. E. Free. "Absorption of CO2 by Moist Oxide," W. 0. Robinson. "Absorption of CO2 by Moist Oxide," W. 0. Robinson. The Production of Low-grade Copper Ore in the WVest: Professor JAMES F. KEMP. The Production of Low-grade Copper Ore in the WVest: Professor JAMES F. KEMP. " Solubility of Gold in Salt Solutions," W. J. McCaughey. " Solubility of Gold in Salt Solutions," W. J. McCaughey. The speaker presented a brief description of the recent development of the so-called " low-grade " copper mines in Bingham Cafnon, Utah, and at Ely, Nev. By means of maps the geographical situation was made clear and the geological rela- tions were outlined. The ores consist of bodies of silicified and brecciated porphyry, impregnated with chalcocite. They are mined by means of g y " The Distribution of NaNO, in the United States," C. E. Munroe. Three members of the society were elected a councilors to the American Chemical Society, vi., L. M. Tolman, E. T. Allen and E. M. Chace. J. A. LECLERC, , Secret
https://openalex.org/W4317352381	https://zenodo.org/records/7546811/files/34.pdf	English	null	Physical and Chemical Properties of Water in Rahuri Tahsil of Ahmednagar District (M.S.)	Zenodo (CERN European Organization for Nuclear Research)	2,023	cc-by	2,036	International Journal of Advance and Applied Research www.ijaar.co.in ISSN – 2347-7075 Impact Factor – 7.328 Peer Reviewed Bi-Monthly Vol.4 No.1 Jan – Feb 2023 Physical and Chemical Properties of Water in Rahuri Tahsil of Ahmednagar District (M.S.) Dr. Sopan N. Shingote1 Dr. Rajendra S. Pawar2 1Arts, Science and Commerce College, Kolhar 2Padmashri Vikhe Patil College of Arts, Science and Commerce College, Pravaranagar Corresponding Author- Dr. Sopan N. Shingote International Journal of Advance and Applied Research www.ijaar.co.in ISSN – 2347-7075 Impact Factor – 7.328 Peer Reviewed Bi-Monthly Vol.4 No.1 Jan – Feb 2023 Physical and Chemical Properties of Water in Rahuri Tahsil of Ahmednagar District (M.S.) Dr. Sopan N. Shingote1 Dr. Rajendra S. Pawar2 1Arts, Science and Commerce College, Kolhar 2Padmashri Vikhe Patil College of Arts, Science and Commerce College, Pravaranagar Corresponding Author- Dr. Sopan N. Shingote International Journal of Advance and Applied Research www.ijaar.co.in ISSN – 2347-7075 Impact Factor – 7.328 Peer Reviewed Bi-Monthly Vol.4 No.1 Jan – Feb 2023 Physical and Chemical Properties of Water in Rahuri Tahsil of Ahmednagar District (M.S.) Dr. Sopan N. Shingote1 Dr. Rajendra S. Pawar2 1Arts, Science and Commerce College, Kolhar 2Padmashri Vikhe Patil College of Arts, Science and Commerce College, Pravaranagar Corresponding Author- Dr. Sopan N. Shingote Introduction Water is the most precious resource because the life of animals and plants depends on it. Most industries also require water for various applications, so the global economy depends on it. Springs are the places where ground water is discharged at specific locations on the earth and they vary dramatically as to the type of water they discharge. Many of the springs are the result of long cracks or joints in sedimentary rock. (Young, 2007) Hot springs are defined as springs where the temperature of water lies significantly above the mean of annual air temperature of that region. (Thompson, 2003 and Young, 2007) Hot ground water can be used to drive turbines and generate electricity, or it can be used directly to heat homes and other buildings. Energy extracted from the Earth’s heat is called geothermal energy. (Thompson and Turk, 2005) The physical, chemical and biological composition of water is influenced to a great extent by different factors including climate, geomorphology and geology. Also the physical variables which include temperature and turbidity; chemical variables in that non- toxic variables such as pH, total dissolved salts, salinity, conductivity, ions, nutrients, organic matter and dissolved gases and toxic variables like biocides and trace metals. The objectives of the present work are to analysis and discuss the suitability of water for drinking and sanitation. Abstract Abstract Water quality assessment of Rahuri tahsil in Ahmednagar district has done in Maharashtra State, India. This paper aims to study the physical and chemical properties of water of Rahuri and its surrounding area. The physical parameters included Temperature, Total dissolved solids and electrical conductivity. The chemical parameters included pH, total hardness, calcium hardness, magnesium hardness, Phenolphthalein alkalinity, total alkalinity. Ionic parameters like chloride, phosphate, sulphate, calcium, magnesium, sodium, potassium, iron, chromium and manganese. Also, the biological parameters studied standard plate count and most probable number. Keywords: Physico-Chemical Parameters, Permissible Limit, Chemical Standards of Drinking Water Keywords: Physico-Chemical Parameters, Permissible Limit, Chemical Standards of Drinking Water. Rainfall, an important and largest source of water, other sources are surface water and sub-surface water or ground water. (Sharma B.K., 2001)Water is mostly important for industrial and municipal purposes. In addition to the direct consumption of water at homes and farms, there are many indirect ways in which water affects our daily life. IJAAR AR Vol.4 No.1 ISSN – 2347-70 The Rahuri tehsil lies in the e of the Western Ghats in Mula and Pravara basin. Figure: Location Map of Study Area Vol.4 No.1 ISSN – 2347-7075 ISSN – 2347-7075 East Longitude. The Rahuri tehsil lies in the rain shadow zone of the Western Ghats in East Longitude. The Rahuri tehsil lies in the rain shadow zone of the Western Ghats in Mula and Pravara basin. Figure: Location Map of Study Area Sampling Methods calcium of the water sample were determined by complex metric titration with EDTA using Murexide as an indicator. Phenolphthalein and Total alkalinities of the water samples were determined by titrating with H2SO4 using phenolphthalein and methyl orange as indicators. The water quality parameters estimated by the standard methods given by APHA (1998). For the present investigation groundwater samples were collected every month during the study year from June 2013 to May 2014 from 32 different sampling stations of Rahuri tehsil. The water samples collected from the Rahuri Tahsil and taken inpre-cleaned polyethylene bottle. Water temperature recorded immediately on the site by mercury thermometer. TDS of water samples measured using gravimetric method. Dissolved oxygen was estimated by the method of Winkler method. EC values of the water sample under investigation were measured using Digital Conductivity meter. The pH value of water sample measured by using Digital pH meter. Study Area Water is one of the abundantly available substances in nature. It is essential constituent of all animal and plants material and forms about 75% of matter of earth crust. It has been argued previously that geochemical energy-yields may be a key determinant of microbial community structure and diversity in thermal environments (Amend and Shock, 2001) The Rahuri Tehsil in Ahmednagar district of Maharashtra has been selected for the present investigation work. The tehsil comprises of 95 villages and two urban centers spread over an area of 1, 00,898 hectares. The geographical extension of the study area is form 19°15' N to 19°34' North latitude and 74°23' E to74°50' Dr. Sopan N. Shingote, Dr. Rajendra S. Pawar Result and Discussion A total of 32 samples were collected from 32 villages of Rahuri tehsil of Ahmednagar. Among these villages, 4% drinking water samples from two locations contain 1 mg/l of fluoride, 96% of the samples contain fluoride 0.5 mg/l. The results indicate that the fluoride content in all the sampling stations was found within the permissible levels as per WHO standards.  Hydrogen Ion Concentration (pH) Temperature On the basis of above discussion, it is concluded that the water quality assessment of Rahuri Tehsil in Ahmednagar district in Maharashtra. It reveals that although the situation is not worst but it has to be maintained. Some of the water characteristics are below the permissible limit in the post-monsoon season and some are above the permissible limits in pre- monsoon season. This may be due to dilution of water by raining. Complete study showed that the water is more polluted in pre- monsoon as compared to post-monsoon. The water temperature noted from 32 villages of Rahuri tehsil, it 28.5oC in pre- monsoon as maximum and 27oC in post- monsoon season. Jaybhaye et al. (2008), reported water temperature ranged from 22.5-32.50C from Kayadhu river, near Hingoli during January-December 2004.  Hydrogen Ion Concentration (pH) y g (p ) The average of pH noted from 32 villages of Rahuri tehsil. Water sample is 8.77as maximum and minimum 5.1 was observed. The total hardness of the water sample was determined by complex metric titration with EDTA using Erichrome black T as an indicator. The calcium hardness and  Electrical conductivity (EC) 204 Dr. Sopan N. Shingote, Dr. Rajendra S. Pawar  Total Dissolved Solids (TDS)  Total Dissolved Solids (TDS) The average total dissolved solids observed from 32 villages of Rahuri tehsil. From water sample are 690 mg/L as maximum in pre-monsoon and 110 mg/L as minimum in post monsoon. Total dissolved solids are above the permissible limiting 500 mg/L recommended by WHO. Approximately of the aquatic characteristics stay lower the accepted edge in the post- monsoon period and some are upstairs the acceptable limits in pre-monsoon season. This might be due to dilution of water by raining. Simmular remarks are observed by Yannawar et al. (2013). Asrari et al. (2008) measured the TDS minimum 50mg/L and maximum 3575 mg/L from Kor River, Iran. The amount of TDS related with increasing dissolved ions. Acknowledgement Acknowledgement We are thankful to the School of Earth Sciences of Swami Raman and Teerth Marathwada University, Nanded for providing laboratory and library facilities. g We are thankful to the School of Earth Sciences of Swami Raman and Teerth Marathwada University, Nanded for providing laboratory and library facilities. Dissolved Oxygen The average dissolved oxygen obtained from 32 villages of Rahuri tehsil of water sample is 0.9 mg/L maximum and 0.2 mg/L minimum with the mean value of 0.49 mg/L. Yannawar VB and Bhosale AB (2013), achieved value of dissolved oxygen varied from 2.0, 1.12, 1.8 and 1.64 in S1, S2, S3 and S4 respectively from the selected sites. The lower dissolved oxygen due to organic contamination near sources to water. References 1. Amend JP, Shock EL, 2001 Energetic of overall metabolic reactions of thermophilic and hyperthermophilic Archaea and Bacteria. FEMS Microbiology Reviews vol.25, pp.175–243. 1. Amend JP, Shock EL, 2001 Energetic of overall metabolic reactions of thermophilic and hyperthermophilic Archaea and Bacteria. FEMS Microbiology Reviews vol.25, pp.175–243. The average hardness obtained from 32 villages of Rahuri tehsil of water sample is 310 mg/L maximum and 80 mg/L minimum with the mean value of 80 mg/L. Singh et al. (2005), found hardness level as 243 mg/L, 180 mg/L and 149 mg/L during June 1999 from the wells, springs and the rivers respectively in Udhampur, Jammu and Kashmir. Also they found hardness 194 mg/L, 179 mg/L and 146 mg/L in October 1999 from same water sampling sites. Singh et al. (2005), found hardness level as 243 mg/L, 180 mg/L and 149 mg/L during June 1999 from the wells, springs and the rivers respectively in Udhampur, Jammu and Kashmir. Also they found hardness 194 mg/L, 179 mg/L and 146 mg/L in October 1999 from same water sampling sites. 2. APHA, 1998, Standard Methods for the Examination of Water and Wastewater. American Public Health Association, 20th edition, Washington. D.C. 3. Asrari E., Madadi M. and Masoudi, 2008, Study of water quality in Kor River, West Southern of Iran, Nature Environment and Pollution Technology, Vol. 7, No. 3, pp. 501-504. ISSN – 2347-7075 The average of Electrical conductivity recorded from 32 villages of Rahuri tahsil. Of it water sample is 4.53 uS/cm as maximum and 0.16 uS/cm as minimum recorded. – 124 mg/L in surface and sub-surface water of Bhadra River respectively. – 124 mg/L in surface and sub-surface water of Bhadra River respectively. – 124 mg/L in surface and sub-surface water of Bhadra River respectively. Phenolphthalein Alkalinity (PA) Dr. Sopan N. Shingote, Dr. Rajendra S. Pawar Phenolphthalein Alkalinity (PA) The phenolphthalein alkalinity of 32 villages of Rahuri tehsil of water sample is below detectable limit in pre-monsoon and 1885 mg/L maximum and minimum 267 mg/L. Average value of phenolphthalein alkalinity 596.9 mg/L. Calcium The value of calcium observed from 32 villages of Rahuri tehsil of water samples are 198 mg/L maximum and 5.6 mg/L minimum in pre and post-monsoon respectively. The mean calcium hardness was 33.1 mg/L. 4. Jaybhaye U. M., Salve B. S. and Pentewar M. S., 2008, Some physico- chemical Aspects of Kayadhu River, District Hingoli, Maharashtra, J. Aqua. Biol., Vol. 23, No.1, pp. 64-68. Vijayakumar et al. (2005), observed calcium ranged from 8.60 – 94.10 mg/L 75.25 205 Dr. Sopan N. Shingote, Dr. Rajendra S. Pawar Vol.4 No.1 ISSN – 2347-7075 ISSN – 2347-7075 IJAAR 5. Sharma B.K., 2001, Environmental chemistry, IV edition, Goel Publication House, Meerut. 6. Singh Omkar, Kumar Vijay and Rai S.P., 2005, Water quality aspects of some wells, springs, and river in Parts of the Udhampur District (J & K), Journal of Environ. Science and Eng., Vol.47, No.1, pp.25-32. 7. Thompson and Turk, 2005, Introduction to physical Geology, Saunders golden sunburst series. 8. Thompson C., 2003, The Arizona Republic, vol.1, pp. 12-03. 9. Vyankatesh B Yannawar, Arjun B Bhosale, Parveen R Shaikh, and Surekha R Gaikwad (2013) Water Quality of Hot Water Unkeshwar Spring of Maharashtra, India. Int J of Innovation and Applied Studies, Vol. 3 No. 2, pp. 541-551. 10. Yannawar Vyankatesh B. and Bhosale Arjun B. (2013) Cultural eutrophication of Lonar Lake, Maharashtra, India. Int J of Innovation and Applied Studies Vol. 3 No. 2, pp. 504-510. 11. Young M.C., 2007, Aqua Thermal Access, vol. 4, pp. 8. 206 Dr. Sopan N. Shingote, Dr. Rajendra S. Pawar
https://openalex.org/W3178980543	https://discovery.ucl.ac.uk/id/eprint/10131589/1/Selai_capr.12438.pdf	English	null	Functional neurological symptoms: Optimising efficacy of inpatient treatment and preparation for change using the Queen Square Guided Self‐Help	Counselling and psychotherapy research	2,021	cc-by	8,636	O R I G I N A L A R T I C L E O R I G I N A L A R T I C L E Couns Psychother Res. 2021;00:1–12. Received: 23 February 2021 \| Revised: 15 June 2021 \| Accepted: 16 June 2021 Received: 23 February 2021 \| Revised: 15 June 2021 \| Accepted: 16 June 2021 DOI: 10.1002/capr.12438 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2021 The Authors. Counselling and Psychotherapy Research published by John Wiley & Sons Ltd on behalf of British Association for Counselling and Psychotherapy Humblestone and Roelofs joint first authors. Selai and Moutoussis joint senior authors. \| 1 wileyonlinelibrary.com/journal/capr Susan Humblestone1 \| Jacob Roelofs2 \| Caroline Selai1,2 \| Michael Moutoussis1,3,4 Susan Humblestone1 \| Jacob Roelofs2 \| Caroline Selai1,2 \| Michael Moutoussis1,3,4 1Neuropsychiatry Department, National Hospital for Neurology and Neurosurgery, London, UK Abstract Objective: Functional neurological symptoms (FNS) are disabling symptoms without macro-structural cause. While inpatient treatment confers important benefits, it is resource-intensive, and hence, it is important to optimise its efficiency. Methods: We developed a brief, Internet-based preparatory therapy based on psych- oeducation and CBT, termed the Queen Square Guided Self-help (QGSH), to maxim- ise the efficacy of the inpatient FNS treatment at the National Hospital for Neurology and Neurosurgery. 3Wellcome Centre for Human Neuroimaging, University College London, London, UK 4Max Planck – University College London Centre for Computational Psychiatry and Ageing Research, London, UK Methods: We developed a brief, Internet-based preparatory therapy based on psych- oeducation and CBT, termed the Queen Square Guided Self-help (QGSH), to maxim- ise the efficacy of the inpatient FNS treatment at the National Hospital for Neurology and Neurosurgery. Results: The QGSH aims to ensure that prior to admission, the patient understands (a) the diagnosis of FNS, (b) the five-areas CBT model and (c) the use of goal setting in rehabilitation. It has now run since 2017, and 191 patients have taken part in the inpatient FNS programme, with 122 of these having participated in the QGSH. It runs for up to 12 weeks and includes original videos and patient worksheets, as well as signposting to existing published resources. Information is sent weekly by email, and content is delivered in the form of 11 modules built around online video sessions. Correspondence Caroline Selai, Department of Clinical and Movement Neurosciences, UCL Queen Square Institute of Neurology, London WC1N 3BG, UK. Email: c.selai@ucl.ac.uk Conclusion: We believe that the set of materials used in QGSH has the potential to benefit patients with FNS and can support clinicians wishing to develop their ex- pertise. It could help with the development of new FNS services, and we are in the process of developing it into a stand-alone service. We hope that the experience of the Queen Square team can be used to help patients and clinicians to improve the provision of FNS services. Implications for practice and policy Implications for practice and policy Functional neurological symptoms (FNS) are disabling symptoms without macro-structural cause (American Psychiatric Association, 2013). Previously, these symptoms have been described using a range of terms including ‘hysterical’, ‘conversion’ and ‘somatisation’, each with implications about the underlying mechanism. The variety of terms has left patients ‘muddled’ and can be seen as offensive (Stone et al., 2002). ‘Functional’ is now preferred as it is acceptable to patients and does not assume an underlying mechanism (Ding & Kanaan, 2016). • The QGSH has the potential to provide significant ben- efit for patients with FNS • Therapy can be delivered remotely • Therapy can be delivered remotely • Clinicians can benefit from the materials and supportive structure • QGSH could be introduced into stepped care for pa- tients with FNS The diagnosis is based on a pattern of positive signs and symp- toms that are characteristic of functional disease and that vary with time or attention (Carson et al., 2016). This is reflected in the DSM-5 and ICD-10 criteria (see Appendix 2). FNS may include motor symp- toms, sensory disturbance or other neurological symptoms such as aphonia. An important subtype of FNS is non-epileptic attack dis- order (NEADs), also known as dissociative or psychogenic seizures, which present with episodes of disrupted consciousness with- out EEG evidence of epileptic brain activity (American Psychiatric Association, 2013). minority of patients are symptom-free after the initial consultation alone (McKenzie et al., 2010). Additionally, patients feeling they are believed by their doctor is an important factor in their recovery (Karterud et al., 2015). Patient acceptance of the diagnosis, acknowl- edgement that emotion may play an important role in symptom pro- duction and a stable social environment all increase the chance of a good recovery (Reuber et al., 2005; Rommelfanger et al., 2017). On the other hand, poor outcomes are associated with expecta- tion of non-recovery, non-attribution of symptoms to psychological factors and receipt of health-related benefits (Sharpe et al., 2010). Psychiatric comorbidity, particularly personality disorder, is also an important negative predictive factor (Gelauff et al., 2014). This may reflect the difficulty of treatment in the presence of another disor- der, and in personality disorders, difficulties with collaboration with treatment. Functional neurological symptoms (FNS) cause a similar level of disability to 'organic' neurological disorders but with a higher rate of psychiatric comorbidity (Sojka et al., 2018). Implications for practice and policy FNS is thought to account for between 6% and 16% of all neurology clinic referrals (Stone et al., 2010) with an incidence of 4–12 per 100,000 (Carson et al., 2012). ‘Functional overlay’, where functional symptoms coex- ist with ‘organic’ illnesses, is thought to occur in up to 30% of neu- rology patients (Stone et al., 2010), and it is thought that 10%–50% of patients with epilepsy may have a combination of epileptic and non-epileptic seizures (Gates, 2002). We place ‘organic’ in quotes to emphasise that all disorders of brain function have an organic substrate at the microscopic level of synaptic connectivity and neu- rotransmitter function. Similarly, we avoid referring to FNS as having ‘no structural cause’, preferring to talk about ‘no macro-structural cause’ as might be detected by current medical imaging. K E Y W O R D S cognitive behavioural therapy, functional neurological symptoms, guided self-help, rehabilitation cognitive behavioural therapy, functional neurological symptoms, guided self-help, rehabilitation \| 1 wileyonlinelibrary.com/journal/capr 1.1 \| The multidisciplinary approach to inpatient treatment Evidence supports both psychological and physical interventions (Conwill et al., 2014; Demartini et al., 2014; Sharpe et al., 2011), helping to overcome the difficulties from diagnosis to effective treatment that have historically troubled the management of FNS (Greiner et al., 2016). Acceptance of this multidisciplinary approach is illustrated by 55% of neurologists and 88% of psychiatrists fa- vouring a combined treatment in one study (Schipper et al., 2014). Using a multidisciplinary team (MDT) of health professionals from a range of backgrounds can maximise the impact of each form of ther- apy by working co-operatively (Demartini et al., 2014; Hubschmid et al., 2015; Jordbru et al., 2014; Saifee et al., 2012). The National Hospital for Neurology and Neurosurgery (NHNN) offers a tertiary care, multidisciplinary treatment package for FNS, whose centre- piece is the inpatient programme. The term ‘organic’ is commonly used to refer to disorders with well-established histopathological or neurochemical substrate, but its simplistic application in the case of FNS has caused many mis- understandings that the programme described in this article often has to address. Importantly, psychological factors and negative life events are thought to be part of a process of symptom emergence, contributing to disrupted attentional and emotional processing to produce and maintain symptoms (Pick et al., 2019). Yet, the role of psychological factors is controversial, as many perceive clinical formulations at the psychological or psychiatric level as discount- ing true distress, disbelieving or stigmatising sufferers as ‘mad’ and denying the role of the brain—while of course, they simply refer to a different level of brain function and structure, that of information processing and learning. 2 \| 1 \| BACKGROUND HUMBLESTONE et al. Implications for practice and policy • The QGSH has the potential to provide significant ben- efit for patients with FNS • Therapy can be delivered remotely • Clinicians can benefit from the materials and supportive structure • QGSH could be introduced into stepped care for pa- tients with FNS 2.1 \| Key therapies within the MDT Our programme comprises the following physical, psychological, oc- cupational, psychiatric and whole-team contributions. Specialist physiotherapy for motor FNS, which focuses on re- training abnormal movements (Nielsen, 2016), can be highly ef- fective (Nielsen et al., 2013). Significant improvements in physical function and quality of life sustained over follow-up have been seen (Jordbru et al., 2014; Nielsen et al., 2015). Cuijpers and Schuurmans reviewed the history of self-help inter- ventions for anxiety disorders and outlined the forms it can take (see Table 1). The only RCT to investigate the efficacy of guided self-help for FNS was performed by Sharpe et al., providing class III evidence. Participants allocated to the UC + GSH condition showed greater improvement in the CGI with an odds ratio of 2.36 (95% CI: 1.17– 4.74, p =.016). There was a 13% absolute improvement in the pro- portion rating their health as ‘better’ or ‘much better’, translating to Psychologically, we use cognitive behavioural therapy (CBT) for FNS (Dallocchio et al., 2016) within a broad biopsychosocial ap- proach. The cognitive component aims to modify the patient's un- helpful beliefs in relation to their illness (O’Neal & Baslet, 2018). CBT has been found to be effective in studies looking at both one-to-one settings (Sharpe et al., 2011) and groups (Conwill et al., 2014). A range of psychological therapies have been used for FNS, including brief psychodynamic interpersonal therapy (BPIP; Sattel et al., 2012). 2 \| METHODS We developed the QGSH through (a) considering the multidisci- plinary approach that patients needed to learn about, (b) adapting existing guided self-help approaches, (c) incorporating an ongoing process of service evaluation and, finally, (d) aiming to provide the resources we developed to the community. 1.2 \| The need for guided self-help While inpatient MDT treatment confers important benefits (Demartini et al., 2014), it is resource-intensive and hence under pressure to minimise its length. The shortening of admissions in Regarding the place of information and learning in treatment, there is significant evidence that the quality of diagnostic explanation impacts the efficacy of treatment (Edwards, 2016) and a substantial HUMBLESTONE et al. \| 3 3 NHNN meant that many patients spent a significant proportion of their admission gaining an understanding of the diagnosis and the rehabilitative approach. Many expected an admission based on ‘or- ganic’ investigations and medical intervention and had doubts about the biopsychosocial, goal-driven approach and how important self- management would be for effective rehabilitation. By the time a col- laborative understanding of FNS has been achieved, there was often little time left for hands-on rehabilitation. Thus, we aimed to develop a preparatory therapy, termed the ‘Queen Square Guided Self-help’ (QGSH), based on psychoeducation and CBT, and to institute it as a key part of the treatment package so that patients would make the best use of the inpatient treatment. 24-hr dynamic risk assessment and so supports a therapeutic reha- bilitative environment. Expert psychiatric understanding is also important. Many of the more disabled FNS patients present with a range of functional symp- toms in the presence of comorbid ‘organic’ diseases. A significant proportion present with psychiatric comorbidity and complex bio- psychosocial presentations. 2.2 \| The development of guided self-help Self-help approaches have been established for many decades in the psychological therapies (2001, 2001). These include, for example, bibliotherapy, where patients read recommended books and forms of computerised CBT delivered by CD or DVD or via the Internet. ‘Book prescription’ schemes where patients can borrow on extended loan specific books via a ‘prescrip- tion’ from a health professional is one way of making bibliotherapy more accessible. In advocating greater accessibility and flexibility in modes of therapy delivery, Lovell and Richard (2000) advocated for Multiple Access Points and Levels of Entry to therapy (MAPLE; Lovell & Richards, 2000). Thus, as well as part of an integrated care package, guided self-help may form one stage in a stepped-care programme. Indeed, NHS Scotland has advocated ‘stepped care’ for patients with FNS. Step 1 is diagnosis; Step 2 is a brief intervention; and Step 3 is complex care with a multidisciplinary team (Healthcare Improvement Scotland, 2012). 3 \| RESULTS Patients are referred to the overall FNS service by neurologists and neuropsychiatrists who have established the diagnosis and are first seen in a neuropsychiatry MDT clinic to assess their suitability for treatment. This clinic consists of a neuropsychiatrist, FNS specialist nurse, FNS specialist occupational therapist and an FNS specialist physiotherapist. When they are first seen in this clinic, some patients have accepted their diagnosis, while other patients report they do not recall their diagnosis or reason for referral. The decision to admit is then made collaboratively based on each patient's needs and the MDT assessment. The patient is encouraged to take responsibility for their own rehabilitation with professional support and guidance. By discussing the treatment programmes on offer, the clinician can gauge the pa- tient's willingness to engage and come up with an estimation of the patient's suitability for treatment. At the end of the assessment, the patients are invited to suggest two or three goals they might like to work on when they start treatment. Patients accepted for the inpa- tient programme are contacted approximately 10–12 weeks prior to their admission date to start the QGSH. The referral process is outlined in Figure 1, and the criteria to be considered for treatment are as follows: 4 \| HUMBLESTONE et al. FI G U R E 1 Flow chart showing the referral pathway for the IP program and GSH a number needed to treat of 8. Much of the recent work on GSH has moved to Internet-based approaches, which can amplify the power of GSH. In this light, the QGSH was developed as a brief therapeutic in- tervention, which aims to ensure that, prior to admission, the patient understands (a) the diagnosis of FNS and how their own diagnosis has been reached; (b) the five-areas CBT model and has started prac- tising it and (c) the use of goal setting in rehabilitation. The ‘five- areas’ approach (Williams et al., 2011) consists of (a) Symptoms, (b) Cognitions/thinking, (c) Feelings, (d) Behaviour and (e) Life situation, and focuses on psychoeducation, explains FNS within a biopsycho- social model and teaches goal-oriented self-management to support engagement with the inpatient programme. The most important aim, however, is to develop a collaborative, trusting alliance with the neu- ropsychiatry multidisciplinary team. 2.3 \| Service evaluation In order to improve the QGSH, we developed an ongoing evalua- tion system based on a Patient-provided Routine Outcome Measure (PROM) and a complementary Clinician-provided Routine Outcome Measure (CROM). Both of these were produced in four stages: as- sembling items, preliminary scale evaluation, analysis of reliability and validity, and final clinical evaluation, in line with standard devel- opment of psychometric scales. FI G U R E 1 Flow chart showing the referral pathway for the IP program and GSH take an inventory of symptoms and explain the diagnosis and the approach to treatment. During the assessment, interviewers and patients can begin to put together elements of a clinical formula- tion. For instance, a patient experiencing intermittent leg weakness might disclose a history of trauma with current symptoms of post- traumatic stress disorder (PTSD), so that ‘leg buckling’ occurs in re- sponse to particular triggers. TA B LE 1 Types of self-help summarised from Cuijpers and Schuurmans (2007) Unguided self-help: Provided by a book or electronically via the Internet or computer programmes. There is no professional support of either the user's understanding of the method or how far to pursue it In QGSH, collaborative work in the therapy relationship is cru- cially informed by a psychodynamic ‘lens’. That is, we think about the feelings of both patients and therapists during the work and how they may depend on past experiences, and discuss these in clinical supervision. This is ‘lens’ rather than ‘technique’, in that we do not use psychoanalytic interventions such as interpretations. Self-help as part of face-to-face therapy: Here, it can be used as part of regular treatment with a professional providing the patient with self-help materials to speed up the treatment process or to give them an opportunity to practise components of the therapy independently. For example, self-help sleep-hygiene guides are commonly used in the standard CBT Occupational therapy for FNS is primarily concerned with func- tion rather than impairment. An in-depth history taken from the in- dividual allows the therapist to place them in the context of their wider biopsychosocial environment. Understanding a person's nar- rative is essential in planning rehabilitation and recovery (Nicholson et al., 2020). The nursing team also provide the detailed knowledge of a person's presentation throughout the day, which allows for a Self-help as an independent intervention: The patient works through a self-help workbook or worksheet with support from a professional at regular times. These are usually brief contacts aimed to provide added explanation about the methods where needed rather than developing a traditional patient–therapist relationship. The capacity for this has expanded significantly with the development of the Internet including the book ‘Overcoming FNS' (Williams et al., 2011), are used according to clinical judgement and patient collaboration within a flexible protocol. Information is sent weekly by email, and content is delivered in the form of 11 modules built around video sessions on YouTube (see Table 2). The YouTube videos are not freely acces- sible; each link is sent to the patient by the QGSH therapist at the appropriate time in the therapy. Patients are supplied with comple- mentary, CBT-based worksheets and exercises and are supported by telephone appointments by experienced clinicians: a senior oc- cupational therapist (SH), a consultant psychiatrist in psychotherapy (MM) and a psychologist (CS). Guidance telephone appointments take place at 2- to 3-week intervals. The therapists are able to adjust the rate and ordering of the modules to best meet the needs of each individual patient. Adjustments such as sending resources by post are often made, as patients' ability to use electronic devices varies, especially if they are older. symptoms and the body'; 'goal setting'; 'helpful and unhelpful think- ing'; 'illness symptoms'; and 'other people managing pain and fatigue management' and 'dealing with low mood'. Setting ‘homework’ tasks is a key area to explore difficulties in collaborative therapy relationships that require the patient to take an active role. Homework raises important issues such as the patient delaying the return of their homework because of concerns about ‘getting it wrong’ leading to criticism or embarrassment. This can be addressed during the GSH to pave the way for the more intensive MDT treatment. The phone calls are also important for bolstering the patient's motivation for change, by engaging them in the clini- cal formulation and collaborative empiricism, that is, devising and testing hypotheses (Beck & Wright, 1997). For patients who are able to work directly with cognitions, during discussion of a Thought Record, the therapist can gently probe and challenge the patient's unhelpful thoughts. The phone calls also provide an opportunity to check in with the patient about their feelings about the therapy and review progress. The patient is then encouraged to set and work to- wards early goals in their rehabilitation. Finally, attention is paid to the ending of this phase of treatment, which includes realisation that it is not a ‘cure all’, and the process of handing over care from the preparatory to the inpatient team. 3.2 \| The GSH modules Guided self-help videos and worksheets are structured into thematic modules. These are provided to patients electronically and supple- mented by phone calls from the GSH therapists. Video clinics are being developed to flexibly replace phone calls. As a patient pro- gresses through available modules, the programme is personalised for each patient by the therapist in terms of module ordering and rate of delivery. This is aimed at providing the best possible experi- ence, while managing the complex needs of this patient group. Each module comprises a video session accessed on YouTube and a set of associated worksheets for the patient to complete. These were pro- duced collaboratively by the therapy team to provide an original set of materials. The worksheets were designed to complement the vid- eos and were based, in part, on the 5-areas approach book (Williams et al., 2011), while respecting the copyright permissions given by its authors. The patients were invited to get hold of a copy of the book, and although this was ‘optional’, most of them did so. We now de- scribe a module that illustrates how a fairly standard CBT approach is finessed to address the needs of this patient group, a philosophy that pervades all preparatory work. The process of handover is one where the patient is encouraged to have a key role: rather than being passively ‘handed over’ within the treatment team, the patient is en- couraged to inform the inpatient clinicians who first meet them what they have learnt and achieved during the preparatory therapy. This is important both for informing the clinicians and, more importantly, for giving the patient a key responsibility for their self-management. The GSH therapists must build trust in a timely manner, explain- ing how the programme works and the importance of a collaborative therapeutic alliance. This also implies a therapy contract and out- lines the boundaries of therapy. During the crucial first few weeks, the GSH therapist must develop a rapport, which can be difficult for patients with a history of negative interactions with healthcare providers. Symptom diaries and worksheet exercises at the start of GSH ensure that the patient's narrative is ‘heard’, and their symp- toms are taken seriously. For patients who feel they have not been believed, this is an important component of the therapy. As well as psychoeducation, the therapist is checking patient acceptance of the diagnosis, since even patients who say they agree with the diagnosis of FNS often harbour doubts that their symptoms are really due to another condition, such as Lyme disease or mast cell activation syn- drome. This is part of the process of exploring the patient's beliefs about what has caused and/or is maintaining their symptoms. They may link them to a recent life event such as bereavement or work- place conflict. There may also be cultural issues such as a belief in evil spirits or that their symptoms are simply ‘God's will’. The topics covered are shown in Table 1 and include the following: introductory sessions on ‘What are FNS and how do they affect your life?’ and further topics including 'What is the five-areas approach?'; 'stress, 3.1 \| The Queen Square Guided Self- help programme 1. Definitive diagnosis of FNS by a neurologist. 2. Acceptance of the diagnosis, with no requests for further diag- nostic investigations. The QGSH is a course of Internet-based guided self-help. It runs for up to 12 weeks and includes original videos and patient work- sheets, as well as signposting to existing published resources such as Neurosymptoms.org. It involves therapists guiding the patient to use a range of psychoeducational resources and guides to simple therapy activities, supported by one-to-one contact, such as brief telephone calls, at sparse intervals (Cuijpers & Schuurmans, 2007). Resources, 3. Willingness to engage in MDT programme. 4. Ability to work with a goal-orientated approach. 5. No current litigation related to symptoms (though this is on a case-by-case basis). The MDT clinic assessment has a number of roles in addition to the usual clinical history taking. The lead clinician must build trust, HUMBLESTONE et al. 5 TA B LE 2 QGSH video titles 1. Introductory Session 1: What are functional neurological symptoms? 2. Introductory Session 2—Body, the role of the autonomic system and of stress, stress and symptoms 3. Goal setting 4. Introduction to the 5-areas approach (symptoms, behaviour and affect) 5. 5-areas approach—focus on cognitions—thinking and feelings 6. Anxiety and FNS 7. Fatigue and pain 8. Presentation of workings of the inpatient therapies and the MDT 9. Thinking about the self and others: Mentalisation for FNS 10. Mood problems 11. The role of medications 12. Avoidance in FNS 1. Introductory Session 1: What are functional neurological symptoms? 2. Introductory Session 2—Body, the role of the autonomic system and of stress, stress and symptoms 3. Goal setting 4. Introduction to the 5-areas approach (symptoms, behaviour and affect) 5. 5-areas approach—focus on cognitions—thinking and feelings 6. Anxiety and FNS 7. Fatigue and pain 8. Presentation of workings of the inpatient therapies and the MDT 9. Thinking about the self and others: Mentalisation for FNS 10. Mood problems 11. The role of medications 12. Avoidance in FNS 1. Introductory Session 1: What are functional neurological symptoms? 3. Goal setting 11. The role of medications In the ‘Anxiety and FNS’ module, the basic principles for dis- cussing anxiety are implemented as follows: (a) collaborative case 12. Avoidance in FNS 6 \| HUMBLESTONE et al. world such as a tree or a leaf or flower. They are asked to focus on this object and observe the fine details, then re-rate their anxiety on a 0–10 scale. The final exercise is a symptom diary that asks the patient to develop the habit of analysing their thoughts, emotions and behaviours at the point of symptom onset. conceptualisation, whereby patient and therapist look beyond the list of current symptoms to determine the predisposing, precipi- tating and perpetuating factors; and (b) collaborative empiricism, whereby patient and therapist pool their experience and knowledge in an ongoing process of generating and testing hypotheses. The ‘Anxiety & FNS’ module has been well received as many pa- tients with FNS have not previously made the link between anxi- ety, stress and their symptoms. Sharing the example of the dentist's waiting room can open up a fruitful discussion. During the course of the QGSH, some patients have expressed a wish to ‘see’ the thera- pist they are interacting with, and the talking head embedded in the video allows them to see the therapist talking through the slides. The format and delivery of this and other modules is subject to ongoing informal reviews. Some patients with FNS describe feelings and behaviours recog- nisable as ‘anxiety’, but they would not describe themselves as ‘anx- ious’, while some symptoms that clinicians recognise as anxiety are simply direct bodily experiences far from psychological concepts. ‘Anxiety’ in the context of FNS is complex and needs to be explored as it can be a triggering factor, or a consequence of symptoms. One patient may recall that everyone in the family was anxious as a re- sult of a tragedy such as the death of a child, while another may recall childhood anxiety alongside other difficulties such as elective mutism. Patients who have lived through trauma may be experienc- ing the symptoms of PTSD, while others may have social anxiety, specific phobias, obsessive–compulsive disorder, or panic disorder. Anxiety can also be a direct consequence of FNS. Symptoms such as intermittent leg weakness, numbness or paralysis can cause em- barrassment, anxiety, panic, and social isolation. Over time, this can drive negative cognitions and low mood. 3.2.1 \| Engagement with the QGSH In the 35 months the programme has run, from January 2017 to December 2019, 191 patients have taken part in the inpatient FNS programme, and 122 of these had taken part in the QGSH. The rate of completion of the QGSH varied between patients but, for these data, ‘taken part’ is defined as at least one email response by the patient. Demographic information is summarised in Table 3. The ‘Anxiety & FNS’ GSH component has two short videos and a worksheet. The videos are a presented as slides and a small ‘talking head’ in the corner (Figure 2). The sections are as follows: (a) What is anxiety and why is it a normal part of life?; (b) how anxiety manifests; (c) When is anxiety helpful and when is it not helpful?; (d) anxiety in people with FNS; (e) ways you can help yourself; (f) an example; and (g) a little exercise for you to do. The first video aims primarily to educate the patient, while the second video is more interactive. The concept of the vi- cious cycle that causes stress is worked through using an example. The viewer of the video is invited to think of the interplay of cog- nitions, moods and bodily symptoms they might experience whilst sitting in a dentist's waiting room. The last section, ‘a little exercise for you to do’, ties in with the accompanying worksheet (Appendix 1) and contains a short mindfulness task. The patient is invited to rate their anxiety on a 0–10 scale, then focus on something in the natural All patients referred to the IP programme were referred to the QGSH, but in small number of patients, there were issues with liter- acy, access or, unusually, urgency of admission, leaving no time for QGSH. Patients who did not respond to the invitation email never- theless progressed to the inpatient programme; that is, QGSH did not have a screening role. 3.2.5 \| The Clinician-Rated Outcome Measure The CROM has 15 items believed to address all elements of patient preparedness. They are as follows: (i) knowledge of FNS, (ii) engage- ment during the preparatory treatment, (iii) handover organisation, and (iv) overall competence for the inpatient therapy. Responses are given on an ordinal 5-point Likert scale ranging from strongly disa- gree (1) to strongly agree (5). attitude of staff. A number of patients talked about their previous negative experiences of diagnosis and unhelpful interactions with healthcare professionals. Conversations with the QGSH therapists explored these experiences and helped to reassure these patients. Although it was an ‘optional’ extra, almost all patients participating in the QGSH got hold of the book. The feedback from patients was overwhelmingly positive; that is, the QGSH had been helpful. CROM: findings The CROM was administered to 29 clinicians. While Sections 1 and 4 were answered positively overall with medians of 4, Section 3, de- scribing handover from the QGSH team, had a low score. It would appear that our handover notes were not received by all members of the inpatient team. The QGSH team is currently addressing this. 3.2.3 \| Preliminary assessment of the outcomes of Queen Square GSH We have embarked on a more comprehensive assessment of out- comes. Unfortunately, data collection was halted due to the COVID-19 pandemic. We present here a summary of our initial as- sessment of the outcome of delivering the QGSH to a sample of pa- tients. We developed two study-specific outcome measures, and the full details of the development and psychometric testing of these scales are outside the scope of this manuscript. We developed a PROM and a CROM. 3.2.4 \| The Patient-Rated Outcome Measure The PROM has 31 items rated using ordinal 5-point Likert scales of ‘strongly disagree’ to ‘strongly agree’, divided into four subsections: (A) knowledge of FNS, (B) experience using the PTP materials, (C) whether PTP helped the patient transition to the inpatient unit and (D) family involvement in FNS. 3.2.6 \| Summary of preliminary outcome data After developing the PROM and the CROM, we collected data from a sample of clinicians (CROM) and patients (PROM). Results for the PROM subsections: (A) knowledge of FNS, (B) experience using the PTP materials, (C) whether PTP helped the patient transition to the inpatient unit, and (D) family involvement in FNS, all were rated posi- tively by most patients except Section D, that is, questions about family involvement. Results for the CROM subsections, (i) knowl- edge of FNS, (ii) engagement during the preparatory treatment, (iii) handover organisation, and (iv) overall competence for the inpatient therapy, were positive except for (iii) handover to the inpatient team. We are making changes to address both of these areas. The QGSH requires fuller assessment of outcomes, and this is the focus of on- going research. TA B LE 3 Demographic data on patients who took part in the FNS programme Patients admitted to inpatient FNS programme 191 Male (%) 63 (33%) Female (%) 128 (67%) Patients who took part in GSH 122 TA B LE 3 Demographic data on patients who took part in the FNS programme most patients reported they were ‘uncertain’ about D, that is, fam- ily involvement in FNS, and this is an area we will address in future. \| Qualitative data When the online service was first used, patients reported informally that interacting with the therapists reduced their anxieties about FNS treatment, in particular their concerns about stigma and the FI G U R E 2 The first video for the ‘Anxiety and FNS’ module FI G U R E 2 The first video for the ‘Anxiety and FNS’ module \| 7 \| 7 HUMBLESTONE et al. REFERENCES American Psychiatric Association. (2013). Diagnostic and Statistical Manual of mental disorders, 5th edn. [Internet]. Arlington, TX: VA. Beck, J. S., & Wright, J. H. (1997). Book reviews journal of psychother- apy practice and research cognitive therapy: Basics and beyond. The Journal of Psychotherapy Practice and Research, 6, 71–72. Carson, A. J., Brown, R., David, A. S., Duncan, R., Edwards, M. J., Goldstein, L. H., Grunewald, R., Howlett, S., Kanaan, R., Mellers, J., Nicholson, T. R., Reuber, M., Schrag, A.-E., Stone, J., & Voon, V. (2012). Functional (conversion) neurological symptoms: Research since the millennium. Journal of Neurology, Neurosurgery and Psychiatry, 83, 842–850. https://doi.org/10.1136/jnnp-2011-301860 Carson, A., Hallett, M., & Stone, J. (2016). Assessment of patients with functional neurologic disorders. In M. Hallett J. Stone & A. Carson (Eds.), Handbook of clinical neurology (pp. 169–188). Elsevier B.V. Since the inception of the QGSH, we have found it to be an im- portant addition to the inpatient programme, providing significant benefits to patients. At the same time, determining the most appro- priate outcome measures for this heterogeneous group of patients is a work in progress. Many patients have reported that they have found the QGSH very helpful both in and of itself and in helping them to get the most out of the inpatient programme. The inpatient staff and therapists have also commented that they feel that pa- tients are better prepared to work with the MDT by the QGSH. Conwill, M., Oakley, L., Evans, K., & Cavanna, A. E. (2014). CBT-based group therapy intervention for nonepileptic attacks and other func- tional neurological symptoms: A pilot study. Epilepsy & Behavior, 34, 68–72. https://doi.org/10.1016/j.yebeh.2014.03.012 Cuijpers, P., & Schuurmans, J. (2007). Self-help interventions for anxi- ety disorders: An overview. Current Psychiatry Reports, 9, 284–290. https://doi.org/10.1007/s11920-007-0034-6 Dallocchio, C., Tinazzi, M., Bombieri, F., Arnó, N., & Erro, R. (2016). Cognitive behavioural therapy and adjunctive physical activity for functional movement disorders (conversion disorder): A pilot, single- blinded, randomized study. Psychotherapy and Psychosomatics, 85(6), 381–383. https://doi.org/10.1159/000446660 In terms of future prospects, the QGSH provides an excellent springboard for service developments. Prior to the inpatient ser- vice being paused because of the COVID-19 pandemic in 2020, a total of 10 to 15 patients were taking part in the QGSH at any one time, across the three therapists currently in the team. PROM: findings Data were collected from 19 patients. Two-thirds of patients re- sponded positively (agree or strongly agree) for sections A, B and C. Section ‘A’ was intended to test ‘knowledge of FNS’. This section was the most consistent across patients, with no scores below 3. Section ‘B’ was intended to test ‘experience using PTP materials’ and included six items that assess the patient's feelings about different elements of the PTP. The overall sectional average was positive. All the patients felt that accessing the videos was easy (item 6). Section ‘C’ had six items aimed at exploring the transition from outpatient to inpatient therapy, and most items were positive, with a median of 4 or 5. Section ‘D’ focused on family involvement in FNS treatment and was the largest section with 12 items. Most patients chose to select 3 (undecided). From additional qualitative comments elicited, The Queen Square Guided Self-Help programme is an Internet-based introduction to rehabilitation for functional neurological symptoms, delivered to a group of tertiary care patients with very significant disabilities, by experienced clinicians. Its main aim is to initiate pa- tients to a CBT-based rehabilitation approach, which offers its own clinical benefits but, crucially, mainly aims to optimise the efficacy of subsequent inpatient multidisciplinary treatment. The QGSH has been developed and applied since 2015 and is being evaluated and developed on an ongoing basis. It has not only a clinical impact, but also an academic and educational one, as its associated projects form an excellent arena for graduate student participation in service evaluation. ORCID Caroline Selai https://orcid.org/0000-0002-9698-3405 ORCID Caroline Selai https://orcid.org/0000-0002-9698-3405 The preparatory therapy includes a number of worksheets and videos, which are continuously reviewed to ensure that they are clear and easily intelligible. As it is common for this patient group to feel that they have been dismissed or rejected by healthcare pro- fessionals in the past, it is particularly important that the videos and worksheets do not use words or phrases which could be interpreted as disrespectful by patients. Part of the review process is there- fore to seek feedback that ensures that the language used helps patients feel understood and believed at all points throughout the programme. In the long term, it is aimed to make the materials easier to use through techniques such as making worksheets electronic for those patients who prefer them. This should keep in mind ease of ac- cess for patients with manual dexterity or (electronic) literacy issues. CONFLICTS OF INTEREST None of the authors have a conflict of interest to declare. None of the authors have a conflict of interest to declare. This consists of a ‘psychodynamic prism’ of thinking and un- derstanding. Taken together, these approaches are particularly im- portant for developing a positive therapeutic relationship, and so promoting patient engagement and satisfaction. 8 \| HUMBLESTONE et al. 8 support clinicians wishing to develop their expertise in treating FNS. It could help with the development of new specialist FNS services, for example for less disabled patients where QGSH can form the basis for completely stand-alone interventions where they have not existed before, something we are keen to support. We hope that the experience of our team can be used to help patients and clinicians to improve the provision of FNS services. Interested clinicians may contact us at UCLH.NHNN-GSHFNS@nhs.net to discuss access and use of the materials with potential for ongoing collaborations. The experience of the QGSH has highlighted how complex FNS can be to treat, particularly in a tertiary referral centre, and has em- phasised the importance of flexible, personalised therapy. To opti- mise patient engagement and treatment, it has been important for the QGSH therapists to be able to individualise the therapy as to the selection, order and rate of delivery of modules to fit the patient's needs. Based on the literature and our clinical experience, we have taken a cognitive rehabilitational approach, but our interactions with patients are also viewed within a psychodynamic lens; i.e. we consider transference and countertransference feelings and discuss them in clinical supervision. support clinicians wishing to develop their expertise in treating FNS. It could help with the development of new specialist FNS services, for example for less disabled patients where QGSH can form the basis for completely stand-alone interventions where they have not existed before, something we are keen to support. We hope that the experience of our team can be used to help patients and clinicians to improve the provision of FNS services. Interested clinicians may contact us at UCLH.NHNN-GSHFNS@nhs.net to discuss access and use of the materials with potential for ongoing collaborations. Gelauff, J., Stone, J., Edwards, M., & Carson, A. (2014). The prognosis of functional (psychogenic) motor symptoms: A systematic review. Journal of Neurology, Neurosurgery and Psychiatry, 85(2), 220–226. https://doi.org/10.1136/jnnp-2013-305321 Rommelfanger, K. S., Factor, S. A., LaRoche, S., Rosen, P., Young, R., & Rapaport, M. H. (2017). Disentangling stigma from functional neurological disorders: Conference report and roadmap for the future. Frontiers in Neurology, 8, 1–7. https://doi.org/10.3389/ fneur.2017.00106 Greiner, C., Schnider, A., & Leemann, B. (2016). Functional neurological disorders: A treatment-focused review. Swiss Archives of Neurology, Psychiatry and Psychotherapy, 167, pp. 234–240. Saifee, T. A., Kassavetis, P., Pareés, I., Kojovic, M., Fisher, L., Morton, L., Foong, J., Price, G., Joyce, E. M., & Edwards, M. J. (2012). Inpatient treatment of functional motor symptoms: A long-term fol- low-up study. Journal of Neurology, 259(9), 1958–1963. https://doi. org/10.1007/s00415-012-6530-6 Healthcare Improvement Scotland (2012). Stepped care for functional neurological symptoms. Heal Improv Scotl [Internet] (February). Hubschmid, M., Aybek, S., Maccaferri, G. E., Chocron, O., Gholamrezaee, Hubschmid, M., Aybek, S., Maccaferri, G. E., Chocron, O., Gholamrezaee, M. M., Rossetti, A. O., Vingerhoets, F., & Berney, A. (2015). Efficacy of brief interdisciplinary psychotherapeutic intervention for motor conversion disorder and nonepileptic attacks. General Hospital Psychiatry, 37(5), 448–455. https://doi.org/10.1016/j.genhosppsy ch.2015.05.007 Sattel, H., Lahmann, C., Gündel, H., Guthrie, E., Kruse, J., Noll-Hussong, M., Ohmann, C., Ronel, J., Sack, M., Sauer, N., Schneider, G., & Henningsen, P. (2012). Brief psychodynamic interpersonal psycho- therapy for patients with multisomatoform disorder: Randomised controlled trial. British Journal of Psychiatry, 200(1), 60–67. https:// doi.org/10.1192/bjp.bp.111.093526 Jordbru, A. A., Smedstad, L. M., Klungsøyr, O., & Martinsen, E. W. (2014). Psychogenic gait disorder: A randomized controlled trial of physi- cal rehabilitation with one -year follow -up. Journal of Rehabilitation Medicine, 46(2), 181–187. https://doi.org/10.2340/16501977-1246 Sharpe, M., Stone, J., Hibberd, C., Warlow, C., Duncan, R., Coleman, R., Roberts, R., Cull, R., Pelosi, A., Cavanagh, J., Matthews, K., Goldbeck, R., Smyth, R., Walker, A., Walker, J., MacMahon, A., Murray, G., & Carson, A. (2010). Neurology out-patients with symptoms unex- plained by disease: Illness beliefs and financial benefits predict 1- year outcome. Psychological Medicine, 40(4), 689–698. https://doi. org/10.1017/S0033291709990717 Karterud, H. N., Risør, M. B., & Haavet, O. R. (2015). The impact of con- veying the diagnosis when using a biopsychosocial approach: A qual- itative study among adolescents and young adults with NES (non- epileptic seizures). Seizure, 24, 107–113. https://doi.org/10.1016/j. seizure.2014.09.006 Sharpe, M., Walker, J., Williams, C., Stone, J., Cavanagh, J., Murray, G., Butcher, I., Duncan, R., Smith, S., & Carson, A. (2011). Guided self- help for functional (psychogenic) symptoms: A randomized controlled efficacy trial. Neurology, 77(6), 564–572. https://doi.org/10.1212/ WNL.0b013e318228c0c7 Lovell, K., & Richards, D. (2000). Multiple access points and levels of entry (MAPLE): Ensuring choice, accessibility and equity for CBT services. Behavioural and Cognitive Psychotherapy, 28(4), 379–391. https://doi.org/10.1017/S1352465800004070 Sojka, P., Bareš, M., Kašpárek, T., & Světlák, M. (2018). Processing of emotion in functional neurological disorder. Frontiers in Psychiatry, 9, 1–13. https://doi.org/10.3389/fpsyt.2018.00479 Marks, I.(2001). Living with fear: Marks, Isaac M.: 9780070403963: Amazon.com: Books, 2nd edn. [Internet]. McKenzie, P., Oto, M., Russell, A., Pelosi, A., & Neurology, R. D. (2010). Early outcomes and predictors in 260 patients with psychogenic nonepileptic attacks. Neurology, 74(1), 64–69. Stone, J., Carson, A., Duncan, R., Roberts, R., Warlow, C., Hibberd, C., Coleman, R., Cull, R., Murray, G., Pelosi, A., Cavanagh, J., Matthews, K., Goldbeck, R., Smyth, R., Walker, J., & Sharpe, M. (2010). Who is referred to neurology clinics? – The diagnoses made in 3781 new pa- tients. Clinical Neurology and Neurosurgery, 112(9), 747–751. https:// doi.org/10.1016/j.clineuro.2010.05.011 Neurosymptoms.org [Internet]. Retrieved from https://www.neurosympt oms.org/ Nicholson, T. R., Carson, A., Edwards, M. J., Goldstein, L. H., Hallett, M., Mildon, B., Nielsen, G., Nicholson, C., Perez, D. L., Pick, S., Stone, J., Anderson, D., Asadi-Pooya, A., Aybek, S., Baslet, G., Bloem, B. R., Brown, R. J., Chalder, T., Damianova, M., … Tinazzi, M. (2020). Outcome measures for functional neurological disorder: A review of the theoretical complexities. The Journal of Neuropsychiatry and Clinical Neurosciences, 32(1), 33–42. https://doi.org/10.1176/appi. neuropsych.19060128 Stone, J., Zeman, A., & Sharpe, M. (2002). Functional weakness and sen- sory disturbance. Journal of Neurology, Neurosurgery & Psychiatry. 73, 241–245. https://doi.org/10.1136/jnnp.73.3.241 Williams, C., Kent, C., Smith, S., Carson, A., Sharpe, M., & Cavanagh, J. (2011). Overcoming functional neurological symptoms: A five areas ap- proach. Overcoming Funct Neurol symptoms A five areas approach [Internet].Routledge. Nielsen, G. (2016). Physical treatment of functional neurologic disorders. Elsevier [Internet]. Nielsen, G., Ricciardi, L., Demartini, B., Hunter, R., Joyce, E., & Edwards, M. J. (2015). Outcomes of a 5-day physiotherapy programme for functional (psychogenic) motor disorders. Journal of Neurology, 262(3), 674–681. https://doi.org/10.1007/s00415-014-7631-1 REFERENCES COVID-19 has demonstrated the need for distanced and virtual approaches to reviewing and treating patients, including in cases where the risks of admission are unacceptable. The QGSH team are in discussions to develop GSH for FNS and medically unexplained symptoms as a stand-alone therapy, to be used as part of a ‘stepped-care’ approach. Even before the COVID-19 pandemic, the need for a service like this was clear from the increasing waiting list for the inpatient treatment, and there is evidence that GSH approaches can be effective in treat- ing anxiety disorders and FNS. de Schipper, L. J., Vermeulen, M., Eeckhout, A. M., & Foncke, E. M. J. (2014). Diagnosis and management of functional neurological symp- toms: The Dutch experience. Clinical Neurology and Neurosurgery, 122, 106–112. https://doi.org/10.1016/j.clineuro.2014.04.020 Demartini, B., Batla, A., Petrochilos, P., Fisher, L., Edwards, M. J., & Joyce, E. (2014). Multidisciplinary treatment for functional neurological symptoms: A prospective study. Journal of Neurology, 261(12), 2370– 2377. https://doi.org/10.1007/s00415-014-7495-4 Ding, J. M., & Kanaan, R. A. A. (2016). What should we say to patients with unexplained neurological symptoms? How explanation affects offence. Journal of Psychosomatic Research, 91, 55–60. https://doi. org/10.1016/j.jpsychores.2016.10.012 org/10.1016/j.jpsychores.2016.10.012 Edwards, M. J. (2016). Functional neurological symptoms: Welcome to the new normal. Practical Neurology, 16, 2–3. https://doi.org/10.1136/ practneurol-2015-001310 Gates, J. R. (2002). Non-epileptic seizures: Classification co-existence with epilepsy: Diagnosis, therapeutic approaches and consensus. Epilepsy and Behavior, 3, 28–33. We believe that the set of materials used in QGSH has the po- tential to provide significant benefits for patients with FNS and can 9 HUMBLESTONE et al. \| 9 AUTHOR BIOGR APHIES Ms. Sue Humblestone is Senior Occupational Therapist working as a mental health specialist in a national neuropsychiatry de- partment for 20 years. She has a special interest and experience in the psychiatric presentations of neurological conditions and the treatment of patients with functional neurological symptoms. Nielsen, G., Stone, J., & Edwards, M. J. (2013). Physiotherapy for func- tional (psychogenic) motor symptoms: A systematic review. Journal of Psychosomatic Research, 75, 93–102. https://doi.org/10.1016/j.jpsyc hores.2013.05.006 O’Neal, M. A., & Baslet, G. (2018). Treatment for patients with a func- tional neurological disorder (conversion disorder): An integrated ap- proach. American Journal of Psychiatry, 175(4), 307–314. https://doi. org/10.1176/appi.ajp.2017.17040450 Dr. Jacob Roelofs is currently Specialist Registrar in Neurology Training in Newcastle. He trained in Cambridge and London, achieving a distinction in clinical practice. He has an interest in functional neurological symptoms. He has worked as a BMA junior doctor representative and chaired a Trust-wide Junior Doctors Forum. He completed an MSc in Clinical Neuroscience at the Institute of Neurology at Queen Square, gaining a distinction. Pick, S., Goldstein, L. H., Perez, D. L., & Nicholson, T. R. (2019). Emotional processing in functional neurological disorder: A review, biopsychoso- cial model and research agenda. Journal of Neurology, Neurosurgery & Psychiatry, 90(6), 704–711 https://doi.org/10.1136/jnnp-2018-319201 Reuber, M., Mitchell, A. J., Howlett, S. J., Crimlisk, H. L., & Grünewald, R. A. (2005). Functional symptoms in neurology: Questions and an- swers. Journal of Neurology, Neurosurgery and Psychiatry, 76, 307–314. https://doi.org/10.1136/jnnp.2004.048280 HUMBLESTONE et al. 10 Dr. Caroline Selai is Chartered Psychologist and Associate Fellow of the British Psychological Society (BPS). She is cur- rently Associate Professor at UCL Queen Square Institute of Neurology and Honorary Psychologist at the National Hospital for Neurology and Neurosurgery, working clinically in a liaison role with the clinical departments of Neuropsychiatry and Uro- neurology. She has an interest in medically unexplained symp- toms and functional neurological symptoms. She is interested in the clinical formulation of complex presentations. She is cur- rently Student Member of the BACP. How to cite this article: Humblestone, S., Roelofs, J., Selai, C., & Moutoussis, M. (2021). Functional neurological symptoms: Optimising efficacy of inpatient treatment and preparation for change using the Queen Square Guided Self-Help. Counselling and Psychotherapy Research, 00, 1–12. https://doi. org/10.1002/capr.12438 Dr. Michael Moutoussis is Clinical Lecturer in Neuroscience and Honorary Consultant Medical Psychotherapist. He has worked for many years in medical psychotherapy. His areas of interest include functional somatic symptoms, psychotic symptoms and so-called personality disorder. He has a special research interest in stakeholder involvement in research, especially in researching engagement and disengagement with therapy. He holds a PhD in the psychology of paranoid ideation and its relationship to how we value ourselves. He has been a key contributor to the new field of computational psychiatry, wherein he strongly advocates for basic research subserving psychological therapies. He is a member of the UKCP. \| 11 11 HUMBLESTONE et al. 11 ANXIETY & FNS: WORKSHEET • Can you think of some thoughts? Please note them down on your worksheet. Th h h l d b dil i Thinking about anxiety: A guide for patients with functional neu- rological symptoms (GNS): To accompany videos 1 & 2 These thoughts lead to bodily sensations. Thinking about anxiety: A guide for patients with functional neu- rological symptoms (GNS): To accompany videos 1 & 2 • Can you think of some of the bodily sensations? Please note them down. down. This might affect your mood. This is a worksheet to accompany a section of our Guided Self-Help (GSH) on the topic: This is a worksheet to accompany a section of our Guided Self-Help (GSH) on the topic: This might affect your mood. • Can you think of ways your mood might be affected? Please note these down. Thinking about anxiety: A guide for patients with Functional Neurological Symptoms (GNS) Finally all this might affect your behaviour. Finally all this might affect your behaviour. There are two videos: Part 1 & Part 2. What might you do..? Please note this down. What might you do..? Please note this down. As you watch the videos you will see reference made to three tasks Task 2 When you are feeling stressed or anxious or panicky: 1. Task 1: Asks you to imagine going to an appointment with your dentist and to write down your thoughts. Rate your feelings on a 0–10 scale: Write down the number………. Spend a few minutes doing something relaxing such as connecting with something in the natural world. Look at a tree or a flower. What do you notice about the leaves, the petals..? Breathe deeply. Hold your breath and count: 1, 2, 3. Now breathe out. Rate your feelings on a 0–10 scale: Write down the number………. Rate your feelings on a 0–10 scale: 2. Task 2: Invites you to rate how you are feeling on a 0–10 scale. Write down the number………. b. There are no right or wrong answers c. You might like to get a pen or pencil now so you have some- thing ready to write with! d. Are you ready to watch the videos..? e. Press Play and let's begin! e. Press Play and let's begin! 12 \| APPENDIX 2 HUMBLESTONE et al. 12 DSM V Criteria DSM V Criteria DSM V Criteria Task 1: In the video you are invited to think of a time when you had an appointment with your dentist. Here is the scenario Please keep a diary over the next week. Each time you feel ‘panicky’ please note down your: appointment with your dentist. Here is the scenario You made an appointment with your dentist because of a problem with your tooth. You are in the dentist's waiting room and you will soon be called in. You can smell the mouthwash and you remem- ber that you are always invited to rinse your mouth at the end of treatment. You start to have automatic thoughts. Time when I felt panicky (date) Thoughts Emotions Physical sensations Behaviour
https://openalex.org/W3198555927	https://www.frontiersin.org/articles/10.3389/fmed.2021.716816/pdf	English	null	Selenium-Enriched Lactobacillus acidophilus Ameliorates Dextran Sulfate Sodium-Induced Chronic Colitis in Mice by Regulating Inflammatory Cytokines and Intestinal Microbiota	Frontiers in medicine	2,021	cc-by	7,569	Selenium-Enriched Lactobacillus acidophilus Ameliorates Dextran Sulfate Sodium-Induced Chronic Colitis in Mice by Regulating Inﬂammatory Cytokines and Intestinal Microbiota Zeyu Wu 1, Dan Pan 2, Min Jiang 1, Lixuan Sang 3 and Bing Chang 1* 1 Department of Gastroenterology, First Afﬁliated Hospital of China Medical University, Shenyang, China, 2 Department of Geriatrics, First Afﬁliated Hospital of China Medical University, Shenyang, China, 3 Department of Gastroenterology, Shengjing Hospital of China Medical University, Shenyang, China Aim: To evaluate the effect of Selenium-enriched Lactobacillus acidophilus (Se-enriched L. acidophilus) on dextran sulfate sodium (DSS)-induced colitis in mice. ORIGINAL RESEARCH published: 31 August 2021 doi: 10.3389/fmed.2021.716816 Citation: Wu Z, Pan D, Jiang M, Sang L and Chang B (2021) Selenium-Enriched Lactobacillus acidophilus Ameliorates Dextran Sulfate Sodium-Induced Chronic Colitis in Mice by Regulating Inﬂammatory Cytokines and Intestinal Microbiota. Front. Med. 8:716816. doi: 10.3389/fmed.2021.716816 Edited by: Methods: Mice were randomly divided into four groups: a control group, a control + Se-enriched L. acidophilus group, a chronic colitis group, and a chronic colitis + Se- enriched L. acidophilus group (n = 10 each group). The mice were sacriﬁced on the 26th day. The disease activity index, survival rates, and histological injury score were determined. Cytokines produced by lamina propria lymphocytes (LPLs), the selenium (Se) concentrations in serum and colon tissue and the mouse intestinal microbiota were evaluated. Yuji Naito, Kyoto Prefectural University of Medicine, Japan Enzo Ieardi, University of Bari Aldo Moro, Italy Chaﬁa Boukoffa Touil-Boukoffa, University of Science and Technology Houari Boumediene, Algeria Yuji Naito, Kyoto Prefectural University of Medicine, Japan Enzo Ieardi, University of Bari Aldo Moro, Italy Chaﬁa Boukoffa Touil-Boukoffa, University of Science and Technology Houari Boumediene, Algeria Results: Se-enriched L. acidophilus can improve histological injury and the disease activity index in mice with chronic colitis and reduce IL-1β, IL-6, IL-12p70, TNF-α, IL-23, IFN-γ, IL-17A, and IL-21 (P < 0.05) and increase IL-10 (P < 0.05) expression levels. Moreover, Se-enriched L. acidophilus can increase the β diversity of intestinal microbiota in mice with chronic colitis, signiﬁcantly reduce the relative abundance of Lactobacillus and Romboutsia (P < 0.05), and signiﬁcantly increase the relative abundance of Parasutterella (P < 0.05). Correspondence: Bing Chang cb000216@163.com Specialty section: This article was submitted to Gastroenterology, a section of the journal Frontiers in Medicine Conclusions: Se-enriched L. acidophilus can improve DSS-induced chronic colitis by regulating inﬂammatory cytokines and intestinal microbiota. Conclusions: Se-enriched L. acidophilus can improve DSS-induced chronic colitis by regulating inﬂammatory cytokines and intestinal microbiota. Received: 29 May 2021 Accepted: 05 August 2021 Published: 31 August 2021 Keywords: inﬂammatory bowel disease, ulcerative colitis, Se-enriched Lactobacillus acidophilus, intestinal ﬂora, molecular pathological epidemiology Keywords: inﬂammatory bowel disease, ulcerative colitis, Se-enriched Lactobacillus acidophilus, intestinal ﬂora, molecular pathological epidemiology Edited by: Giuseppe Losurdo, University of Bari Medical School, Italy Reviewed by: Yuji Naito, Kyoto Prefectural University of Medicine, Japan Enzo Ieardi, University of Bari Aldo Moro, Italy Chaﬁa Boukoffa Touil-Boukoffa, University of Science and Technology Houari Boumediene, Algeria Correspondence: Bing Chang cb000216@163.com Cell Preparation, Culture, and Activation Cell Preparation, Culture, and Activation The large intestine of each mouse was cut into 1- to 2-mm small pieces. The pieces were stirred twice in PBS containing 3 mmol/L EDTA for 15 min each and twice in RPMI 1640 (HyClone) containing 1 mmol/L EGTA for 20 min each to remove epithelium at 37◦C. The remaining pieces were stirred in RPMI 1640 (HyClone) containing 20% fetal bovine serum, 100 U/ml collagenase (C2139; Sigma-Aldrich Corp., St. Louis, MO, United States) and 5 U/ml DNase1 (Sigma-Aldrich Corp) at 37◦C for 90 min. The suspensions were centrifuged, and the particles were cleaned. Lamina propria lymphocytes (LPLs) were isolated from lamina propria (LP) cell preparations by 45-66.6% Induction of Chronic DSS Colitis Colitis was induced in the mice by oral administration of 1.5% DSS (molecular mass 36-50 kDa; MP Biomedicals, Solon, OH, United States) on days 0-5, 10-15, and 20-25 d and tap water on the other days (26). The mice were sacriﬁced on 26th day. Selenium (Se) is an important trace element in the human body that has antioxidant and anti-inﬂammatory eﬀects and has an important inﬂuence on human immunity (16, 17). Clinical studies have found that compared with healthy people, CD patients exhibit signiﬁcantly reduced concentrations of selenoprotein P and Se (18, 19), and the concentration of Se in UC patients is also signiﬁcantly reduced (20). Moreover, it was reported that sodium selenite can alleviate DSS-induced colitis in mice (21). INTRODUCTION Inﬂammatory bowel disease (IBD) is a chronic recurrent inﬂammatory disease of the intestine that mainly includes two forms, ulcerative colitis (UC) and Crohn’s disease (CD) (1), and its prevalence is increasing annually (2). The pathogenesis of IBD is not fully understood. Genes, immunity, intestinal ﬂora, and the environment are all involved in the pathogenesis of IBD (3). There are a August 2021 \| Volume 8 \| Article 716816 Frontiers in Medicine \| www.frontiersin.org Colitis and Selenium-Enriched Lactobacillus acidophilus Wu et al. large number of microbiota in the human intestine, which has an important impact on the human body, and disorders of the intestinal ﬂora are considered to be closely related to the occurrence and development of IBD (4). Studies have shown that the treatment methods for regulating the intestinal ﬂora such as fecal bacteria transplantation (FMT) (5–7), and VSL#3 probiotic treatment (8, 9) could be used in the treatment of ulcerative colitis. Animal Ethics Committee and Animal Care Committee of China Medical University. Ethics batch number: 2019069. Experimental Design g Forty mice were randomly divided into four groups: 10 in the control group (group A), 10 in the control + Se-enriched L. acidophilus group (group B), 10 in the chronic colitis group (group C), and 10 in the chronic colitis + Se-enriched L. acidophilus group (group D). The control group was given a normal diet and tap water, with normal saline gavage once a day. The control + Se-enriched L. acidophilus group was given a normal diet and tap water, with Se-enriched L. acidophilus (100 mg/kg) gavage once a day. The chronic colitis group was induced colitis by 1.5% DSS and given a normal diet with saline gavage once a day. The chronic colitis + Se-enriched L. acidophilus group was induced colitis by 1.5% DSS and given a normal diet with Se-enriched L. acidophilus (100 mg/kg) gavage once a day. Weight and disease activity index were recorded every day. Lactobacillus acidophilus (L. acidophilus) is an important probiotic (10) that has a certain therapeutic eﬀect on many diseases. L. acidophilus can alleviate the pain caused by osteoarthritis and delay the progression of osteoarthritis by reducing the destruction of cartilage and inhibiting the production of proinﬂammatory cytokines (11). It also has a certain relieving eﬀect on type 2 diabetes (12). Obesity and fatty liver caused by diet can also be relieved by L. acidophilus through improving fat metabolism and insulin sensitivity (13). L. acidophilus can also inhibit endoplasmic reticulum stress (ER), thereby alleviating intestinal inﬂammation (14). In addition, evodiamine can relieve dextran sulfate sodium (DSS)-induced colitis by increasing L. acidophilus in the intestine (15). EXPERIMENTAL METHOD Experimental Animals and Probiotics Disease Activity Index The disease activity index was used to assess the severity of colitis in mice. It consists of three parts, the percentage of weight loss (0-4 points), stool consistency (0-4 points), and intestinal bleeding (0-4 points) (26), as shown in Table 1. After the mice were sacriﬁced, the colon tissue was ﬁxed with 4% paraformaldehyde, embedded in paraﬃn, cut into 4-µm sections, stained with hematoxylin and eosin, and scored for histological damage. Histological scores were assessed by two pathologists independently in a blinded fashion. The histological scores were obtained by calculating the sum of scores of inﬂammation severity, degree of mucosal damage, percentage of crypt damage, and pathological change range. The none, mild, moderate, or severe inﬂammation was quantiﬁed as to the percentage involvement by the inﬂammation (none, 0-33%, 33-67%, 67- 100%). Depth of inﬂammation (none, mucous layer, submucosa, muscularis, and serosa) represented the mucosal damage, as shown in Table 2 (26). The preparation of Se-enriched probiotics adopts the biological transformation method, in which inorganic Se is added during the probiotic culture process, and the probiotics take up inorganic Se and convert it into organic Se, which is then transformed into Se-enriched probiotics (22). Studies have found that Se-enriched probiotics can reduce liver damage induced by carbon tetrachloride (23, 24). The anti-inﬂammatory and antioxidant eﬀects of Se-enriched probiotics can also improve the liver damage induced by heat stress in rats (25). Since both Se and probiotics alleviate intestinal inﬂammation, Se-enriched probiotics may alleviate intestinal inﬂammation. Therefore, our study established a DSS-induced mouse colitis model to study the eﬀect of Se-enriched L. acidophilus on intestinal inﬂammation and its possible mechanism. DNA Sequencing and Analysis According to the manufacturer’s instructions, cell culture supernatants were collected after centrifugation at 1,000 rpm for 10 min, and cytokine concentrations were measured using mouse immunoassay kits (R&D Systems Inc., Minneapolis, MN, United States). The levels of TNF-α, IL-1β, IL-6, IL-23, and IL-12p70 were measured in the supernatants without anti- CD3/anti-CD28 monoclonal antibody stimulation. The levels of IFN-γ, IL-17A, IL-22, IL-21, and IL-10 were measured in the supernatants with or without anti-CD28/anti-CD3 monoclonal antibody stimulation (26). Realbio Genomics Institute (Shanghai,China) performed DNA sequencing and analysis. According to the manufacturer’s instructions, the PCR products were extracted from 2% agarose gels and puriﬁed using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, U.S.). Amplicons were quantiﬁed using Qubit 2.0 (Invitrogen, U.S.). All quantiﬁed amplicons were pooled to equalize concentrations in order to sequence using Illumina HiSeq/MiSeq (Illumina, Inc., CA, USA) and PANDAseq (https://github.com/neufeld/ pandaseq, version 2.9) was used to overlap the paired end reads of 250bp on their 3 ends for concatenation into original longer tags. Determination of Selenium in Serum and Colon Tissue g g g OTUs were clustered according to 97% similarity using UPARSE (http://drive5.com/uparse/), and USEARCH (version 7.0.1090) was used to identify and remove chimeric sequences. Each representative sequence was annotated by RDP Classiﬁer (http://rdp.cme.msu.edu/) based on RDP Database. The OTU proﬁling table and alpha diversity indices (including Chao1 index, Shannon index, Simpson index, observed species index, and PD-whole-tree diversity index) were achieved by the Python scripts of QIIME (version 1.9.1). Principal coordinate analysis (PCoA) based on weighted UniFrac distance and the Adonis test were implemented by R software (version 3.5.1). The microbiota diﬀerences between diﬀerent groups were analyzed with linear discriminant analysis eﬀect size (LEfSe) analysis software. The The selenium content in colon tissue was determined by ﬂuorescence atomic absorption spectrometry. Serum selenium concentrations were detected in duplicate by inductively coupled plasma mass spectrometry (ICP-MS, Perkin-Elmer SCIEX ElAN 6000, US) (21). Experimental Animals and Probiotics p Forty 8-week-old speciﬁc pathogen-free C57BL/6 male mice were purchased from Liaoning Changsheng Biology, each weighing 22 ± 2 g and bred under speciﬁc pathogen-free conditions (temperature 21-25◦C, humidity 50-60%, and a 12 h light/12 h dark-light regimen). Se-enriched L. acidophilus is a freeze-dried powder produced by the Immunology Laboratory of China Medical University. Each gram of freeze-dried powder contains Se-enriched L. acidophilus 5 × 10 ∼9 cfu, and the selenium content is 0.30 mg/g. The research protocol was approved by the August 2021 \| Volume 8 \| Article 716816 Frontiers in Medicine \| www.frontiersin.org 2 Colitis and Selenium-Enriched Lactobacillus acidophilus Wu et al. TABLE 1 \| Disease activity index (DAI) score chart. Score Weight loss (%) Stool property Bleeding 0 0 Normal Normal 1 >0-5 2 >5-10 Loose Fecal occult blood 3 >10-15 4 >15 Diarrhea Bleeding TABLE 2 \| Histological injury score chart. Grade 0 1 2 3 4 Inﬂammation None Mild Moderate Severe - Mucosal damage None Mucous layer Submucosa Muscularis and serosa - Crypt damage None 1/3 2/3 100% 100% with epithelium loss Pathological change range None 0-25% 26-50% 51-75% 76-100% discontinuous Percoll (Solarbio) gradient centrifugation at 2,500 rpm for 20 min (26). FAST DNA Stool Mini Kit (Item No. 51604, Qiagen, Germany) according to the instructions. The integrity and concentration of total DNA were quality tested by a Thermo NanoDrop 2000 UV spectrophotometer and 1% agarose gel electrophoresis. Primers 341F 5′-CCTACGGGRSGCAGCAG-3′ and 806R 5′- GGACTACVVGGGTATCTAATC-3′ (with a speciﬁc barcode in the primer) were used to amplify the V3-V4 region of the bacterial 16 s ribosomal RNA gene by PCR(95◦C for 3 min, followed by 30 cycles at 98◦C for 20 s, 58◦C for 15 s, and 72◦C for 20 s and a ﬁnal extension at 72◦C for 5 min), and ampliﬁed fragments of approximately 500 bp were obtained. In an atmosphere containing 5% CO2, LPLs(1 × 105/well in 0.2 ml of RPMI 1640 medium containing 10% fetal bovine serum, 1% penicillin, and 1% streptomycin) were cultured in 96-well plates coated with anti-CD3 (10 µg/ml e-Bioscience, San Diego, CA, United States) and soluble anti-CD28 (1 µg/ml, e- Bioscience) mAbs for 48 h at 37◦C. After 48 h, the supernatants were collected, and the cytokine concentrations were determined by enzyme-linked immunosorbent assay (26). DNA Extraction and Ampliﬁcation The fecal samples of mice were transported to laboratory within 2 h with an ice pack. All samples were frozen immediately then and stored at −80◦C. Realbio Genomics Institute (Shanghai, China) performed DNA extraction and ampliﬁcation. The microbial DNA of the samples was extracted by a QIAamp August 2021 \| Volume 8 \| Article 716816 Frontiers in Medicine \| www.frontiersin.org 3 Wu et al. Colitis and Selenium-Enriched Lactobacillus acidophilus FIGURE 1 \| (A) Survival rates between the chronic colitis group and the chronic colitis + Se-enriched L. acidophilus group (n = 10); (B) Chronic colitis group and chronic colitis + Se-enriched L. acidophilus group DAI scores (n = 8); (C) H&E staining of colon tissue of four groups. (200×) (Control) There were no inﬂammatory cells inﬁltration. (Control+ Se-enriched L. acidophilus) There were no inﬂammatory cells inﬁltration. (Chronic colitis) Numerous neutrophil and mononuclear cells inﬁltration could be found. (Chronic colitis + Se-enriched L. acidophilus) There were fewer neutrophil and mononuclear cells inﬁltration than chronic colitis group. (D) Histological injury scores between the chronic colitis group and the chronic colitis + Se-enriched L. acidophilus group (n = 5). Data are expressed as the mean ± standard error (P < 0.05). FIGURE 1 \| (A) Survival rates between the chronic colitis group and the chronic colitis + Se-enriched L. acidophilus group (n = 10); (B) Chronic colitis group and chronic colitis + Se-enriched L. acidophilus group DAI scores (n = 8); (C) H&E staining of colon tissue of four groups. (200×) (Control) There were no inﬂammatory cells inﬁltration. (Control+ Se-enriched L. acidophilus) There were no inﬂammatory cells inﬁltration. (Chronic colitis) Numerous neutrophil and mononuclear cells inﬁltration could be found. (Chronic colitis + Se-enriched L. acidophilus) There were fewer neutrophil and mononuclear cells inﬁltration than chronic colitis group. (D) Histological injury scores between the chronic colitis group and the chronic colitis + Se-enriched L. acidophilus group (n = 5). Data are expressed as the mean ± standard error (P < 0.05). score of the chronic colitis + Se-enriched L. acidophilus group signiﬁcantly decreased (P < 0.05), as shown in Figure 1. correlations between microbiota and cytokines were analyzed by R software (version 3.5.1). Data Analysis The data are expressed as the mean ± standard error, and the Shapiro Wilk test was used for normality analysis. If the data conformed to a normal distribution and homogeneity of variance, analysis of variance or t-test was used. If the data conformed to a normal distribution and uneven variance, the Welch test or t’ test was used. If the data did not conform to a normal distribution, a non-parametric test was used. P < 0.05 indicated that the diﬀerence was statistically signiﬁcant. SPSS version 22.0 (SPSS, Inc., Chicago, IL, United States) was used for data analysis, and GraphPad Prism 6.0 (GraphPad Software, Inc., La Jolla, CA, United States) was used for drawing. y Detection of the cytokine concentrations in the supernatant (ﬁve samples per group) revealed that IL-1β, IL-6, IL-12p70, TNF- α, and IL-23 were signiﬁcantly decreased in the chronic colitis + Se-enriched L. acidophilus group compared with the chronic colitis group (P < 0.05). Regardless of whether there was anti- CD3/CD28 antibody stimulation, IFN-γ, IL-17A, and IL-21 were signiﬁcantly decreased (P < 0.05), and IL-10 was signiﬁcantly increased in the chronic colitis + Se-enriched L. acidophilus group compared with the chronic colitis group (P < 0.05). Only the concentration of IL-22 between the two groups was not statistically signiﬁcant. Compared with the chronic colitis group, serum and colon tissue Se concentrations were signiﬁcantly higher in the chronic colitis + Se-enriched L. acidophilus group (P < 0.01), as shown in Figure 2. Se-Enriched L. acidophilus Regulates Intestinal Microbiota The eﬀect of Se-enriched L. acidophilus on DSS colitis was compared by the diﬀerences in survival rate, DAI score and colon histology between the two groups. The chronic colitis group and the chronic colitis + Se-enriched L. acidophilus group had similar survival rates (P > 0.05). The DAI score of the chronic colitis + Se-enriched L. acidophilus group decreased signiﬁcantly on the 25th and 26th days (P < 0.05), and the histological injury Se-enriched L. acidophilus has regulatory eﬀects on the intestinal microbiota. The feces of mice in the control, control + Se- enriched L. acidophilus, chronic colitis, and chronic colitis + Se- enriched L. acidophilus groups were collected (ﬁve samples per group). There were no signiﬁcant diﬀerences in the α diversity August 2021 \| Volume 8 \| Article 716816 Frontiers in Medicine \| www.frontiersin.org 4 Colitis and Selenium-Enriched Lactobacillus acidophilus Wu et al. FIGURE 2 \| Cytokine concentrations produced by LPL cells and Se concentrations in serum and colon tissue. (A) Unstimulated cells; (B) LPL cells with or without anti-CD3 and anti-CD28 mAbs; (C) Se concentrations in serum and colon tissue. The values are expressed as the mean ± standard error. (P < 0.01) (n = 5). FIGURE 2 \| Cytokine concentrations produced by LPL cells and Se concentrations in serum and colon tissue. (A) Unstimulated cells; (B) LPL cells with or without anti-CD3 and anti-CD28 mAbs; (C) Se concentrations in serum and colon tissue. The values are expressed as the mean ± standard error. (P < 0.01) (n = 5). among the four groups, Chao1 index (P > 0.05), Shannon index (P > 0.05), Simpson index (P > 0.05), observed species index (P > 0.05), or PD−whole−tree diversity index (P > 0.05), as shown in Figure 3. There was a signiﬁcant diﬀerence in microbial β diversity between the control group and the chronic colitis group (P < 0.05). There was also a signiﬁcant diﬀerence in microbial β diversity between the chronic colitis group and the chronic colitis + Se-enriched L. acidophilus group (P < 0.05), as shown in Figure 4. rich in Bacteroidetes (P < 0.05), and the chronic colitis group was rich in Firmicutes and Tenericutes (P < 0.05), as shown in Figure 6. Frontiers in Medicine \| www.frontiersin.org Correlation Analysis Indicated That Many Species Were Correlated With Cytokines acidophilus could alleviate DSS-induced colitis in mice, reduce inﬂammatory cytokines produced by LPL cells, decrease the relative abundance of Romboutsia and Lactobacillus. In order to identify whether the alteration of microbiota were related to the cytokines, correlation analysis was performed. Akkermansia was positive related to IL-10 (P < 0.05). Romboutsia was positive related to TNF-α, IL-1β, IL-6, IL-23, IL-12p70, IFN-γ, IL-17A, IL-22, IL-21, and IL-10, as shown in Figure 8. The serum Se concentration of IBD patients decreased (27). A Korean study showed that 30.9% of IBD patients had Se deﬁciency (28). Although the details of the relationship between Se and IBD still need further elucidation, animal studies have found that Se can increase CD4 (+) CD25 (+) regulatory T cells and reduce Th1, Th17, and γδ T cells, thus alleviating DSS- induced colitis (26). Se can transform M1 macrophages into M2 macrophages (29). M1 macrophages promote the development of inﬂammation, and M2 macrophages have anti-inﬂammatory eﬀects (30). Se-Enriched L. acidophilus Regulates Intestinal Microbiota (A) The difference in β diversity between the control group (group A) and the chronic colitis group (group C) was statistically signiﬁcant (P = 0.032 < 0.05); (B) The difference in β diversity between the chronic colitis group (group C) and the chronic colitis + Se-enriched L. acidophilus group (group D) was statistically signiﬁcant (P = 0.019 < 0.05). FIGURE 5 \| The relative abundance of each microorganism in each group. (A) Genus level; (B) Phylum level. FIGURE 4 \| Comparison of β diversity among the control group (group A), the chronic colitis group (group C), and the chronic colitis + Se-enriched L. acidophilus group (group D). (A) The difference in β diversity between the control group (group A) and the chronic colitis group (group C) was statistically signiﬁcant (P = 0.032 < 0.05); (B) The difference in β diversity between the chronic colitis group (group C) and the chronic colitis + Se-enriched L. acidophilus group (group D) was statistically signiﬁcant (P = 0.019 < 0.05). FIGURE 4 \| Comparison of β diversity among the control group (group A), the chronic colitis group (group C), and the chronic colitis + Se-enriched L. acidophilus group (group D). (A) The difference in β diversity between the control group (group A) and the chronic colitis group (group C) was statistically signiﬁcant (P = 0.032 < 0.05); (B) The difference in β diversity between the chronic colitis group (group C) and the chronic colitis + Se-enriched L. acidophilus group (group D) was statistically signiﬁcant (P = 0.019 < 0.05). FIGURE 5 \| The relative abundance of each microorganism in each group. (A) Genus level; (B) Phylum level. FIGURE 5 \| The relative abundance of each microorganism in each group. (A) Genus level; (B) Phylum level. FIGURE 5 \| The relative abundance of each microorganism in each group. (A) Genus level; (B) Phylum level. Se-Enriched L. acidophilus Regulates Intestinal Microbiota At the genus level, compared with the chronic colitis group, the control group was rich in Helicobacter, Rikenella, Barnesiella, and Enterorhabdus, while Turicibacter, Romboutsia, Escherichia_Shigella, Clostridium sensu stricto, Butyricimonas, Parasutterella, Biﬁdobacterium, Allobaculum, Clostridium IV, Anaeroplasma, Intestinimonas, and Clostridium XVIII were signiﬁcantly reduced (P < 0.05). The relative abundances of Lactobacillus and Romboutsia in the chronic colitis + Se- enriched L. acidophilus group signiﬁcantly decreased (P < 0.05), and the relative abundance of Parasutterella signiﬁcantly increased (P < 0.05), as shown in Figure 6. The relative abundance of Akkermansia increased, although the diﬀerence was not statistically signiﬁcant [LDA score(log10) <2], as shown in Figures 5, 7. To identify the diﬀerences in microbiota between diﬀerent groups, we conducted LEfSe analysis of the dominant ﬂora between diﬀerent groups. There were diﬀerences in the composition of intestinal microbiota between the control group and the chronic colitis group and between the chronic colitis group and the chronic colitis + Se-enriched L. acidophilus group, as shown in Figure 5. At the phylum level, the control group was August 2021 \| Volume 8 \| Article 716816 5 Colitis and Selenium-Enriched Lactobacillus acidophilus Wu et al. 3 \| α diversity index. (A) Chao1 index: the differences between the four groups were not statistically signiﬁcant; (B) Shannon index: the differences between ups were not statistically signiﬁcant; (C) Simpson index: the differences between four groups is not statistically signiﬁcant; (D) Observed-species diversity: the es between four groups were not statistically signiﬁcant; (E) PD-whole-tree index: the differences between the four groups were not statistically signiﬁcant. n Medicine \| www.frontiersin.org 6 August 2021 \| Volume 8 \| Article 716816 FIGURE 3 \| α diversity index. (A) Chao1 index: the differences between the four groups were not statistically signiﬁcant; (B) Shannon index: the differences between four groups were not statistically signiﬁcant; (C) Simpson index: the differences between four groups is not statistically signiﬁcant; (D) Observed-species diversity: the differences between four groups were not statistically signiﬁcant; (E) PD-whole-tree index: the differences between the four groups were not statistically signiﬁcant. August 2021 \| Volume 8 \| Article 716816 Frontiers in Medicine \| www.frontiersin.org 6 Colitis and Selenium-Enriched Lactobacillus acidophilus Wu et al. FIGURE 4 \| Comparison of β diversity among the control group (group A), the chronic colitis group (group C), and the chronic colitis + Se-enriched L. acidophilus group (group D). DISCUSSION The beneﬁcial eﬀects of Se and L. acidophilus on IBD have been reported. Se-enriched L. acidophilus may have a certain therapeutic eﬀect on IBD. We found that Se-enriched L. August 2021 \| Volume 8 \| Article 716816 Frontiers in Medicine \| www.frontiersin.org 7 Wu et al. Colitis and Selenium-Enriched Lactobacillus acidophilus FIGURE 6 \| Se-enriched L. acidophilus changes the intestinal microbiota. (A) Marker bacteria (LDA score > 2) between the control group (group A) and the (Continued) FIGURE 6 \| chronic colitis group (group C); (B) A LEfSe cladogram shows the dominant species in the control group (group A) and the chronic colitis group (group C); (C) Marker bacteria (LDA score > 2) between the chronic colitis group (group C) and the chronic colitis + Se-enriched L. acidophilus group (group D); (D) A LEfSe cladogram shows the dominant species of the chronic colitis group (group C) and the chronic colitis + Se-enriched L. acidophilus (group D). Lactobacillus has long been considered a probiotic. Many studies have reported the therapeutic eﬀects of diﬀerent Lactobacillus strains on IBD. A randomized clinical trial showed that Lactobacillus reuteri ATCC 55730 enema combined with oral mesalazine can improve the intestinal inﬂammation of children with mild to moderate active distal ulcerative colitis (31). The Lactobacillus rhamnosus GG strain (LGG) plays a certain role in maintaining the remission stage of ulcerative colitis (32). Animal experiments showed that two Lactobacillus reuteri strains had therapeutic eﬀects on colitis in mice (33). Lactobacillus plantarum 06CC2 has anti-inﬂammatory eﬀects (34). At the same time, it was found that the intestinal Lactobacillus of mice with DSS-induced colitis decreased (35). This suggested that probiotics belonging to Lactobacillus may be beniﬁcial to colitis treatment. However, some studies also found the opposite result: Lactobacillus increased in IBD (36, 37), which is consistent with our result, which may suggest that the intestinal microbiota may have diﬀerent changes in diﬀerent stages of IBD. Our study found that Se-enriched L. acidophilus can reduce the relative abundance of Lactobacillus. A previous study also found that Lactobacillus plantarum could reduce the relative abundance of intestinal Lactobacillus in DSS-induced colitis mice (38). These indicate that although the changes in Lactobacillus in IBD need to be further clariﬁed, some probiotics belonging to Lactobacillus may always have certain beneﬁts for the treatment of IBD. DISCUSSION Changes in the ﬂora of IBD may be inﬂuenced by diﬀerent situations (gene, diet, immunity, etc.) Therefore, molecular pathological epidemiology (MPE) may be useful for IBD research, and MPE can help doctors better understand the relationship between the ﬂora and the disease. The changes in diﬀerent ﬂora may be used to distinguish diﬀerent subtypes of IBD to facilitate more precise and eﬀective treatments. FIGURE 6 \| Se-enriched L. acidophilus changes the intestinal microbiota. (A) Marker bacteria (LDA score > 2) between the control group (group A) and the (Continued) Se-enriched L. acidophilus can reduce the relative abundance of Romboutsia. It has been reported that the relative abundance of Romboutsia in the intestinal microbiota of patients with the autoimmune disease Hashimoto’s thyroiditis is increased (39). Our study also found that the relative abundance of Romboutsia in DSS-induced colitis was increased, suggesting that Romboutsia may play a role in promoting autoimmune diseases, which needs further research. Se-enriched L. acidophilus also increased the abundance of Akkermansia in mice with colitis, although there was no signiﬁcant diﬀerence between the chronic colitis group and the chronic colitis + Se-enriched L. acidophilus group. Our study found that the relative abundance of Akkermansia was similar between the chronic colitis group and the control group. However, increase of the relative abundance of Akkermansia in the intestines of mice with DSS-induced colitis was also reported (40). Akkermansia plays an important role in the intestine. FIGURE 6 \| Se-enriched L. acidophilus changes the intestinal microbiota. (A) Marker bacteria (LDA score > 2) between the control group (group A) and the (Continued) August 2021 \| Volume 8 \| Article 716816 Frontiers in Medicine \| www.frontiersin.org 8 Colitis and Selenium-Enriched Lactobacillus acidophilus Wu et al. FIGURE 7 \| Relative abundance of species among groups. (A) Lactobacillus; (B) Romboutsia; (C) Parasutterella; (D) Akkermansia; (E) Firmicutes; (F) Bacteroidetes; (G) Verrucomicrobia (P < 0.05). Chlorogenic acid and polyphenol-rich cranberry extract have been reported to alleviate colitis by increasing the abundance of Akkermansia (41, 42). Akkermansia muciniphila, a strain of Akkermansia, can maintain intestinal barrier function, reduce Se-enriched L. acidophilus also aﬀects inﬂammatory cytokines. Cytokines play an important role in the pathogenesis of IBD. The expression levels of TNF-α, IL-1β, and IFN-γ in patients with IBD increased (46). L. acidophilus has inhibitory eﬀects FIGURE 7 \| Relative abundance of species among groups. DISCUSSION (A) Lactobacillus; (B) Romboutsia; (C) Parasutterella; (D) Akkermansia; (E) Firmicutes; (F) Bacteroidetes; (G) Verrucomicrobia (P < 0.05). FIGURE 7 \| Relative abundance of species among groups. (A) Lactobacillus; (B) Romboutsia; (C) Parasutterella; (D) Akkermansia; (E) Firmicutes; (F) Bacteroidetes; (G) Verrucomicrobia (P < 0.05). of species among groups. (A) Lactobacillus; (B) Romboutsia; (C) Parasutterella; (D) Akkermansia; (E) Firmicutes; (F) Bacteroidetes FIGURE 7 \| Relative abundance of species among groups. (A) Lactobacillus; (B) Romboutsia; (C) Parasutterella; (D) Akkermans (G) Verrucomicrobia (P < 0.05). FIGURE 7 \| Relative abundance of species among groups. (A) Lactobacillus; (B) Romboutsia; (C) Parasutterella; (D) Akkermansia; (E) Firmicutes; (F) Bacteroidetes; (G) Verrucomicrobia (*P < 0.05). Chlorogenic acid and polyphenol-rich cranberry extract have been reported to alleviate colitis by increasing the abundance of Akkermansia (41, 42). Akkermansia muciniphila, a strain of Akkermansia, can maintain intestinal barrier function, reduce the inﬂammatory response, and alleviate DSS-induced colitis in mice (43). Akkermansia muciniphila extracelluar vesicles help to alleviate the progression of DSS-induced colitis (44). Se also has a certain eﬀect on intestinal microﬂora. Zhai et al. reported that Se can increase the abundance of Akkermansia in the intestines of mice (45). Se-enriched L. acidophilus also aﬀects inﬂammatory cytokines. Cytokines play an important role in the pathogenesis of IBD. The expression levels of TNF-α, IL-1β, and IFN-γ in patients with IBD increased (46). L. acidophilus has inhibitory eﬀects on the proinﬂammatory factors IL-6, IL-17, IL-1β, and TNF- α (47). Our study also found that Se-enriched L. acidophilus can inhibit the above proinﬂammatory cytokines. Another study found that L. acidophilus can improve endoplasmic reticulum stress and induce IL-10 production (14). IL-10 is an important anti-inﬂammatory cytokine in the human August 2021 \| Volume 8 \| Article 716816 Frontiers in Medicine \| www.frontiersin.org 9 Colitis and Selenium-Enriched Lactobacillus acidophilus Wu et al. FIGURE 8 \| Correlation analysis between microbiota and cytokines. FIGURE 8 \| Correlation analysis between microbiota and cytokines. There are still some limitations in this study. The speciﬁc mechanisms by which Se-enriched L. acidophilus regulates inﬂammatory cytokines are still unclear. 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Geerling BJ, Badart-Smook A, Stockbrügger RW, Brummer RJ. Comprehensive nutritional status in recently diagnosed patients with inﬂammatory bowel disease compared with population controls. DATA AVAILABILITY STATEMENT DP performed animal and molecular biology experiments. ZW and LS analyzed and interpreted the data and wrote the manuscript. MJ conceived and designed the study. All authors approved the ﬁnal manuscript. The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s. DP performed animal and molecular biology experiments. ZW and LS analyzed and interpreted the data and wrote the manuscript. MJ conceived and designed the study. All authors approved the ﬁnal manuscript. FUNDING This work was Supported by the innovative talent support program of the Institute of Higher Learning of Liao Ning Province (No. 2018-478) and the 2020 Shenyang Science and Technology Plan (Second Batch) (20-205-4-094). All specimens from the mice were taken after ethical permission was obtained for participation in the study. The experimental protocols were approved by the Institutional Animal Care and Use Committee of China Medical University. DISCUSSION This mechanism may be related to Akkermansia. It has been reported that Akkermansia is positively correlated with IL-10 (43). We also found that Akkermansia is positively associated with IL-10. IL-21 can induce the initial T cells to diﬀerentiate into Th17 cells and produce IL-17 (51). IL- 17 can promote the production of other inﬂammatory cytokines (52) and then promote the occurrence and development of inﬂammation. IL-23 can expand Th17 cells responses (53). IL-1β in synergy with IL-6 can promote the diﬀerentiation of Th17 cells (54). Therefore, inhibition of these inﬂammatory cytokines helps to alleviate inﬂammation. In general, Se-enriched L. acidophilus can reduce the production of proinﬂammatory cytokines in DSS-induced colitis in mice, regulate the intestinal microbiota, and alleviate DSS- induced chronic colitis in mice. Therefore, Se-enriched L. acidophilus may have certain therapeutic eﬀects on IBD, especially for patients with reduction of Akkermansia and IL-10, and clinical multicenter studies could be conducted to further study its eﬃcacy in humans. August 2021 \| Volume 8 \| Article 716816 Frontiers in Medicine \| www.frontiersin.org 10 Colitis and Selenium-Enriched Lactobacillus acidophilus Wu et al. REFERENCES (2012) 35:327-34. doi: 10.1111/j.1365-2036.2011.04939.x 47. Park JS, Choi JW, Jhun J, Kwon JY, Lee BI, Yang CW, et al. 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(2018) 28:175-86. doi: 10.1089/thy.2017.0395 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. 40. Berry D, Schwab C, Milinovich G, Reichert J, Ben Mahfoudh K, Decker T, et al. Phylotype-level 16S rRNA analysis reveals new bacterial indicators of health state in acute murine colitis. ISME J. (2012) 6:2091-106. doi: 10.1038/ismej.2012.39 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. 41. Zhang Z, Wu X, Cao S, Cromie M, Shen Y, Feng Y, et al. Chlorogenic acid ameliorates experimental colitis by promoting growth of akkermansia in mice. Nutrients. (2017) 9:677. doi: 10.3390/nu9070677 42. Anhê FF, Roy D, Pilon G, Dudonné S, Matamoros S, Varin TV, et al. A polyphenol-rich cranberry extract protects from diet-induced obesity, insulin resistance and intestinal inﬂammation in association with increased Akkermansia spp. population in the gut microbiota of mice. Gut. (2015) 64:872-83. doi: 10.1136/gutjnl-2014-307142 Copyright © 2021 Wu, Pan, Jiang, Sang and Chang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). Frontiers in Medicine \| www.frontiersin.org REFERENCES The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. 43. Bian X, Wu W, Yang L, Lv L, Wang Q, Li Y, et al. Administration of Akkermansia muciniphila ameliorates dextran sulfate sodium- induced ulcerative colitis in mice. Front Microbiol. (2019) 10:2259. doi: 10.3389/fmicb.2019.02259 August 2021 \| Volume 8 \| Article 716816 Frontiers in Medicine \| www.frontiersin.org 12
https://openalex.org/W2410783462	https://zenodo.org/records/579341/files/ZK_article_7241.pdf	English	null	Barcoding Chrysomelidae: a resource for taxonomy and biodiversity conservation in the Mediterranean Region	ZooKeys	2,016	cc-by	5,202	http://zoobank.org/4D7CCA18-26C4-47B0-9239-42C5F75E5F42 Citation: Magoga G, Sassi D, Daccordi M, Leonardi C, Mirzaei M, Regalin R, Lozzia G, Montagna M (2016) Barcoding Chrysomelidae: a resource for taxonomy and biodiversity conservation in the Mediterranean Region. In: Jolivet P, Santiago-Blay J, Schmitt M (Eds) Research on Chrysomelidae 6. ZooKeys 597: 27–38. doi: 10.3897/ zookeys.597.7241 * these authors contributed equally Copyright Giulia Magoga et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. onservation... 27 Launched to accelerate biodiversity research A peer-reviewed open-access journal onservation... 27 Launched to accelerate biodiversity research A peer-reviewed open-access journal Barcoding Chr ZooKeys 597: 27–38 (2016) doi: 10.3897/zookeys.597.7241 http://zookeys.pensoft.net Barcoding Chry ZooKeys 597: 27–38 (2016) doi: 10.3897/zookeys.597.7241 http://zookeys.pensoft.net esource for taxonomy a RESEARCH ARTICLE Barcoding Chrysomelidae: a resource for ta and biodiversity conservation in the Mediterranean Region Giulia Magoga1,, Davide Sassi2, Mauro Daccordi3, Carlo Leonardi4, Mostafa Mirzaei5, Renato Regalin6, Giuseppe Lozzia7, Matteo Montagna7, Giulia Magoga1,, Davide Sassi2, Mauro Daccordi3, Carlo Leonardi4, Mostafa Mirzaei5, Renato Regalin6, Giuseppe Lozzia7, Matteo Montagna7, 1 Via Ronche di Sopra 21, 31046 Oderzo, Italy 2 Centro di Entomologia Alpina–Università degli Studi di Milano, Via Celoria 2, 20133 Milano, Italy 3 Museo Civico di Storia Naturale di Verona, lungadige Porta Vittoria 9, 37129 Verona, Italy 4 Museo di Storia Naturale di Milano, Corso Venezia 55, 20121 Milano, Italy 5 Department of Plant Protection, College of Agriculture and Natural Resources–University of Tehran, Karaj, Iran 6 Dipartimento di Scienze per gli Alimenti, la Nutrizione e l’Ambiente–Università degli Studi di Milano, Via Celoria 2, 20133 Milano, Italy 7 Dipartimento di Scienze Agrarie e Ambientali–Università degli Studi di Milano, Via Celoria 2, 20133 Milano, Italy Corresponding authors: Matteo Montagna (matteo.montagna@unimi.it) Corresponding authors: Matteo Montagna (matteo.montagna@unimi.it) Corresponding authors: Matteo Montagna (matteo.montagna@unimi.it) cademic editor: J. Santiago-Blay \| Received 20 November 2015 \| Accepted 30 January 2016 \| Published 9 June Academic editor: J. Santiago-Blay \| Received 20 November 2015 \| Accepted 30 January 2016 \| Published 9 Ju http://zoobank.org/4D7CCA18-26C4-47B0-9239-42C5F75E5F42 Keywords f b l y eaf beetles, molecular taxonomy, DNA barcoding, Cytochrome c oxidase subunit 1, C-bar project Leaf beetles, molecular taxonomy, DNA barcoding, Cytochrome c oxidase subunit 1, C-bar project Abstracth The Mediterranean Region is one of the world’s biodiversity hot-spots, which is also characterized by high level of endemism. Approximately 2100 species of leaf beetle (Coleoptera; Chrysomelidae) are known from this area, a number that increases year after year and represents 5/6% of the known species. These features, associated with the urgent need to develop a DNA-based species identification approach for a broad spectrum of leaf beetle species, prompted us to develop a database of nucleotide sequences, with a solid taxonomic background, for all the Chrysomelidae Latreille, 1802 sensu latu inhabiting the Mediter ranean region. The Mediterranean Chrysomelidae Barcoding project, which has started in 2009, involves more than fifty entomologists and molecular biologists from different European countries. Numerous collecting campaigns have been organized during the first seven years of the project, which led to the col Giulia Magoga et al. / ZooKeys 597: 27–38 (2016) 28 lection of more than 5000 leaf beetle specimens. In addition, during these collecting campaigns two new allochthonous species for Europe, namely Ophraella communa LeSage, 1986 and Colasposoma dauricum Mannerheim, 1849, were intercepted and some species new to science were discovered (e.g., Pachybrachis sassii Montagna, 2011 and Pachybrachis holerorum Montagna et al., 2013). DNA was extracted from 1006 specimens (~13% of the species inhabiting the Mediterranean region) and a total of 910 cox1 gene sequences were obtained (PCR amplification efficiency of 93.8%). Here we report the list of the bar coded subfamilies, genera and the number of species for which cox1 gene sequences were obtained; the metadata associated with each specimen and a list of problematic species for which marker amplification failed. In addition, the nucleotide divergence within and between species and genera was estimated and values of intraspecific nucleotide divergence greater than the average have been discussed. Cryptocephalus quadripunctatus G. A. Olivier, 1808, Cryptocephalus rugicollis G. A. Olivier, 1791 and Exosoma lusitanicum Linnaeus, 1767) are representatives of these cases. Introduction In the last decades we have witnessed what has been defined as the “taxonomy impedi ment” (Rodman and Cody 2003) indicating the crisis in taxonomic studies due primar ily to a shortage of time and taxonomists (Wheeler 2004, Wheeler et al. 2004, Wilson 2004), a situation that is made even more critical due to the decrease in the funding of natural history studies. The causes of the taxonomy crisis are many and complex, and a comprehensive analysis of this situation is beyond our purpose (see as example Boero 2001, Tautz et al. 2003). In our view, the causes can be described by the sentence …a lack of prestige and resources that is crippling the continuing cataloguing of biodiversity (God fray 2002). If we consider the increased rate of species extinction (Thomas et al. 2004) amplified by climate change and habitat erosion due to exploitation by human beings the situation is worsened. A DNA-based strategy, which plays a central role in modern taxo nomic studies, has been proposed by different authors as a methodology to overcome the identified problems (Tautz et al. 2002, Tautz et al. 2003, Hebert et al. 2004, Goldstein and DeSalle 2010) whilst maintaining the importance of a traditional approach mainly based on morphology. Interestingly, in a survey conducted among Coleopteran taxono mists, taxonomic initiatives based on DNA have been regarded of potential utility in solving the “taxonomy impediment”, even if a few consider it absolutely useless (Löbl 2005). Currently, in the scientific world, an agreement on the correct approach to be adopted has not yet been reached. The “gold standard” for species identification studies based on molecular markers (e.g. mitochondrial cytochrome oxidase subunit I–cox1, or the nuclear small ribosomal subunit–SSU 18S rRNA) is to develop sequence databases used as a reference, beginning with DNA extracted from type and type series specimens preserved in Museum dry collections. The main problem with this strategy is related to the conservation status of the old dry specimens; 18th and 19th century specimens have fragmented DNA (not easily amplified through standard PCR approaches targeting Barcoding Chrysomelidae: a resource for taxonomy and biodiversity conservation... 29 fragments of 500-700 bp) and are often infested by fungal hypha, which contaminate the insect’s genomic DNA. Even with the advent of high-throughput sequencing tech nologies to solve the problem of fragmented sequences, the contamination due to fungal DNA remains. Introduction Developing strategies for the acquisition and storage of molecular data to address molecular taxonomy purposes, we face another problem, which affects the DNA sequences deposited in publicly available databases, i.e. the accuracy of specimen identi fication. In light of these issues, an alternative strategy has been adopted in the Mediter ranean Chrysomelidae Barcoding project (C-Bar). The aim of the C-Bar project is to develop a reference database of cox1 gene sequences for all the Chrysomelidae (excluding Bruchinae Latreille, 1802), the Megalopodidae Latreille, 1802 and the Orsodacnidae Thomson, 1859 (hereafter indicated as Chrysomelidae or leaf beetles sensu latu – s. l.) inhabiting the Mediterranean region. The study area of C-Bar includes all the states that possess coastline on the Mediterranean Sea or territories characterized by Mediterranean- type habitat plus Romania and Switzerland (Figure 1). Starting from the Catalogue of Palaearctic Coleoptera (Löbl and Smetana 2010), about 2100 species of Chrysomelidae s. l. (corresponding to an estimated 5/6% of all described species) are present in this area. The Mediterranean Region is one of the world’s biodiversity “hot-spots” (Myers et al. 2000, Cuttelod et al. 2008), which is characterized by exceptional concentrations of species with high levels of endemism that inhabit one of the most populated areas. The assumption of high levels of endemic species inhabiting the Mediterranean Region is also valid for leaf beetles (Biondi et al. 2013, Sassi 2006). Although the Mediterranean region has been the subject of investigation by generations of entomologists, knowledge of Chrysomelidae inhabiting this area is far from being fully known. The number of leaf beetle species new to science described from the Mediterranean region in the last dec ades, associated with the fact that they are widespread among different genera, confirms the need to increase the effort in biodiversity-based studies (e.g. Cryptocephalus O.F. Muller, 1764, Chrysolina Motschulsky, 1860, Gonioctena Motschulsky, 1860, Longitar sus Berthold, 1827, Psylliodes Berthold, 1827, Colaspidea Laporte de Castelnau, 1833; Bastazo 1997, Biondi 1997, Sassi 2001, Leonardi 2007, Daccordi and Ruffo 2005, Bavi era 2007, Vela and Bastazo 2012, Zoia 2014). In this project are involved taxonomists, specialized in different leaf beetle clades, in order to guarantee the accurate specimen identification. Introduction In our view, the adoption of this strategy is a way to bring together traditional (intended as based on morphology) and molecular taxonomy in order to tentatively overcome the “taxonomy impedi ment” (Rodman and Cody 2003).h The purpose of this paper is to report the preliminary results achieved during the first seven years of the project in order to show the potential of a cooperation between molecular biologists and traditional taxonomists. In particular, we report: i) the meth od adopted and issues arisen in the development of the sequence dataset; ii) the list of subfamilies, genera and the number of species for which cox1 gene sequences were obtained; iii) the metadata associated with the processed organisms; iv) mean values of intraspecific and interspecific nucleotide divergence v) the new species described and the important faunistic findings. Giulia Magoga et al. / ZooKeys 597: 27–38 (2016) 30 Figure 1. Area investigated by the Chrysomelidae Barcoding project. The countries in which were per formed the collecting campaigns are reported in dark grey. The percentage of the total processed speci mens is reported for each country. Figure 1. Area investigated by the Chrysomelidae Barcoding project. The countries in which were per formed the collecting campaigns are reported in dark grey. The percentage of the total processed speci mens is reported for each country. Specimen collection and identification More than 50 entomologists, from different European Countries, have joined the C- Bar project and have actively participated in samples collection. During the first seven years of the project (from 2009 to 2015) numerous collecting campaigns were organ ized from March to September of each year. The specimens were collected using dif ferent methods: from the vegetation by sweep net or by beating sheet, and directly by hand in specific habitats (e.g. under stones or digging the host plant roots). All the collected specimens were placed in 5 ml vials filled with absolute ethanol in order to preserve the genomic DNA. Within an hour of specimen collection, the mixture in the vials was replaced with fresh absolute ethanol in order to obtain better sample dehydra tion and preservation for long-term storage. Each vial was preserved at -20°C and was labeled by a unique identifier plus other metadata related to the sampling locality (i.e. Country, Province, Region, exact site, latitude, longitude and elevation), the date of collection, the collector/s and other ecological information related to the specimens. Specimen manipulation and dissection (when necessary) were completed with the auxiliary use of a stereomicroscope. Images of the specimen habitus were acquired by a reflex camera (Canon EOS 450D, macro objective 60 mm or 100 mm with a set of Barcoding Chrysomelidae: a resource for taxonomy and biodiversity conservation... 31 macro extension tubes) or with Axiocam 506 mounted on Zeiss Axio Zoom V16. The specimens were morphologically identified by Italian taxonomists expert in different leaf beetle clades (most of them are listed among the authors of the present article). The nomenclature adopted in the C-bar project follows the work of Bouchard et al. (2011) at the levels of family and subfamily, while at the levels of genus and species was adopted the recently published Catalogue of Palaearctic Coleoptera–Chrysomeloidea (Löbl and Smetana 2010). DNA extraction, PCRs and sequence quality control Giulia Magoga et al. / ZooKeys 597: 27–38 (2016) 32 DNA extraction, PCRs and sequence quality control DNA extraction was performed in two different ways since it took place in different laboratories (Biodiversity Institute of Ontario, University of Guelph and Department of Agricultural and Environmental Sciences, Università degli Studi di Milano): for 950 samples the DNA was extracted from one hind leg while for the 56 remaining samples the DNA was extracted from the whole specimen, after the removal of the abdomen. The latter procedure ensures to keep specimen morphology intact. In both cases, DNA was purified using the Qiagen DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany). Here we describe the adopted non-destructive procedure: the specimen was taken off from absolute ethanol and dried in single 1.5 ml vials for 45 minutes at 30°C; after the removal of the abdomen with the use of sterile pins and tweezers the specimen was placed in 180 µL of ATL lysis buffer (Qiagen) with 200 ng/mL proteinase K (Sigma Aldrich, St. Louis, MO, USA) at 56°C for 12 hours. The following steps of the DNA extraction were performed according to the manufac turer’s instructions of Qiagen DNeasy Blood and Tissue Kit. After DNA extraction, the specimens were dry mounted on pins together with genitalia and kept for future reference. A quote of the extracted DNA was preserved in the C-bar DNA library at -80°C for long term storage and a rate was preserved at -20° in order to perform the following amplifications. A fragment of 658 bp at the 5’-end of the mitochondrial cytochrome c oxidase subunit 1 gene (cox1) was amplified with primers LCO1490 5’-GGT CAA CAA ATC ATA AAG ATA TTG G / HCO2198 5’-TAA ACT TCA GGG TGA CCA AAA AAT CA (Folmer et al. 1994). When this pair of primers resulted in unsuccessful amplification of the target marker, other primers amplify ing the same gene region were used, i.e. LepF1 5’-ATT CAA CCA ATC ATA AAG ATA TTG G / LepR1 5’-TAA ACT TCT GGA TGT CCA AAA AAT CA (Hebert et al. 2004). Successful amplifications were determined by gel electrophoresis. PCR products were directly sequenced on both strands using the marker-specific primers from ABI technology (Applied Biosystems, Foster City, CA, USA). The obtained sequences were edited using Geneious R8 (Biomatters Ltd., Auckland, New Zealand) and primers, pseudogenes and contaminations removed. Finally, they were deposited in the Bold Systems (Ratnasingham and Hebert 2007) and in the European Nucleo tide Archive (Montagna et al. under revision). Intraspecific and intrageneric nucleotide divergence The obtained cox1 gene sequences were aligned at codon level using MUSCLE (Edgar 2004) with default parameters. A pairwise nucleotide distance matrix was estimated starting from the aligned sequences implementing the Kimura-two-parameter (K2P) model (Kimura 1980), considered as an adequate evolutionary nucleotide model when p-distances between sequences are low (Nei and Kumar 2000). The nucleotide distance matrix was used for the calculation of the mean intraspecific and interspecific nu cleotide distances and for the calculation of mean intrageneric distance; these analyses were performed using the R package Spider (Brown et al. 2012). We also calculated nucleotide intraspecific distances for some species with a wide range of distribution. Results and discussion Until now, C-Bar collecting campaigns have investigated some areas of Bulgaria, France, Greece, Italy, Morocco, Romania, Spain, Switzerland, Turkey and Tunisia (Figure 1). The sampling efforts that have been accomplished until now led to the collection of more than 5000 Chrysomelidae specimens. During the identification process, some specimens of previously unknown species were recognized, these samples were used for the description of the following species: Pachybrachis sassii (Montagna 2011) from the Giglio Island in the Tuscan Archipelago; Pachybrachis holerorum (Montagna et al. 2013) from the Northern Apennines and Oulema mauroi Bezděk & Baselga, 2015, from Northen Italy. Other samples collected during the C-Bar collecting campaigns were used in a revision of Colaspidea genus that led to the description of seven new species (Zoia 2014). All these new taxa were formally described by a traditional mor phological approach, in some cases molecular data were added to confirm the existence of the new species. Besides the discovery of new taxa, two allochthonous species new to Europe, namely Ophraella communa (Boriani et al. 2013) and Colasposoma dauricum (Montagna et al. in press), were intercepted. O. communa is a leaf beetle of Nearctic origin accidentally introduced in 1996 in Taiwan (Wang and Chiang 1998) and Japan (Takizawa et al. 1999); the species rapidly spread in East Asia and few years ago we in tercepted it in the Northern part of Italy (Boriani et al. 2013). C. dauricum is a species originally present in the North and Central-East of Asia, it has never been observed out of its original range until our interception in 2011 in Piedmont (North of Italy). Among the collected samples, the DNA was extracted from 1006 specimens and PCRs targeting a fragment of the cox1 gene performed. PCRs with the selected primer pairs lead to successful amplification in 93.8% of the cases (62 specimens failed the amplification). Among the specimens for which the amplification failed, 43 specimens belong to the subfamily Cryptocephalinae Gyllenhaal, 1813: 18 species of Crypto cephalus (40 specimens); interestingly cox1 sequences have never been obtained for Cryptocephalus therondi Franz, 1949, Cryptocephalus cantabricus Franz, 1958 and Cryp tocephalus etruscus Sassi, 1995. We can hypothesize the presence of mutations in the Barcoding Chrysomelidae: a resource for taxonomy and biodiversity conservation... 33 annealing region of the used primers. Results and discussion Sequences obtained from Clytra laeviuscula Rat zeburg, 1837, Clytra quadripunctata Linnaeus, 1758, Cryptocephalus cristula Dufour, 1843, Cryptocephalus octoguttatus Linnaeus, 1767, Lachnaia tristigma Lacordaire, 1848 and Oomorphus concolor Sturm, 1807 did not possess an open reading frame and were thus considered as nuclear pseudogenes. Twenty-seven sequences were discarded be cause of contamination from exogenous DNA. A total of 910 cox1 sequences (267 species corresponding to ~13% of those inhabiting the Mediterranean region) were obtained, the size of the sequences was > 400 bp in ~99% of the cases. q p We observed that only two species, namely Cryptocephalus violaceus Laicharting, 1781 and Cryptocephalus duplicatus Suffrian, 1845, sharing the same haplotype can not be discriminated through DNA barcoding. In this and in similar cases a barcod ing failure can be confirmed only ensuring the correct identification of the samples by expert taxonomists. Therefore 99.3% of the species (265) for which we obtained cox1 sequences possessed unique haplotypes, allowing their molecular identification. The mean intraspecific nucleotide distance value is of 2%, while the mean interspecific and intrageneric distances result of, respectively, 25.2% and of 19.8%. The obtained intraspecific value are higher than that inferred in a previous study on Coleoptera (Pentinsaari et al. 2014). This results might be the effect of geographical distances among localities of collection of co-specific specimens; a possible alternative explana tion is the presence of cryptic species. Among the species showing high intraspecific nucleotide distance noteworthy are the cases of Cryptocephalus rugicollis (2.8% [0%, 5.5%]), Exosoma lusitanicum (6.7% [0.2%, 9.2%]) and Cryptocephalus quadripuncta tus that shows a mean intraspecific distance (3% [0%, 4.9%]). To test the formulated hypotheses further analyses, including the use of other mitochondrial and nuclear markers as well as a wider sample of specimens, are required. Among the nine subfamilies for which cox1 sequences were obtained (Table 1), Cryptocephalinae and Galerucinae Latreille, 1802 were better represented. In the first subfamily are listed 111 species (83 species of Cryptocephalini Gyllenhaal, 1813 and 28 of Clytrini Lacordaire, 1848, 426 specimens in total) while the second counts 88 species (24 species of Galerucini Latreille, 1802 and 64 of Alticini Spinola, 1844, 274 specimens in total). Results and discussion The unbalanced sampling towards Cryptocephalini, which in some way might affect the obtained results, could be explained by the fact that most of the C-bar speci mens have been collected by Sassi and Montagna, which mainly work on this clade and are likely to have developed collecting strategies that increase their sampling (Figure 1).hi The metadata related to the specimens (i.e., specimen identification, collection identifier, collecting date, state, province, exact site of collection, latitude, longi tude, elevation and collector/s) from which cox1 gene sequences were obtained, are available in a web site dedicated to the project (http://www.c-bar.org). Regarding the specimens collected within Italian administrative boundaries the metadata as sociated with the specimens are also available in the Biodiversity Database and GIS platform of the Italian National Network of Biodiversity. These faunistic data are useful because increase the awareness of species presence and distribution in the sampled area. Giulia Magoga et al. / ZooKeys 597: 27–38 (2016) 34 Table 1. List of the barcoded subfamilies and genera with the number of species and specimens belonging to each taxon. to each taxon. Results and discussion Subfamily Genus Ns a bNspec Nb Zeugophorinae Böving and Craighead, 1931 Zeugophora Kunze, 1818 1 1 1 Orsodacninae Thomson, 1859 Orsodacne Latreille, 1802 3 7 2.3 Donacinae Kirby, 1837 Donacia Fabricius, 1775 2 6 3 Criocerinae Latreille, 1804 Crioceris Muller, 1764 3 18 3 Lilioceris Reitter, 1912 1 Lema Fabricius, 1798 1 Oulema Gozis, 1886 1 Cassidinae Gyllenhal, 1813 Cassida Linnaeus, 1758 14 61 3.4 Hypocassida Weise, 1893 2 Hispa Linnaeus, 1767 1 Dicladispa Gestro, 1897 1 Chrysomelinae Latreille, 1802 Chrysolina Motschulsky, 1860 13 117 3.4 Chrysomela Linnaeus, 1758 3 Entomoscelis Chevrolat, 1836 1 Gastrophysa Chevrolat, 1836 1 Gonioctena Motschulsky, 1860 3 Oreina Chevrolat, 1836 6 Plagiosterna Motschulsky, 1860 1 Phratora Chevrolat, 1836 1 Plagiodera Chevrolat, 1836 1 Prasocuris Latreille, 1802 1 Timarcha Latreille, 1829 3 Galerucinae Latreille, 1802 Agelastica Chevrolat, 1836 1 274 3.1 Arima Chapuis, 1875 1 Calomicrus Stephens, 1831 3 Exosoma Jacoby, 1903 2 Diabrotica Chevrolat, 1836 1 Galeruca Geoffroy, 1762 5 Galerucella Crotch, 1873 3 Lochmaea Weise, 1883 2 Luperus Geoffroy, 1762 6 Nymphius Weise, 1900 2 Sermylassa Reitter, 1913 1 Altica Muller, 1764 4 Aphthona Chevrolat, 1842 6 Argopus Fischer von Waldheim, 1824 1 Arrhenocoela Foudras, 1860 1 Chaetocnema Stephens, 1831 2 Crepidodera Chevrolat, 1836 5 Derocrepis Weise, 1886 2 Dibolia Latreille, 1829 2 Epitrix Foudras, 1860 1 Hermaeophaga Foudras, 1860 1 Hippuriphila Foudras, 1860 1 Galerucinae Latreille, 1802 Barcoding Chrysomelidae: a resource for taxonomy and biodiversity conservation... 35 Subfamily Genus Ns a bNspec Nb Longitarsus Berthold, 1827 9 Lythraria Bedel, 1897 1 Neocrepidodera Heikertinger, 1911 6 Phyllotreta Chevrolat, 1836 4 Podagrica Chevrolat, 1836 1 Psylliodes Berthold, 1827 12 Sphaeroderma Stephens, 1831 2 Cryptocephalinae Gyllenhal, 1813 Cryptocephalus Geoffroy, 1762 73 426 3.8 Pachybarchis Chevrolat, 1836 8 Stylosomus Suffrian, 1848 2 Clytra Laicharting, 1781 4 Coptocephala Chevrolat, 1836 3 Labidostomis Chevrolat, 1836 10 Lachnaia Chevrolat, 1836 3 Macrolenes Chevrolat, 1836 1 Smaragdina Chevrolat, 1836 7 Tituboea Lacordaire, 1848 1 Eumolpinae Hope, 1840 Chrysochus Chevrolat, 1836 1 5 1.7 Colaspidea Laporte de Castelnau, 1833 1 Macrocoma Chapuis, 1874 1 aNs indicates the number of barcoded species; bNspec and N indicates respectively the total number and the average number of barcoded specimens belonging to each subfamily aNs indicates the number of barcoded species; bNspec and N indicates respectively the total number and the average number of barcoded specimens belonging to each subfamily Conclusion In this paper, we report that C-Bar project, besides having produced useful data for molecular taxonomy (cox1 sequences were obtained for about 13% of the species in habiting the investigated area), has obtained important results also from the viewpoint of the classical taxonomy leading to the morphological description of same new species of Chrysomelidae. A further important achievement has been the interception of al lochthonous species. These results have been obtained only thanks to the cooperation amongst the taxonomists specialized in different leaf beetle clades, which have ensured the correct identification of samples, the people involved in the extensive collecting campaigns and the molecular biologists. g g The promising preliminary results that have been obtained encourage us to contin ue with this project since they strongly confirm the urgent need to increase the efforts in faunistic studies to uncover the real biodiversity of leaf beetles inhabiting the Mediter ranean region. For these reasons, we are confident that the aim of C-bar project of de veloping a repository of cox1 sequences for the majority of the species of Chrysomelidae s. l. inhabiting the Mediterranean region may be achieved in the near future.i In conclusion, as demonstrated by the relevant results obtained during the first years of the project, we believe that DNA barcoding projects, when developed with the participation of taxonomists and molecular biologists, represent an opportunity Giulia Magoga et al. / ZooKeys 597: 27–38 (2016) 36 to bring together two different worlds and may be considered the driving force able to revive interest in what can be regarded as the milestone of biological studies that is a-taxonomy, helping to fill the “taxonomy impediment”. Acknowledgements The Authors would like to thank Dr. Stefano Zoia for the work performed and pre cious suggestions. 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https://openalex.org/W2265382194	http://journals.krc.karelia.ru/index.php/biology/article/download/219/154	Russian	null	STIMULATION OF REPRODUCTIVE FUNCTION OF FEMALES AND MALES OF FUR ANIMALS	Trudy Karelʹskogo naučnogo centra Rossijskoj akademii nauk	2,015	cc-by	4,234	Труды Карельского научного центра РАН № 11. 2015. С. 56–61 DOI: 10.17076/eb219 Труды Карельского научного центра РАН № 11. 2015. С. 56–61 DOI: 10.17076/eb219 УДК 636.93 В условиях Кировской области изучали влияние гуминового препарата лигногумат на репродуктивную функцию пушных зверей при введении в рацион. У самок но- рок лигногумат увеличивает количество зарегистрированных щенков к отсадке на 0,8 головы при включении в рацион в течение месяца до гона, число благополуч- но ощенившихся самок и их плодовитость, количество зарегистрированных щен- ков к отсадке на 0,7–1,5 головы при включении в рацион в течение месяца до гона и во вторую половину беременности. У самок лисиц препарат снижает количество пропустовавших и неблагополучно родивших самок в 3–4 раза, увеличивает коли- чество благополучно ощенившихся самок на 16–17 %, сохранность щенков на 19 % и количество зарегистрированных щенков на 0,8–1,5 головы. У самок песца лигно- гумат оказывает негативное влияние на репродуктивную функцию молодых самок (в возрасте до года) и положительное воздействие на репродуктивную функцию старых самок (в возрасте 2–4 лет). У последних он увеличивал количество благопо- лучно ощенившихся самок на 8 % и их плодовитость на 31 %. У самцов норки и ли- сицы препарат увеличивал количество зарегистрированных щенков, полученных в расчете на благополучно ощенившуюся и на племенную самку на 0,5–1,5 головы. Таким образом, лигногумат стимулирует репродуктивную функцию самок пушных зверей при введении в рацион в течение месяца до гона и во вторую половину бе- ременности в дозе 0,3 мл/кг массы тела, а также репродуктивную функцию самцов при введении в рацион в течение месяца до гона в той же дозе. Исключение состав- ляют молодые самки песца. К л ю ч е в ы е с л о в а: пушные звери; норка; лисица; песец; стимуляция; репродук- тивная функция; лигногумат. O. Yu. Bespyatykh, N. V. Pronina, O. N. Sukhikh, A. E. Kokorina. STIMULATION OF REPRODUCTIVE FUNCTION IN FEMALE AND MALE FUR ANIMALS The effect of humic product Lignohumate addition to the diet on the reproduction of fur animals was studied in the Kirov region. Lignohumate increases the number of registered pups at separation time by 0.8 pups when included in the diet of female mink during the month before estrus, and it increases the number of successfully whelped females, their fertility and the number of registered pups at separation time by 0.7–1.5 pups when in- cluded in the diet of female mink during the month before estrus and during the sec- ond half of gestation. The product reduces the number of unproductive female foxes and defective litter by 3–4 times, increases the number of successfully whelped female 56 56 foxes by 16–17 %, the survival rate of pups by 19 % and the number of registered pups by 0.8–1.5 pups. Lignohumate has a negative impact on the reproductive function of young female arctic foxes (aged up to one year) and a positive effect on the reproductive func- tion of older female arctic foxes (aged 2–4 years). It increased the number of successfully whelped older female foxes by 8 % and their fertility by 31 %. The product increases the reproductive function in male mink and fox resulting in the growth of the number of regis- tered pups by 0.5–1.5 pups per female. Thus, Lignohumate stimulates the reproductive function of female fur animals when added to the diet during the month before estrus and during the second half of gestation in a dose of 0.3 ml/kg of body weight, and the repro- ductive function of males when introduced to the diet during the month before estrus in the same dose. The exception is young female arctic fox. K e y w o r d s: fur animals; mink; fox; arctic fox; stimulation; reproductive function; ligno- humate. Введение животных, повышает общую резистентность организма, улучшает обмен веществ, облада- ет высокими антитоксическими и антистрес- совыми свойствами. Гуминовые препараты безвредны для животных и человека. Они не вызывают аллергии, не имеют канцерогенных, тератогенных и эмбриотоксических свойств. Повышение репродуктивной функции жи- вотных является одной из важнейших задач звероводства, не потерявшей свою актуаль- ность до настоящего времени. Основными причинами снижения репро- дуктивной способности являются негатив- ное воздействие на организм стресс-факто- ров, эмбриональная смертность, физическая и физиологическая неподготовленность зве- рей к спариванию, неправильное кормление, содержание и другие. Следует отметить, что в промышленном звероводстве большее вни- мание уделяется селекции животных по пока- зателям продуктивности и меньшее – отбору зверей по устойчивости к производственным стресс-факторам. Из пушных зверей наиболее чувствительной к стрессам является красная лисица, наименее – песец. В звероводстве лигногумат не использовали до настоящего времени, поэтому изучение его влияния на репродуктивную функцию пушных зверей представляет несомненный интерес для специалистов-практиков. Материалы и методы р р Из племенных самцов норки браун и плати- новой лисицы также формировали две груп- пы: контрольная – звери получали общехозяй- ственный рацион и опытная – дополнительно в рацион вводился лигногумат КД-Б в дозе 0,3 мл/кг массы тела в течение месяца до гона. В ходе исследований у пушных зверей оце- нивали общее физиологическое состояние и показатели, характеризующие репродуктив- ную функцию. Результаты исследований обра- ботаны статистически с использованием про- граммы «Biostat», при этом достоверность раз- личий между группами считали с р < 0,05. Из племенных самцов норки браун и плати- новой лисицы также формировали две груп- пы: контрольная – звери получали общехозяй- ственный рацион и опытная – дополнительно в рацион вводился лигногумат КД-Б в дозе 0,3 мл/кг массы тела в течение месяца до гона. В ходе исследований у пушных зверей оце- нивали общее физиологическое состояние и показатели, характеризующие репродуктив- ную функцию. Результаты исследований обра- ботаны статистически с использованием про- граммы «Biostat», при этом достоверность раз- личий между группами считали с р < 0,05. Увеличение числа благополучно ощенив- шихся самок после дополнительного введения препарата во вторую половину беременности связано с его оптимизирующим влиянием на организм зверей, которое способствует сни- жению эмбриональной смертности, так как из- вестно, что около 30 % эмбрионов норки гибнет в латентную фазу, когда эмбрионы свободно перемещаются, мигрируют из рога в рог матки, а 70 % эмбрионов гибнет после имплантации [Абрамов и др., 1970; Абрамов, 1976; Колпов ский, 1982; Murphy, Mead, 1983]. Результаты и обсуждение Включение лигногумата в рацион самок нор- ки в течение месяца только до гона способству- ет повышению плодовитости самок, что в ре- зультате увеличивает выход щенков к отсадке на племенную самку на 0,8 гол. в сравнении с контрольной группой (табл. 1). Введение пре- парата в рацион зверей в течение месяца до гона и во вторую половину беременности кроме Введение препарата в рацион самок лиси- цы способствовало снижению числа пропус- товавших и неблагополучно родивших самок в среднем в 3–4 раза, увеличению количества благополучно ощенившихся самок в среднем на 16–17 % и сохранности щенков в среднем на 58 Таблица 1. Репродуктивная функция племенных самок норки Показатели воспроизводства Контрольная группа Опытная группа, получавшая препарат: до гона до гона и во время беременности Кол-во самок, гол. 145 123 34 Покрыто самок, % 100 100 100 Пало самок, % 7,59 8,13 8,82 Пропустовало самок, % 70,34 58,54 23,53 Благополучно ощенилось самок, % 22,07 33,33 67,65 Плодовитость самок, гол. 3,53 ± 0,5 # 4,47 ± 0,6 5,04 ± 0,4 Сохранность щенков, % 64,53 63,60 61,98 Зарегистрировано щенков: – на благополучно ощенившуюся самку, гол. – на племен. самку, гол. 2,12 ± 0,4 0,63 ± 0,3 # 2,84 ± 0,4 1,18 ± 0,3 # 2,88 ± 0,5 2,21 ± 0,4 Примечание. # – различия с 3-й группой достоверны (р < 0,05). Таблица 2. Репродуктивная функция племенных самок лисицы Показатели воспроизводства Контрольная группа Опытная группа молодые самки старые самки молодые самки старые самки Кол-во самок, гол. 12 13 10 14 Покрыто самок, % 100 100 100 100 Пропустовало самок, % 16,7 7,7 0 7,1 Неблагополучно родившие самки, % 8,3 7,7 0 0 Благополучно ощенилось самок, % 75,0 84,6 100 92,9 Плодовитость самок, гол. в т. ч. мертворожд. щенков, гол. 7,3 ± 0,5 0,1 ± 0,1 7,2 ± 0,3 0,3 ± 0,2 6,2 ± 0,6 0,2 ± 0,1 6,8 ± 0,4 0 Сохранность щенков, % 66,2 77,6 91,7 90,9 Зарегистрировано щенков: – на благополучно ощенившуюся самку, гол. – на племенную самку, гол. 4,8 ± 0,6 3,6 ± 0,6 5,4 ± 0,6 4,5 ± 0,5 5,5 ± 0,4 5,5 ± 0,4 # 6,2 ± 0,3 5,8 ± 0,3 # Примечание. # – различия с контрольной группой достоверны (р < 0,05). Таблица 1. Репродуктивная функция племенных самок норки Показатели воспроизводства Контрольная группа Опытная группа, получавшая препарат: до гона до гона и во время беременности Кол-во самок, гол. Материалы и методы Исследования проводили в 2013–2014 гг. на племенном поголовье пушных зверей ООО «Зверохозяйство «Вятка» и ООО «Русский ве- люр» (Кировская обл.). Из самок норки браун (Neovison vison, Schr.), с сомнительной реак- цией к вирусу алеутской болезни, по принципу групп-аналогов сформировали три группы: 1 – контрольная – звери получали общехозяйствен- ный рацион, 2 – дополнительно в рацион вво- дился лигногумат КД-Б в дозе 0,3 мл/кг массы тела в течение месяца до гона, 3 – дополнитель- но в рацион включался лигногумат КД-Б в дозе 0,3 мл/кг массы тела в течение месяца до гона и во вторую половину беременности. В звероводстве активно применяются био- логически активные вещества различной при- роды. В последнее время разработаны новые отечественные препараты гуминового ряда, соответствующие мировому уровню, в частно- сти лигногумат. Лигногумат – это кормовая добавка на ос- нове калиевых солей гуминовых кислот, полу- ченных методом окислительно-гидролитичес- кой деструкции лигносодержащего сырья от переработки древесины хвойных и лиственных пород. Выпускается в виде порошка и 20%-го раствора. Препарат содержит не менее 58 % органических веществ (от сухого вещества), 60 % высокомолекулярных гуминовых кис- лот (от органических веществ), не более 40 % фульвовых и низкомолекулярных кислот (от ор- ганических веществ). Лигногумат КД-Б представляет собой 20%-й раствор, который перед применени- ем взбалтывают, затем вводят в кормосмесь и перемешивают. Из племенных самок серебристо-черной ли- сицы (Vulpes vulpes L.) и из самок песца шэдоу (Alopex lagopus L.) было сформировано по две группы: контрольная – звери получали общехо- зяйственный рацион и опытная – дополнитель- но в рацион вводился лигногумат КД-Б в дозе 0,3 мл/кг массы тела в течение месяца до гона и во вторую половину беременности. Кро- ме того, в каждой группе при анализе данных Препарат положительно зарекомендовал себя в птицеводстве и свиноводстве [Бессара- бов и др., 2006, 2007; Сечин и др., 2014; Топу- рия и др., 2014]. Лигногумат стимулирует рост 57 повышения плодовитости самок (р < 0,05) еще и уменьшает количество пропустовавших, а также увеличивает число благополучно още- нившихся самок. Поэтому в данной группе выход щенков на благополучно ощенившу- юся самку и на племенную самку больше на 0,76 и 1,6 гол. (р < 0,05) соответственно, чем в контрольной группе. выделяли молодых самок – в возрасте до 1 года и старых – в возрасте 2–4 лет. Результаты и обсуждение # – различия с 4-й группой достоверны (р < 0,05). Примечание. Данные приведены в расчете на 1-го самца по результатам щенения самок, покрытых им. # – различия с 4-й группой достоверны (р < 0,05). Данные приведены в расчете на 1-го самца по результатам щенения самок, покрытых им. # – й группой достоверны (р < 0,05). 19 %, что в итоге привело к повышению числа зарегистрированных щенков в расчете на бла- гополучно ощенившуюся и племенную самку в среднем на 0,8–1,5 щенка (р < 0,05) соот- ветственно, в сравнении с контрольной груп- пой (табл. 2). При этом лигногумат оказывал стимулирующее влияние на репродуктивную функцию как молодых (в возрасте до года), так и старых самок (в возрасте 2–4 лет). условиям Заполярья [Sillero-Zubiri et al., 2004]. Подобная реакция организма молодняка песца на введение препаратов (селенит натрия, янтар- ная кислота и др.), эффективность которых до- казана на других животных, отмечена и другими исследователями [Сергина и др., 2009; Беспя- тых и др., 2011; Кокорина, Беспятых, 2011]. Для объективности необходимо отметить, что негативное действие препарата на моло- дых самок песца частично могло быть обуслов- лено климатическим фактором, так как во вре- мя их щенения (позже старых самок) наступи- ло похолодание. Возраст оказался важен при введении лиг- ногумата в рацион самок песца. Препарат нега- тивно влиял на репродуктивную функцию моло- дых самок (в возрасте до года) и положительно воздействовал на воспроизводительную спо- собность старых самок (в возрасте 2–4 лет). У последних он способствовал увеличению количества благополучно ощенившихся са- мок на 8 % и плодовитости – на 31 % (р < 0,05) (табл. 3), что, несмотря на уменьшение сохран- ности щенков, в итоге привело к повышению числа зарегистрированных щенков в расчете на благополучно ощенившуюся и племенную самку на 0,8–0,9 щенка (р < 0,05), в сравнении с контрольной группой. Включение лигногумата в рацион племенных самцов норки и лисицы способствовало увели- чению количества щенков, полученных в расчете на благополучно ощенившуюся и на племенную самку на 0,5–1,5 гол. соответственно (р < 0,05), в сравнении с контрольной группой (табл. 4). Более эффективное действие лигногумата при введении в рацион зверей в течение меся- ца до гона и во вторую половину беременнос- ти по сравнению с его введением только в те- чение месяца до гона согласуется с данными, полученными нами ранее при изучении влия- ния янтарной кислоты на воспроизводительную способность племенных самок пушных зверей [Беспятых, 2010, 2011]. Вероятно, это связа- но со снижением эмбриональной смертности в результате оптимизации обмена веществ и повышения стрессоустойчивости животных. Результаты и обсуждение # – различия с 4-й группой достоверны (р < 0,05), * различия с 1-й группой достоверны (р < 0,05). Таблица 4. Репродуктивная функция племенных самцов норки и лисицы Показатели воспроизводства Норка Лисица Контроль Опыт Контроль Опыт Покрыто самок, гол. 4,23 ± 0,5 4,33 ± 0,5 3,0 ± 0,5 3,3 ± 0,5 Благополучно ощенилось самок, гол. 1,55 ± 0,5 1,5 ± 0,4 2,4 ± 0,5 3,0 ± 0,3 Рождено щенков на благополучно ощенившуюся самку, гол. 4,69 ± 0,5 5,46 ± 0,3 6,75 ± 0,6 7,6 ± 0,4 Рождено щенков на племенную самку, гол. 1,98 ± 0,5 2,52 ± 0,5 5,4 ± 0,5 # 6,9 ± 0,4 Примечание. Данные приведены в расчете на 1-го самца по результатам щенения самок, покрытых им. # – различия с 4-й группой достоверны (р < 0,05). Таблица 3. Репродуктивная функция племенных самок песца Таблица 3. Репродуктивная функция племенных самок песца Показатели воспроизводства Контрольная группа Опытная группа молодые самки старые самки молодые самки старые самки Кол-во самок, гол. 10 20 8 18 Покрыто самок, % 100 100 100 100 Пропустовало самок, % 10,0 25,0 12,5 11,1 Неблагополучно родившие самки, % 0 0 12,5 5,6 Благополучно ощенилось самок, % 90,0 75,0 75,0 83,3 Плодовитость самок, гол. в т. ч. мертворожд. щенков, гол. 10,3 ± 0,7 0,7 ± 0,3 11 ± 0,5 # 0,7 ± 0,2 11,8 ± 0,8 # 2,2 ± 0,4 14,4 ± 0,5 1,7 ± 0,3 Сохранность щенков, % 72,4 89,1 65,5 78,9 Зарегистрировано щенков: – на благополучно ощенившуюся самку, гол. – на племенную самку, гол. 7,0 ± 0,5 6,3 ± 0,6 9,2 ± 0,5 * 7,4 ± 0,3 # 6,3 ± 0,6 # 4,8 ± 0,7 # 10,0±0,4 8,3 ± 0,3 Примечание. # – различия с 4-й группой достоверны (р < 0,05), * различия с 1-й группой достоверны (р < 0,05). Таблица 4. Репродуктивная функция племенных самцов норки и лисицы Показатели воспроизводства Норка Лисица Контроль Опыт Контроль Опыт Покрыто самок, гол. 4,23 ± 0,5 4,33 ± 0,5 3,0 ± 0,5 3,3 ± 0,5 Благополучно ощенилось самок, гол. 1,55 ± 0,5 1,5 ± 0,4 2,4 ± 0,5 3,0 ± 0,3 Рождено щенков на благополучно ощенившуюся самку, гол. 4,69 ± 0,5 5,46 ± 0,3 6,75 ± 0,6 7,6 ± 0,4 Рождено щенков на племенную самку, гол. 1,98 ± 0,5 2,52 ± 0,5 5,4 ± 0,5 # 6,9 ± 0,4 Примечание. Данные приведены в расчете на 1-го самца по результатам щенения самок, покрытых им. Результаты и обсуждение 145 123 34 Покрыто самок, % 100 100 100 Пало самок, % 7,59 8,13 8,82 Пропустовало самок, % 70,34 58,54 23,53 Благополучно ощенилось самок, % 22,07 33,33 67,65 Плодовитость самок, гол. 3,53 ± 0,5 # 4,47 ± 0,6 5,04 ± 0,4 Сохранность щенков, % 64,53 63,60 61,98 Зарегистрировано щенков: – на благополучно ощенившуюся самку, гол. – на племен. самку, гол. 2,12 ± 0,4 0,63 ± 0,3 # 2,84 ± 0,4 1,18 ± 0,3 # 2,88 ± 0,5 2,21 ± 0,4 Примечание. # – различия с 3-й группой достоверны (р < 0,05). Таблица 1. Репродуктивная функция племенных самок норки Таблица 2. Репродуктивная функция племенных самок лисицы Показатели воспроизводства Контрольная группа Опытная группа молодые самки старые самки молодые самки старые самки Кол-во самок, гол. 12 13 10 14 Покрыто самок, % 100 100 100 100 Пропустовало самок, % 16,7 7,7 0 7,1 Неблагополучно родившие самки, % 8,3 7,7 0 0 Благополучно ощенилось самок, % 75,0 84,6 100 92,9 Плодовитость самок, гол. в т. ч. мертворожд. щенков, гол. 7,3 ± 0,5 0,1 ± 0,1 7,2 ± 0,3 0,3 ± 0,2 6,2 ± 0,6 0,2 ± 0,1 6,8 ± 0,4 0 Сохранность щенков, % 66,2 77,6 91,7 90,9 Зарегистрировано щенков: – на благополучно ощенившуюся самку, гол. – на племенную самку, гол. 4,8 ± 0,6 3,6 ± 0,6 5,4 ± 0,6 4,5 ± 0,5 5,5 ± 0,4 5,5 ± 0,4 # 6,2 ± 0,3 5,8 ± 0,3 # Примечание. # – различия с контрольной группой достоверны (р < 0,05). Таблица 2. Репродуктивная функция племенных самок лисицы 58 Таблица 3. Репродуктивная функция племенных самок песца Показатели воспроизводства Контрольная группа Опытная группа молодые самки старые самки молодые самки старые самки Кол-во самок, гол. 10 20 8 18 Покрыто самок, % 100 100 100 100 Пропустовало самок, % 10,0 25,0 12,5 11,1 Неблагополучно родившие самки, % 0 0 12,5 5,6 Благополучно ощенилось самок, % 90,0 75,0 75,0 83,3 Плодовитость самок, гол. в т. ч. мертворожд. щенков, гол. 10,3 ± 0,7 0,7 ± 0,3 11 ± 0,5 # 0,7 ± 0,2 11,8 ± 0,8 # 2,2 ± 0,4 14,4 ± 0,5 1,7 ± 0,3 Сохранность щенков, % 72,4 89,1 65,5 78,9 Зарегистрировано щенков: – на благополучно ощенившуюся самку, гол. – на племенную самку, гол. 7,0 ± 0,5 6,3 ± 0,6 9,2 ± 0,5 * 7,4 ± 0,3 # 6,3 ± 0,6 # 4,8 ± 0,7 # 10,0±0,4 8,3 ± 0,3 Примечание. Литература Сечин В. А., Топурия Г. М., Семенов С. В. Влия- ние Лигногумата КД-А на продуктивность свинома- ток // Достижения науки и техники АПК. 2014. № 5. С. 45–46. Абрамов М. Д., Бернацкий В. Г., Носова Н. Г. О причинах пропустования и малоплодия норок // Науч. тр. НИИПЗК. М., 1970. Т. 9. С. 129–132. Абрамов М. Д. Причины бесплодия и меры повы- шения репродуктивных свойств норок // Интенси- фикация производства клеточной пушнины. М.: Рос- сельхозиздат, 1976. С. 57–67. Топурия Г. М., Топурия Л. Ю., Семенов С. В. Фи- зиологический статус организма свиней при исполь- зовании в рационе Лигногумата КД-А // Ветерина- рия Кубани. 2014. No 3. Беспятых О. Ю. Использование янтарной кислоты с целью улучшить хозяйственно полезные признаки у лисиц // Кролиководство и звероводство. 2010. № 1. С. 8–9. Murphy B. D., Mead R. A. Luteal contribution to the termination of preimplantation delay in mink // Biol. Re- prod. 1983. Vol. 28, No 2. Р. 497–503. Sillero-Zubiri C., Hoffmann M., McDonald D. W. Ca- nids: foxes, wolves, jackals and dog. N. Y., USA, 2004. 430 р. Беспятых О. Ю., Кокорина А. Е., Тебенькова Т. В. Рост и качество шкурок молодняка пушных зве- рей при использовании добавки янтарной кисло- ты // Проблемы биологии продуктивных животных. 2011. № 3. С. 91–97. Поступила в редакцию 26.06.2015 Выводы 1. Лигногумат эффективно стимулирует ре- продуктивную функцию самок при введении в рацион в течение месяца до гона и во вто- рую половину беременности. Бессарабов Б. Ф., Мельникова И. И., Дугин А. В. и др. Применение Лигногумата КД в птицеводстве: Методические рекомендации. М. 2007. 15 с. 2. Препарат оказывает стимулирующее влия- ние на самок независимо от их возраста, за исключением молодых самок песца. Бессарабов Б., Мельникова И., Фомин А. Эффек- тивность Лигногумата КД-А при выращивании цып- лят-бройлеров // Птицеводство. 2006. № 6. С. 15–16. Кокорина А. Е., Беспятых О. Ю. Эффективность применения янтарной кислоты на племенных самках лисиц и песцов // Зоотехния. 2011. № 8. 36 с. 3. Лигногумат при введении в рацион самцов в течение месяца до гона способствует по- вышению их репродуктивной функции. Колповский В. М. Эмбриональная смертность у американской норки, ВНИИОЗ // Обогащение фау- ны и разведение охотничьих животных. Киров, 1982. 179 с. 4. Препарат целесообразно вводить в дозе 0,3 мл/кг массы тела в рацион племенных самок в течение месяца до гона и во вторую половину беременности, в рацион племен- ных самцов – в течение месяца до гона. Сергина С. Н., Ильина Т. Н., Илюха В. А. и др. Особенности функционирования антиоксидантной системы хищных млекопитающих под влиянием се- ленита натрия // Сельскохозяйственная биология. 2009. № 6. С. 66–72. Результаты и обсуждение Отсутствие стимулирующего влияния лиг- ногумата на молодых самок песца, вероятно, обусловлено более высокой гомеостатирован- ностью организма песца, то есть меньшей плас- тичностью к изменяющимся условиям сущест- вования в сравнении с другими пушными зверя- ми, что обеспечивает его адаптацию к суровым 59 Беспятых О. Ю. Повышение воспроизводитель- ной способности пушных зверей // Доклады Россий- ской академии сельскохозяйственных наук. 2011. № 2. С. 49–50. References succinic acid additive on the growth of young fur animals and the quality traits of pelts]. Problemy biologii produk- tivnykh zhivotnykh [Problems of productive animal biolo- gy]. 2011. No 3. P. 91–97. succinic acid additive on the growth of young fur animals and the quality traits of pelts]. Problemy biologii produk- tivnykh zhivotnykh [Problems of productive animal biolo- gy]. 2011. No 3. P. 91–97. Abramov M. D., Bernatskii V. G., Nosova N. G. O prichinakh propustovaniya i maloplodiya norok [Some causes of mink barrenness and low fertility]. Nauch. tr. NIIPZK [Proc. Res. Inst. of fur farming and rabbit breed- ing]. Moscow, 1970. Vol. 9. P. 129–132. Bespyatykh O. Yu. Povyshenie vosproizvoditel’noi sposobnosti pushnykh zverei [Improving the reproduc- tive characteristics of fur-bearing animals]. Doklady Rossiiskoi akademii sel’skokhozyaistvennykh nauk [Pro- ceedings of the Russian Academy of Agricultural Scien- ces]. 2011. No 2. P. 49–50. Abramov M. D. Prichiny besplodiya i mery povyshe- niya reproduktivnykh svoistv norok [Causes of infertil- ity and ways to enhance reproductive characteristics of mink]. Intensifikatsiya proizvodstva kletochnoi pushniny [Intensification of fur-bearing animals husbandry]. Mos- cow: Rossel’khozizdat, 1976. P. 57–67. Bessarabov B. F., Mel’nikova I. I., Dugin A. V., Sad- chikov S. Yu., Fomin A. V. Primenenie Lignogumata KD v ptitsevodstve: Metodicheskie rekomendatsii [Applica- tion of Lignohumate KD in poultry farming. Methodical recommendation]. Moscow, 2007. 15 p. Bespyatykh O. Yu. Ispol’zovanie yantarnoi kisloty s tsel’yu uluchshit’ khozyaistvenno poleznye priznaki u lisits [The effect of succinic acid on the improvement of economically valuable characteristics in foxes]. Kroliko- vodstvo i zverovodstvo [Rabbit breeding and farming]. 2010. No 1. P. 8–9. Bespyatykh O. Yu. Ispol’zovanie yantarnoi kisloty s tsel’yu uluchshit’ khozyaistvenno poleznye priznaki u lisits [The effect of succinic acid on the improvement of economically valuable characteristics in foxes]. Kroliko- vodstvo i zverovodstvo [Rabbit breeding and farming]. 2010. No 1. P. 8–9. Bessarabov B., Mel’nikova I., Fomin A. Effektivnost’ Lignogumata KD-A pri vyrashchivanii tsyplyat-broilerov [Efficiency of Lignohumate-CD-A application in broiler chicken farming]. Ptitsevodstvo [Poultry farming]. 2006. No 6. P. 15–16. Bespyatykh O. Yu., Kokorina A. E., Teben’kova T. V. Rost i kachestvo shkurok molodnyaka pushnykh zve- rei pri ispol’zovanii dobavki yantarnoi kisloty [Effect of 60 Kokorina A. E., Bespyatykh O. Yu. Effektivnost’ pri- meneniya yantarnoi kisloty na plemennykh samkakh li- sits i pestsov [Effect of succinic acid on breeding female foxes and polar foxes]. Zootekhniya [Zootechny]. 2011. No 8. 36 p. Sechin V. A., Topuriya G. M., Semenov S. V. References Vliyanie Lignogumata KD-A na produktivnost’ svinomatok [Ef- fect of Lignohumate-CD-A on breeding sows productivi- ty]. Dostizheniya nauki i tekhniki APK [Achievements of science and technology in agricultural complex]. 2014. No 5. P. 45–46. Kolpovskii V. M. Embrional’naya smertnost’ u ameri- kanskoi norki, VNIIOZ [Embryonic death in American mink. All-Union Res. Inst. of hunting and fur farming]. Obogashchenie fauny i razvedenie okhotnich’ikh zhi- votnykh [Enrichment of the fauna and breeding of game animals]. Kirov, 1982. 179 p. Topuriya G. M., Topuriya L. Yu., Semenov S. V. Fizio- logicheskii status organizma svinei pri ispol’zovanii v ra- tsione Lignogumata KD-A [Physiological status of pigs at the use of Lignohumate-CD-A in the ration]. Veteri- nariya Kubani [Veterinary of Kuban]. 2014. No 3. Sergina S. N., Il’ina T. N., Ilyukha V. A., Fatyshe- va M. V., Podlepina L. G. Osobennosti funktsioniro- vaniya antioksidantnoi sistemy khishchnykh mleko- pitayushchikh pod vliyaniem selenita natriya [Fea- tures of antioxidant system functioning in carnivorous mammals under the influence of sodium selenite]. Sel’skokhozyaistvennaya biologiya [Agricultural biolo- gy]. 2009. No 6. P. 66–72. Murphy B. D., Mead R. A. Luteal contribution to the termination of preimplantation delay in mink. Biol. Re- prod. 1983. Vol. 28, No 2. P. 497–503. Sillero-Zubiri C., Hoffmann M., McDonald D. W. Ca- nids: foxes, wolves, jackals and dog. N. Y., USA, 2004. 430 р. Received June 26, 2015 Received June 26, 2015 Received June 26, 2015 Беспятых Олег Юрьевич Pronina, Natalia Russian Research Institute of Game Management and Fur Farming 79 Preobrajenskaia St., Kirov, Russia e-mail: bio.vniioz@mail.ru Pronina, Natalia Russian Research Institute of Game Management and Fur Farming 79 Preobrajenskaia St., Kirov, Russia e-mail: bio.vniioz@mail.ru CONTRIBUTORS: Bespyatykh, Oleg Russian Research Institute of Game Management and Fur Farming 79 Preobrajenskaia St., Kirov, Russia Vyatka State University of Humanities 26 Krasnoarmeyskaia St., Kirov, Russia e-mail: b__oleg@mail.ru tel.: 89226626820 Bespyatykh, Oleg Russian Research Institute of Game Management and Fur Farming 79 Preobrajenskaia St., Kirov, Russia Vyatka State University of Humanities 26 Krasnoarmeyskaia St., Kirov, Russia e-mail: b__oleg@mail.ru tel.: 89226626820 Беспятых Олег Юрьевич ведущий научный сотрудник, к. б. н. Всероссийский научно-исследовательский институт охотничьего хозяйства и звероводства им. проф. Б. М. Житкова ул. Преображенская, 79, Киров, Россия, 610000 доцент Вятский государственный гуманитарный университет ул. Красноармейская, 26, Киров, Россия, 610002 эл. почта: b__oleg@mail.ru тел.: 89226626820 Пронина Наталья Владимировна Sukhikh, Olesia Russian Research Institute of Game Management and Fur Farming 79 Preobrajenskaia St., Kirov, Russia e-mail: bio.vniioz@mail.ru Сухих Олеся Николаевна Kokorina, Anastasia Russian Research Institute of Game Management and Fur Farming 79 Preobrajenskaia St., Kirov, Russia e-mail: bio.vniioz@mail.ru
https://openalex.org/W2791055636	https://europepmc.org/articles/pmc5798757?pdf=render	English	null	Overexpression of ubiquitin carboxyl-terminal hydrolase 1 (UCHL1) in boys with cryptorchidism	PloS one	2,018	cc-by	5,220	RESEARCH ARTICLE Results Copyright: © 2018 Toliczenko-Bernatowicz et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The median concentration of UCHL1 in the blood plasma of boys with cryptorchidism, was 5-folds higher than in boys with inguinal hernia, whose testicles were located in the scrotum. We also noticed statistically significant difference between UCHL1 levels in boys with crypt- orchidism up to 2 years old, and above 2 years old. Older boys, whose testicles since birth were located in the inguinal pouch or in the abdominal cavity, had higher concentration of UCHL1 in their blood plasma, than boys from younger group. In the group of cryptorchid boys, we also found slightly lower concentrations of INSL3, without statistical significance and no correlation with UCHL1 levels. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: The authors received no specific funding for this work. Methods Patients—50 boys aged 1–4 years (median = 2,4y.) with unilateral cryptorchidism. Exclusion criteria were: previous human chorionic gonadotropin treatment, an abnormal karyotype, endo- crine or immunological disorders or any long-term medication. The control group—50 healthy, age matched boys (aged 1–4 years, median = 2,1y.), admitted to the Pediatric Surgery Depart- ment for planned herniotomy. To investigate UCHL1 in blood plasma of boys with cryptorchi- dism, we used a novel technique Surface PLASMON RESONANCE Imaging (SPRI). OPEN ACCESS Citation: Toliczenko-Bernatowicz D, Matuszczak E, Tylicka M, Szymańska B, Komarowska M, Gorodkiewicz E, et al. (2018) Overexpression of ubiquitin carboxyl-terminal hydrolase 1 (UCHL1) in boys with cryptorchidism. PLoS ONE 13(2): e0191806. https://doi.org/10.1371/journal. pone.0191806 Background The ubiquitin-proteasome system regulate p53, caspase and Bcl-2 family proteins, and is crucial for the degradation of the defective germ cells in testes. Purpose: to evaluate the concentration of ubiquitin carboxyl-terminal hydrolase 1 (UCHL1) in the blood plasma of boys with cryptorchidism and if there is any correlation with patient age. Overexpression of ubiquitin carboxyl-terminal hydrolase 1 (UCHL1) in boys with cryptorchidism Dorota Toliczenko-Bernatowicz1, Ewa Matuszczak1, Marzena Tylicka2, Beata Szymańska3, Marta Komarowska1, Ewa Gorodkiewicz3, Wojciech Debek1, Adam Hermanowicz1 Dorota Toliczenko-Bernatowicz1, Ewa Matuszczak1, Marzena Tylicka2, Beata Szymańska3, Marta Komarowska1, Ewa Gorodkiewicz3, Wojciech Debek1, Adam Hermanowicz1 1 Paediatric Surgery Department,Medical University of Bialystok, Bialystok, Poland, 2 Biophysics Department Medical University of Bialystok, Bialystok, Poland, 3 Electrochemistry Department, University of Bialystok, Bialystok, Poland a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 * ewamat@tlen.pl Introduction Testicles which did not descend to the scrotum, are at risk of disturbed spermatogenesis and testicular cancer, caused by elevated temperature in the abdominal cavity or in the inguinal pouch. According to some authors, early surgical orchidopexy does not change the risk of malignant transformation [2]. Contrary to that, the United Kingdom Testicular Cancer Study Group concluded that, orchidopexy before the age of 10 years, reduced the risk of testicular cancer [3]. Still, the surgical treatment of innate cryptorchidism is advocated in early infancy [1]. Spermatogenesis is a complex process, during which apoptosis controls the amount of germ cells and eliminates impaired germ cells [4]. According to recent studies, ubiquitin-pro- teasome system is crucial to the regulation of this process. The levels of ubiquitin are under control of ubiquitinating enzymes and deubiquitinating enzymes (DUBs)[5]. DUBs are divided into ubiquitin C-terminal hydrolases (UCHs) and ubiquitin specific proteases (UBPs) [5]. Kwon et al. suggested that one of those enzymes—ubiquitin Carboxyl-terminal hydrolase 1 (UCHL1) “plays a role in balancing the expression of apoptosis-inducing and apoptosis-pro- tecting proteins”[6]. UCHL1 is expressed specifically in neurons and testes or ovaries [7]. In mouse testes, UCHL1 is expressed in gonocytes, already at 14 days of gestation [8]. Postnatally, between 8th and 16th day, UCHL1 is localized in the spermatogonia, but in mature mice it appeared in spermatogonia, and in Sertoli cells [9]. Undescended testes exposed to higher body temperature, show degeneration of germ cells which occurs via apoptosis [10]. It is already known, that apoptosis of germ cells in unde- scended testes involves many genes including Bcl-2 proteins, p53 and caspases [6]. The ubiqui- tin-proteasome system regulate p53, caspase and Bcl-2 family proteins, and is crucial for the degradation of the defective germ cells in testes [6,11]. According to recent theories “ubiquiti- nation in the epididymis may trigger apoptotic mechanisms that recognize and eliminate abnormal spermatozoa” and control the sperm quality [12]. In animal study, Du et al. showed that in undescended testes, UCHL1 concentrated into spermatocytes with apoptotic appearance to response to hyperthermia [13]. It is hypothesized, that UCHL1 accumulates in the spermatocytes due to the blockade of degradation or the up- regulation of its expression. The proof of this theory, can be found in animal study, in which spermatogenesis in UCHL1 deficient gad mice was resistant to apoptotic stress caused by heat [10]. Conclusions Uchl1 concentrations in the blood plasma of boys with cryptorchidism, may reflect the heat- induced apoptosis of germ cells. Higher UCHL1 concentrations in older boys with Competing interests: The authors have declared that no competing interests exist. 1 / 10 PLOS ONE \| https://doi.org/10.1371/journal.pone.0191806 February 5, 2018 UCHL1 in cryptorchidism undescended testicles, probably express intensity of germ cell apoptosis, more extensive when testicles are subjected to heat-stress for longer period. Further analyses of UCHL1 may help to elucidate its role in mechanisms influencing spermatogenesis. PLOS ONE \| https://doi.org/10.1371/journal.pone.0191806 February 5, 2018 Methods Blood samples were drawn from the antecubital vein. Blood was collected in tubes containing EDTA. The serum was separated from cells by centrifugation. Serum samples were made anonymous and stored at –20oC. Patients The study population comprised 50 boys aged 1–4 years (median = 2,4y.) with unilateral crypt- orchidism. Exclusion criteria were: previous human chorionic gonadotropin treatment, an abnormal karyotype, endocrine or immunological disorders or any long-term medication. All of them were operated on and underwent successful orchidopexy. During the operation, testes were measured, and their the position and morphology evaluated. Control group The control group represented 50 healthy, age matched boys (aged 1–4 years, median = 2,1y.), admitted to the Pediatric Surgery Department for planned herniotomy. All boys in the control group had their testes in the scrotum. They also underwent karyotyping before their surgical treatment. The study was approved by the Ethics Committee of Medical University of Bialystok Poland. All parents gave their informed, written consent for the inclusion to the study, clinical and biochemical follow-up and for surgical treatment. Introduction To confirm findings from animal models, we wanted to evaluate the concentration of UCHL1 in the blood plasma of boys with cryptorchidism and if there is any correlation with patient age. To our knowledge this is the first human study assessing UCHL1 in connection with undescended testes. To investigate the ubiquitin carboxyl-terminal hydrolase 1 (UCHL1) in blood plasma of boys with cryptorchidism, we used a novel technique Surface PLASMON RESONANCE Imag- ing (SPRI). So far several SPRI biosensors were used for clinical research to determinate e.g. 20S proteasome, matrix metalloproteinase-2, ubiquitin carboxyl-terminal hydrolase L1 (UCHL1), and immunoproteasome [14,15,16]. 2 / 10 PLOS ONE \| https://doi.org/10.1371/journal.pone.0191806 February 5, 2018 UCHL1 in cryptorchidism PLOS ONE \| https://doi.org/10.1371/journal.pone.0191806 February 5, 2018 Procedure of UCHL1 determination The ubiquitin carboxy-terminal hydrolase L1 (human recombinant UCH-L1, R&D System. Inc.) concentration was determined using the SPRI (Surface Plasmon Resonance Imaging) biosensor as described in the previous paper [18]. Gold chips were manufactured as described in other papers [17,18]. The gold surface of the chip was covered with photopolymer and hydrophobic paint. According to Sankiewicz et al.: “Chips were rinsed with ethanol and water and dried under a stream of nitrogen. They were then immersed in 20mM of cysteamine ethanolic solutions for 2h and after rinsing with ethanol and water dried again under a stream of nitrogen. The rabbit monoclonal IgG2A antibody specific for human UCHL (R&D System. Inc.) were immobilized on the thiol monolayer under the suitable conditions. The antibody solution in a PBS buffer was activated with NHS (250 mM) and EDC (250 mM). Activation of the antibody was done by adding the mixture of NHS and EDC (1:1) in a carbonate buffer solution (pH 8.5) into the antibody solution and with vigorous stirring for 5 min at the room temperature. 3μl of this solution was placed on the active places with the amine-modified surface, and incubated at 37oC for 1h” [18]. “After this time the biosensor were rinsed with water. Next, serum samples (10 x diluted) were placed directly on the prepared biosensor. The volume of the sample applied on each measuring field was 3 μl. Time of the interaction with antibody was max 10 minutes. The bio- sensor was washed with water and HBS-ES buffer solution pH = 7.4 (0.01 M 4-(2-hydro- xyethyl) piperazine-1-ethanesulfonic acid, 0.15 M sodium chloride, 0.005% Tween 20, 3 mM EDTA), BIOMED, Lublin, Poland) to remove unbound molecules from the surface” [18]. SPRI measurements for protein array were performed as described in the previous papers:”The SPRI signal was measured at a fixed SPR angle on the basis of registered images. The first image after immobilization of the antibody was taken. Then, the second image after PLOS ONE \| https://doi.org/10.1371/journal.pone.0191806 February 5, 2018 3 / 10 UCHL1 in cryptorchidism interaction with UCH-L1 was taken. The SPRI signal was obtained by subtraction of the signal after and before interaction with a biomolecule, for each spot separately. The contrast values obtained for all pixels across a particular sample single spot were integrated. Then, the SPRI signal was integrated over the spot area. Procedure of UCHL1 determination NIH Image J version 1.32 software was used to evalu- ate the SPRI images in 2D form and to convert of numerical signal to a quantitative signal (A. U.)” [17,18]. The results were assessed using a linear section of the calibration graph (0.1–2.5 ng/ml, R2 = 0.9926) after an adequate sample dilution with PBS buffer. The curve was made just before the assessment. The precision of measurements was approximately 10%.The levels of INSL-3, AMH and inhibin B were assessed using commercial enzyme-linked immunosorbent assay ELISA kits Beckman Coulter and Uscn Life Science Inc. Statistical analysis Statistical analysis was performed using the STATISTICA PL release 12.5 Program. All the results are presented as median with 25th and 75th percentiles. Because tested parameters in plasma of patients did not pass the normality Shapiro-Wilk test continuous variables were compared by the nonparametric Mann-Whitney U test (two-sided nature)between study groups. Correlations were studied by using the Spearman Correlation Test. A p value of p<0.05 indicated a statistically significant difference. There was no statistically significant difference in the age distribution of the group of boys with cryptorchidism and boys with inguinal hernia, who served as controls. All boys had kar- yotypes 46XY. In most cases of cryptorchidism, the undescended testes were found in the superficial inguinal pouch (n = 46), but in two boys were located in the external ring of ingui- nal canal, and also two patients had their testicles located in the abdomen. Generally, unde- scended testes were smaller in comparison to the testes positioned normally (mean 1cm and mean 1,5cm respectively). The median concentration of UCHL1 in the blood plasma of boys with cryptorchidism, was 5-folds higher than in boys with inguinal hernia, whose testicles were located in the scrotum (Fig 1, S1 Fig). The difference was statistically significant (p< 0.001). We also noticed statistically significant difference between UCHL1 levels in boys with cryptorchidism up to 2 years old, and above 2 years old. Older boys, whose testicles since birth were located in the inguinal pouch or in the abdominal cavity, had higher concentration of UCHL1 in their blood plasma, than boys from younger group. The difference was statistically significant (p = 0.038) (Fig 2). In the group of cryptorchid boys, we also found slightly lower concentrations of INSL3, without statistical significance, and without correlation with UCHL1 levels. We did not find statistically significant differences in the levels of AMH and inhibin B in the group of boys with cryptorchidism and boys with inguinal hernia, and there were no correlations between UCHL1 and AMH or UCHL1 and inhibin B in those groups (Table 1). Discussion Sperm production in the testis depends on equilibrium between division and loss of germ cells. During spermatogenesis, apoptosis limits the quantity of germ cells and eliminates those which carry DNA mutations, occurring through chromosomal crossing over during the first meiotic division [19]. Kwon et al. indicate that ubiquitination targets proteins for degradation, and by this, takes part in the control of sperm quality during sperm maturation [10]. UCHL1 is expressed at high levels in both testis and epididymis, and defective spermatozoa and pro- teins in these cells become ubiquitinated during transit through the epididymis [6,10,19]. According to Wang et al. “overexpression of UCHL1 affects spermatogenesis during meiosis and, induces apoptosis in primary spermatocytes” [20]. Contrary to that, Kwon et al. reported PLOS ONE \| https://doi.org/10.1371/journal.pone.0191806 February 5, 2018 4 / 10 Fig 1. Plasma UCHL-1 concentration in children with hernia and cryptorchidism (N = 50). UCHL1 in cryptorchidism Fig 1. Plasma UCHL-1 concentration in children with hernia and cryptorchidism (N = 50). UCHL1 in cryptorchidism UCHL1 in cryptorchidism Fig 1. Plasma UCHL-1 concentration in children with hernia and cryptorchidism (N = 50). https://doi org/10 1371/journal pone 0191806 g001 Fig 1. Plasma UCHL-1 concentration in children with hernia and cryptorchidism (N = 50). https://doi.org/10.1371/journal.pone.0191806.g001 https://doi.org/10.1371/journal.pone.0191806.g001 that in mice, UCH-L1 can be detected mainly in spermatogonia and somatic Sertoli cells, so it might function in mitotic proliferation in spermatogenesis [6,10,19]. Still, the spermatocytes are the main type of germ cells undergoing apoptosis in cryptorchidism [21]. Apoptosis of germ cells caused by elevated temperature in undescended testes, involves many determinants, such as Bcl-2 famliy proteins, the tumor suppressor protein p53, and cas- pases [22,23]. P53 protein is highly expressed in the testis, increase of its level caused by high temperature, results in intensified germ cell apoptosis of germ cells and less production of spermatozoa [24]. In addition Bcl-2 family and IAP (inhibitor of apoptosis protein) family are other important factors regulating apoptosis, and together with caspase members, are targets for ubiquitination [25]. UCHL1 occurred abnormally in the spermatocytes in response to abdominal hyperthermia, and in association with Jab1 and p27kipl [13]. Under the physiologi- cal conditions, UCHL1, Jab1 and p27kipl were not expressed in spermatocytes, Du et al found that cyclin-dependent kinase inhibitor p27kipl increased in some spermatocytes, just paral- leled to Jab1 and UCHL1, in response to heat stress, and apparently concentrated in nuclei of apoptotic spermatocytes [13]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0191806 February 5, 2018 Discussion who compared fertility potential of undescended testes, by age groups in children revealed, that “the higher the testicular position at the time of treatment, the fewer the number of germ cells” [26] According to human studies “spermatogenic index decreases significantly by 9 months of age”‘ so operation of undescended testes at this age or before, may stop testicu- lar degeneration and improve chances for future fertility [26]. Moreover, Tasian et al. reported “a significant 2% risk per month of severe germ cell loss and 1% risk per month Leydig cell depletion for each month a testis remains undescended” [27]. According to the same research “the odds of germ cell loss almost double for each age range at the time of orchidopexy” [27]. UCHL1 was first identified in the brain and testes, and accounts for 1–2% of brain soluble proteins [28,29]. Studies on adults demonstrated that increased concentrations of UCHL1 can be found in serum after traumatic brain injury [28,29]. Also in children after head trauma, increased serum levels of UCHL1 correlated with worse outcome [30]. In control groups, levels of UCH-L1 were significantly higher in the first year of life, than those measured in older chil- dren, the highest levels found below 3 months of age. Most probably the cause of that is under- development of the blood–brain barrier in infants. Contrary to this observation, according to Ferguson et al. serum levels of UCH-L1 increase with age in young healthy adults [31]. Heat induced alterations in tight-junction proteins in undescended testis proteins cause higher permeability of blood-testis barrier [32]. We speculate, that it can be the source of UCHL1 in blood plasma originating from the apoptotic germ cells. p g g p p g We believe, that the results of our study, indicate that Uchl1 concentrations in the blood plasma of boys with cryptorchidism, reflect the heat-induced apoptosis of germ cells. The levels of UCHL1 in the blood plasma of our patients with cryptorchidism, were 5-folds higher than in controls, whose testicles were located in the scrotum. Furthermore, we also noticed that UCHL1 concentrations differed between the group of boys with cryptorchidism up to 2 years old, and above 2 years old. Older boys, whose testicles since birth were located in the inguinal pouch or in the abdominal cavity, had higher concentration of UCHL1 in their blood plasma, than boys from the younger group. Discussion Probably p27kipl mediate the UCHL1-involved apoptosis PLOS ONE \| https://doi.org/10.1371/journal.pone.0191806 February 5, 2018 5 / 10 Fig 2. Plasma UCHL-1 concentration in children with cryptorchidism of age up to (N = 20) and above 2 years old (N = 30). UCHL1 in cryptorchidism UCHL1 in cryptorchidism Fig 2. Plasma UCHL-1 concentration in children with cryptorchidism of age up to (N = 20) and above 2 years old (N = 30). https://doi.org/10.1371/journal.pone.0191806.g002 on in children with cryptorchidism of age up to (N = 20) and above 2 years old (N = 30). Fig 2. Plasma UCHL-1 concentration in children with cryptorchidism of age up to (N = 20) and above 2 years old (N = 30). directly through its abnormal increase in spermatocytes in response to heat-stress [13]. All this findings suggest apoptotic cell death is regulated mainly by ubiquitination. directly through its abnormal increase in spermatocytes in response to heat-stress [13]. All this findings suggest apoptotic cell death is regulated mainly by ubiquitination. In human males, spermatogenesis begins at puberty in seminiferous tubules in the testicles and go on continuously [1]. In prepubertal boys, immature germ cells–spermatogonia, Table 1. The statistic parameters of UCH-L1, INSL3, AMH and inhibin B concentrations in plasma of children diagnosed with hernia and cryptorchidism. Cryptorchid n = 50 Control group n = 50 P value UCHL1 ng/ml 5.83 (±2.02) 1.04 (±0.33) <0.05 INSL3 pg/ml 2672.000 (±1334.4) 3267.500 (±1505.9) = 0.05 AMH ng/ml 118.200 (± 59.996) 111.500 (±59.5) >0.05 Inhibin B pg/ml 113.770 (± 54.241) 129.575 (±78.646) >0.05 A p value <0.05 is considered to show a significant difference between groups https://doi.org/10.1371/journal.pone.0191806.t001 Table 1. The statistic parameters of UCH-L1, INSL3, AMH and inhibin B concentrations in plasma of children diagnosed with hernia and cryptorchidism. 6 / 10 PLOS ONE \| https://doi.org/10.1371/journal.pone.0191806 February 5, 2018 PLOS ONE \| https://doi.org/10.1371/journal.pone.0191806 February 5, 2018 6 / 10 PLOS ONE \| https://doi.org/10.1371/journal.pone.0191806 February 5, 2018 UCHL1 in cryptorchidism proliferate continuously by mitotic divisions around the outer edge of the seminiferous tubules, next to the basal lamina [1]. In boys with cryptorchidism, according to Chung: “The reduction in germ cell count starts as early as 6 months of age and is dependent on the position of the testis” [1]. Study by Wilker- son et al. PLOS ONE \| https://doi.org/10.1371/journal.pone.0191806 February 5, 2018 Discussion This observation exclude potential bias of serum con- centrations of UCHL1 correlating with age in young children, as according to Berger et al. “UCH-L1 concentrations during the first year of age were significantly higher than those mea- sured at later ages” [30]. In the group of cryptorchid boys, we also found slightly lower concen- trations of INSL3, without statistical significance and without correlation with UCHL1 levels. INSL3 is expressed in Leydig cells in testis, shortly after the onset of testicular development, and controls the thickening of the gubernaculum anchoring the testis to the inguinal region. Kumagai et al. in animal model showed that “deficiency of INSL3 resulted in cryptorchidism or defects in testis descent due to abnormal gubernaculum development” [33]. We did not find statistically significant differences in the levels of AMH and inhibin B in the group of boys with cryptorchidism and boys with inguinal hernia and it also did not correlate with UCHL1 concentration. AMH is secreted by immature Sertoli cells during the 8th week of gestation, and is responsible for the regression of Mullerian ducts in the male fetus [34]. Mutations in the the AMH receptor cause the persistent Mullerian duct syndrome in males, and disrupts the descend of the testis [34]. Surprisingly in this group of patients, the AMH levels did not differ significantly between cryptorchid boys and boys with inguinal hernia, contrary to the results of previous studies [34]. As to Inhibin B, it is produced by the Sertoli cells in the prepubertal testis, and controls FSH secretion by a negative feedback mechanism [34]. Before puberty lev- els of inhibin B “reflect the presence of Sertoli cells, and not the germ cells” [34]. We did not PLOS ONE \| https://doi.org/10.1371/journal.pone.0191806 February 5, 2018 7 / 10 UCHL1 in cryptorchidism assessed the testosterone levels, although an infant boy may have slightly elevated its levels, but at the age of six months it will drop to a range of undetectable to 20 ng per dL of blood until a boy reaches puberty [35]. Most probably, higher UCHL1 concentrations in older boys with undescended testicles, express intensity of apoptosis of germ cells, more extensive when testicles are subjected to heat-stress for longer period. The aetiology of cryptorchidism is multifactorial, genetic, hormonal and environmental factors playing a role [36–40]. Author Contributions Conceptualization: Ewa Matuszczak, Ewa Gorodkiewicz. Conceptualization: Ewa Matuszczak, Ewa Gorodkiewicz. Data curation: Dorota Toliczenko-Bernatowicz, Marzena Tylicka, Beata Szymańska, Marta Komarowska. Formal analysis: Ewa Matuszczak, Marzena Tylicka, Beata Szymańska. Formal analysis: Ewa Matuszczak, Marzena Tylicka, Beata Szymańska. Investigation: Dorota Toliczenko-Bernatowicz, Ewa Matuszczak. Investigation: Dorota Toliczenko-Bernatowicz, Ewa Matuszczak. Software: Ewa Matuszczak, Marzena Tylicka. Software: Ewa Matuszczak, Marzena Tylicka. Supervision: Ewa Matuszczak, Ewa Gorodkiewicz. Supervision: Ewa Matuszczak, Ewa Gorodkiewicz. Writing – original draft: Ewa Matuszczak. Writing – original draft: Ewa Matuszczak. Writing – review & editing: Ewa Gorodkiewicz, Wojciech Debek, Adam Hermanowicz. Writing – review & editing: Ewa Gorodkiewicz, Wojciech Debek, Adam Hermanowicz. Discussion We believe, that our study may help to understand its molecular consequences, influencing future fertility in patients with undescended testes.Uchl1 concen- trations in the blood plasma of boys with cryptorchidism, may reflect the heat-induced apopto- sis of germ cells. Higher UCHL1 concentrations in older boys with undescended testicles, probably express intensity of germ cell apoptosis, more extensive when testicles are subjected to heat-stress for longer period. Further analyses of UCHL1 may help to elucidate its role in mechanisms influencing spermatogenesis. S1 Fig. Blood plasma levels of UCHl1 in boys with cryptorchidism and inguinal hernia. (XLSX) S1 Fig. Blood plasma levels of UCHl1 in boys with cryptorchidism and inguinal hernia. (XLSX) PLOS ONE \| https://doi.org/10.1371/journal.pone.0191806 February 5, 2018 References Sutovsky P: Ubiquitin-dependent proteolysis in mammalian spermatogenesis, fertilization, and sperm quality control: killing three birds with one stone. Microsc Res Tech 2003; 61: 88–102. https://doi.org/10. 1002/jemt.10319 PMID: 12672125 13. DU P, Yao YW, Shi Y, Gu Z, Wang J, Sun ZG et al.: Uchl1 and its associated proteins were involved in spermatocyte apoptosis in mouse experimental cryptorchidism. 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https://openalex.org/W4292648133	https://www.frontiersin.org/articles/10.3389/fpsyg.2022.893469/pdf	English	null	A systematic review of interventions to support adults with ADHD at work—Implications from the paucity of context-specific research for theory and practice	Frontiers in psychology	2,022	cc-by	12,120	attention deﬁcit hyperactivity disorder, ADHD, workplace, systematic review, interventions, treatment, work TYPE Systematic Review PUBLISHED 22 August 2022 DOI 10.3389/fpsyg.2022.893469 TYPE Systematic Review PUBLISHED 22 August 2022 DOI 10.3389/fpsyg.2022.893469 A systematic review of interventions to support adults with ADHD at work—Implications from the paucity of context-speciﬁc research for theory and practice OPEN ACCESS EDITED BY Roberto Truzoli, University of Milan, Italy REVIEWED BY Joseph Sadek, Dalhousie University, Canada Andreas Conca, Bolzano Central Hospital, Italy CORRESPONDENCE Kirsty Lauder kirstymlauder@gmail.com SPECIALTY SECTION This article was submitted to Psychology for Clinical Settings, a section of the journal Frontiers in Psychology RECEIVED 16 March 2022 ACCEPTED 19 July 2022 PUBLISHED 22 August 2022 CITATION Lauder K, McDowall A and Tenenbaum HR (2022) A systematic review of interventions to support adults with ADHD at work—Implications from the paucity of context-speciﬁc research for theory and practice. Front. Psychol. 13:893469. doi: 10.3389/fpsyg.2022.893469 Kirsty Lauder1, Almuth McDowall1 and Harriet R. Tenenbaum2 1C t f N di it R h t W k Bi kb k C ll L d U it d Ki d 2S h l Kirsty Lauder1, Almuth McDowall1 and Harriet R. Tenenbaum2 Attention Deﬁcit Hyperactivity Disorder (ADHD) is estimated to afect 3.5% of the global workforce. Despite the high prevalence rate, little is known about how best to support adults with ADHD (ADHDers) at work. Relevant research is dispersed across diferent disciplines such as medicine, health studies and psychology. Therefore, it is important to synthesize interventions aimed at ADHDers to examine what learning can be gleaned for efective workplace support. We conducted a systematic review of relevant interventions framed by realist evaluation and the Context-Intervention-Mechanism-Outcome classiﬁcation to identify key mechanisms of efectiveness for workplace interventions. We searched 10 databases including a range of journals from medical science to business management applying predetermined inclusion criteria and quality appraisal through a risk of bias assessment for quantitative and qualitative methods. We synthesized 143 studies with realist evaluation. Most studies evaluated the efectiveness of pharmacological interventions highlighting the dominance of the medical approach to supporting ADHDers. Key mechanisms of efectiveness were identiﬁed from psychosocial interventions including group therapy, involvement of people in the ADHDers network, and the importance of the client-patient relationship. Overall, there is limited research that examines the efectiveness of workplace interventions for ADHDers. Furthermore, much of the existing research evaluates pharmacological interventions which is difcult to transfer to the workplace context. It is recommended that future research and practice consider the key mechanisms identiﬁed in this review when designing interventions as well as barriers to accessing support such as disclosure and self-awareness. Introduction and thriving careers, such as concentration challenges being interpreted as a sign of underperformance, rather than supported through adjustments, it is important to revisit the existing literature to identify studies that examine relevant interventions to recognize any eﬀective mechanisms transferable to a work context. Somewhat contrary to the broad NICE guidance, existing reviews have suggested psychosocial interventions as more eﬀective than pharmacological when addressing functional outcomes such as quality of life or co-occurring challenges associated with ADHD such as anxiety or depression (Linderkamp and Lauth, 2011; Lopez et al., 2018), and thus may be more pertinent and appropriate in a work context. and thriving careers, such as concentration challenges being interpreted as a sign of underperformance, rather than supported through adjustments, it is important to revisit the existing literature to identify studies that examine relevant interventions to recognize any eﬀective mechanisms transferable to a work context. Somewhat contrary to the broad NICE guidance, existing reviews have suggested psychosocial interventions as more eﬀective than pharmacological when addressing functional outcomes such as quality of life or co-occurring challenges associated with ADHD such as anxiety or depression (Linderkamp and Lauth, 2011; Lopez et al., 2018), and thus may be more pertinent and appropriate in a work context. Attention deﬁcit hyperactivity disorder (ADHD) is a multidimensional neurodevelopmental condition that has only recently been considered to impact the lifespan (Caye et al., 2016). In the UK, ADHD is formally diagnosed by a psychiatrist where marker symptoms are inattention and hyperactivity/impulsivity (Tatlow-Golden et al., 2016) in varied constellations. Other symptoms experienced are diﬃculties with emotional regulation and challenges with social interactions (Pitts et al., 2015; Corbisiero et al., 2017). The conceptualization of ADHD is debated and often pathologized in line with the core symptoms. However, recent conceptualisations consider ADHD to be part of neurodiversity and conceptualized through a biopsychosocial model shifting the focus to understanding diﬀerence (Doyle, 2020) rather than framing ADHD as a burden (Asherson et al., 2012). Regardless of conceptualization of ADHD, the range of symptoms associated with the condition impacts on all functional domains from personal life to the workplace; for a formal diagnosis these need to be present in more than one life domain (Davidson, 2008; American Psychiatric Association, 2013). A systematic review of interventions to support adults with ADHD at work—Implications from the paucity of context-speciﬁc research for theory and practice Lauder K, McDowall A and Tenenbaum HR (2022) A systematic review of interventions to support adults with ADHD at work—Implications from the paucity of context-speciﬁc research for theory and practice. © 2022 Lauder, McDowall and Tenenbaum. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Frontiers in Psychology Frontiers in Psychology 01 frontiersin.org 10.3389/fpsyg.2022.893469 Lauder et al. Introduction The estimated 3.5% of the global workforce who are likely to have ADHD (de Graaf et al., 2008) are likely to report issues with work performance, diﬃculties in job retention, under- and unemployment, and negative work-related well-being (Küpper et al., 2012; Adamou et al., 2013; Painter et al., 2017). Therefore, it is imperative that adequate workplace support is in place to mitigate such likely negative outcomes (Adamou et al., 2013). Objectives Using realist synthesis and evaluation, the present review aims to synthesize, (a) the respective types of support/ interventions available to adult ADHDers and (b) the evidence for their eﬀectiveness in workplace contexts or on any work-relevant outcomes. Review approach As it was our aim to take a cross disciplinary review including organizational and management literature as well as complex interventions derived from clinical and health context, we took a realist evaluative approach (Pawson and Tilley, 1997). With foundations in programme theory (identifying the underlying theory about why an intervention is eﬀective), realist evaluation emphasizes the importance of context where researchers are advised to focus on “what works for whom, in what circumstances, in what respects and over which duration” (p. 15) rather than surmising whether the type of intervention is eﬀective or not (Pawson, 2013). Drawing on the premise that underlying theory provides the answers to why and how interventions work in some circumstances, but not in others (Astbury and Leeuw, 2010) we applied the CIMO-logic (Denyer et al., 2008). The context (C) is deﬁned as the environment or human factors, the intervention (I) as speciﬁed in the research question, the mechanisms (M) created by the intervention as the key components for its eﬃcacy, and the outcomes (O) ranging from performance to cost reduction (Denyer et al., 2008) also encompassing potential interactions between respective units of analysis (for an example see Doyle and McDowall, 2019) throughout the review process including the review question, data extraction, quality assessment, and interpretation of ﬁndings. The National Institute for Health Care Excellence (2019) guidelines that clinicians apply when recommending how to manage ADHD state that medications (referred to in the present review as pharmacological interventions) are the ﬁrst line of treatment once environmental modiﬁcations, through reasonable adjustments, have been implemented and reviewed. Non-pharmacological (also referred to as psychosocial) interventions are only recommended if the ADHDer does not want to use medication, has diﬃculty adhering to medication, or found it ineﬀective (National Institute for Health Care Excellence, 2019). Gaining and sustaining good work is imperative for well-being for everyone, including neurominority populations. However, little is known about what workplace environmental modiﬁcations are eﬀective and whether pharmacological or psychosocial interventions are at all eﬀective in workplace contexts to enhance work outcomes, including individual performance. Thus far, no comprehensive review of interventions for adult ADHD has focused on work-related studies or scrutinized the organizational/management literature (De Crescenzo et al., 2017; Fullen et al., 2020). Given habitual challenges experienced by ADHDers regarding eﬀective functioning Frontiers in Psychology frontiersin.org 02 10.3389/fpsyg.2022.893469 Lauder et al. on and if necessary suggest amends for the protocol review questions. Review approach Their responses were analyzed using content analysis to identify common themes as summarized in Table 1 (Krippendorﬀ, 2004). Adult ADHD was conceptualized by the panel to encompass multiple domains and contexts which aﬀect the life span. The panel stressed diﬃculties beyond the core symptoms such as working memory, self-regulation, and the high prevalence of co-occurrences. Strengths were also highlighted in some panel members’ answers including enthusiasm, passion, and loyalty. Regarding the most eﬀective intervention, psychoeducation and coaching were the most common response, emphasizing that these required delivery from a trained specialist working with ADHDers. The experts identiﬁed broad research gaps including interventions such as diet and exercise, managing speciﬁc behaviors and relationships, and the impact of diagnosis and stigma on the individual. Finally, workplace related research was identiﬁed as a priority for academia in the next 5 years given the paucity of primary evidence. FIGURE 1 Systematic review method. Review questions The panel agreed our review questions and scope; following some discussion we agreed to keep the focus broad and international notwithstanding diﬀerences in legislative frameworks which may impact on how any interventions are delivered. Guided by the CIMO framework, the ﬁnal overarching review questions were: Which interventions, documented in the literature, aim to support adult ADHDers? (a) In which contexts have any studies been conducted, (b) How can we classify types of intervention, (c) What are the mechanisms in the interventions, and (d) What are the outcomes addressed? (a) In which contexts have any studies been conducted, (b) How can we classify types of intervention, (c) What are the mechanisms in the interventions, and (d) What are the outcomes addressed? Frontiers in Psychology Study design Our inclusion criteria stipulated that participants in primary studies had to be over the age of 18 years and received a formal ADHD diagnosis using the DSM 3, 4, or 5 criteria in the treatment/intervention group prior or at the beginning of the intervention from a clinical practitioner. The intervention had to meet the following deﬁnition: “...activities, techniques, or strategies that target biological, behavioral, cognitive, emotional, interpersonal, social, or environmental factors with the aim of improving health functioning and well-being” (Institute of Medicine of the National Academies, 2015, p. 31). In line with best practice guidelines and prior research (Gough et al., 2012; Beauséjour et al., 2013), the current review incorporated an expert panel consultation (N = 4) at multiple points in the review process (see Figure 1). The panel included practitioners working in employment support for ADHDers, an academic whose research focuses on support for ADHDers, and a psychoanalyst who works therapeutically with ADHDers. Two members of the expert panel disclosed that they had received a diagnosis of ADHD and hence took the dual role of potential ‘service users’ as recommended in reviews where the public may be impacted by the ﬁndings (Gough et al., 2012). Furthermore, the interventions had to be preventative or therapeutic and not be purely diagnostic or prognostic (Santos et al., 2007) including combinations of pharmacological and psychological treatments. Interventions based on altered brain stimulation were excluded because (1) they tend to have a The experts were invited to answer nine review scoping questions including their views on deﬁnitions for ADHD, the most eﬀective intervention for ADHDers is and comment Frontiers in Psychology frontiersin.org 03 Lauder et al. 10.3389/fpsyg.2022.893469 TABLE 1 Findings from the expert panel at the research question stage. Deﬁnition of adult ADHD Eﬀective interventions Eﬃcacy of psychological interventions Research gaps • Lifelong • Working memory • Concentration • Strengths- enthusiasm, passion, loyalty, novelty. Study design • Invisible disability • Self-regulation • Aﬀects multiple domains/ cross-contextual • Co-morbid • Coaching- the coach must be experienced with ADHD • Technology • Exercise • Medication, initially rather than long-term and • Psychoeducation • Interventions involving the support network around the person • Separate treatment for co-morbidities • Coaching is eﬀective in boosting work-related performance • Group sessions • Diet and exercise • Managing hoarding and compulsive behaviors • Improving awareness among public- body decision-makers and GPs • ADHD presentation in females • ADHD in relationships • Workplace support • Stigma and marginalization • Targeted at organizational challenges- developing strategies in a job that matches interests • Success narratives • Impact of diagnosis on career success guidelines • Diet and exercise • Managing hoarding and compulsive behaviors • Improving awareness among public- body decision-makers and GPs • ADHD presentation in females • ADHD in relationships • Workplace support • Stigma and marginalization • Success narratives • Impact of diagnosis on career success guidelines TABLE 2 Databases. Medical, science, psychology and business databases ADHD speciﬁc journals Academic search complete ADHD Attention Deﬁcit and Hyperactivity Disorders Business source premier Journal of Attention Disorders Criminal justice abstracts with full text Library, information science and technology abstracts PsycARTICLES PsycINFO MEDLINE ProQuest Business collection Scopus Web of science TABLE 2 Databases. diagnostic purpose, and (2) they are not currently recommended for supporting adult ADHD (Kooij et al., 2010; National Institute for Health Care Excellence, 2019). We excluded pilots, protocols, systematic reviews and meta-analyses because of our focus on speciﬁc primary interventions. The outcomes or ﬁndings from the study had to be measurable and deﬁned as an “expected result” consistent with the PICO framework (Santos et al., 2007, p. 510). We excluded interventions that did not assess the “expected result” was excluded, for example, interventions solely assessing adverse eﬀects of the drug treatments as well as studies solely assessing outcomes non-transferable to the workplace such as physiological changes. Qualitative primary studies were included where relevant as they are suited to eliciting process focused evidence (“how”). Finally, no date restriction was placed on the searches, studies could be published or unpublished but had to be written or translated into the English language. Systematic review protocol agreed by dividing the research question into its individual elements with consultation from a specialist subject librarian and are shown in Table 3 (Petticrew and Roberts, 2006). Our review protocol was registered with PROSPERO, an international register of prospective systematic reviews (registration number CRD42018092237). Frontiers in Psychology Systematic mapping EPPI Reviewer was used to manage the data and record the decision making (Thomas et al., 2020). The ﬁrst step involved screening the study titles and removing any duplicates. The lead author screened the title and abstracts against the inclusion criteria. A member of the review team then screened 5% of the title and abstracts and any disagreements were discussed and resolved. Following a percentage agreement of 97%, Cohen’s kappa was calculated across the two reviewers and they had a score of κ = 0.86 indicating strong agreement (McHugh, 2012). The full text versions were retrieved and screened accordingly. If the full texts were not available, the reviewer emailed the author to request a copy. Figure 2 displays the PRISMA ﬁgure of the screening and selection process (Page et al., 2021). We synthesized 143 articles. Each study is listed in Table 4 (Supplementary material) with its representative (a) year of publication, (b) intervention type, (c) country, (d) total sample, (e) gender ratio, (f) design, and (g) length of follow- up in weeks. The studies were published from 1996 to 2021 as presented in Figure 3. There are two peaks in publications that reﬂect prior systematic review ﬁndings and represent the contextual shifts in understanding and diagnosis of adult ADHD. In 2008, the National Institute of Clinical Excellence (NICE) guidelines were released which unlike previous versions, included methods to support adult ADHDers. The second and most signiﬁcant peak in publications is after 2013 where the upgraded criteria for ADHDers was published in the Diagnostic Statistical Manual (DSM) version ﬁve (2013), which was the ﬁrst time ADHD symptoms and experiences in adults were noted as part of the diagnostic criteria. Then a third peak occurred in 2019, when the NICE guidelines were updated to highlight the importance of environmental modiﬁcations and pharmacological intervention rather than previously combined pharmacological and psychosocial interventions. Search strategy We undertook an inductive open-coding approach during the data extraction (Oliver and Sutcliﬀe, 2012) including some predetermined categories such as study design and participant gender ratios. During extraction we identiﬁed additional We searched databases from a variety of disciplines, including organizational and management journals and those speciﬁc to ADHD as shown in Table 2. Our search terms were Frontiers in Psychology 04 frontiersin.org Lauder et al. 10.3389/fpsyg.2022.893469 TABLE 3 Search terms. FIGURE 2 Flow chart of the reviews screening process using the PRISMA guidelines. Element Variations Adult ADHD Adult ADHD, Adult ADD, Adult Attention Deﬁcit Hyperactivity Disorder, Adult Attention Deﬁcit Disorder, adults with ADHD, adults with attention deﬁcit hyperactivity disorder, adults with ADD, adults with attention deﬁcit disorder Interventions Intervent, treat, manag, program, counsel, coach, therapy, trial, train *represents the wildcard in searches. categories including the intention-to-treat analysis (deﬁned as a type of analysis that includes data from participants who have dropped out in the latter stages) and placebo-controlled interventions. We developed further criteria for extracting information about study quality including ratings according to whether primary studies had answered their research question (Jarde et al., 2012). FIGURE 2 Flow chart of the reviews screening process using the PRISMA guidelines. Findings Our ﬁndings are presented in two parts. Part one provides a systematic map of the studies with their representative characteristics to give an overview of the interventions documented in the literature and the ﬁeld in line with the aim of the review and the overarching review question. The second part contains a realist evaluative synthesis of the interventions discussing the contexts in which they have been studied, the mechanisms in the interventions, and the outcomes addressed. A total of 22,132 participants were involved in the studies. The mean sample per study was 155. Studies had typically high levels of drop-out rates at follow up, ranging from 0 to 90% (Johnson et al., 2010). Follow-up length varied greatly, ranging from the same day to 4 years. The mean follow-up length was Frontiers in Psychology frontiersin.org 05 Lauder et al. 10.3389/fpsyg.2022.893469 FIGURE 3 Publication dates of included studies. representative underpinning theory and oﬀers a breakdown of the sub-classiﬁcations of the interventions. 16 weeks. Follow-up length was further classiﬁed into long-term and short-term with long term being 6 months or more (23% of studies) and short term <6 months (all other studies). Most studies evaluated pharmacological interventions, assessing the eﬃcacy of the three common drug treatments used to treat adult ADHD; Methylphenidate (MPH), Atomoxetine (ATX) and Lisdexamfetamine dimesylate (LDX) (k = 87). Pharmacological interventions can be further categorized according to their drug type as stimulants or anti-depressants. Caye et al. (2018) explain that psychostimulants such as Methylphenidate and Amphetamines are the ﬁrst line of treatment for ADHD because they are the most researched. Second line treatments involve Atomoxetine and anti- depressants that are often prescribed if psychostimulants are contraindicated or not tolerated or when co-occurrence is present, especially in cases of ADHD and Bipolar Disorder, any substance abuse or Tourette’s Syndrome (Caye et al., 2018). From the 143 studies, 60.8% were evaluating the eﬃcacy of pharmacological interventions, 28.7% were classiﬁed as psychosocial interventions and the remaining 10.5% evaluated the eﬃcacy of pharmacological combined with psychosocial interventions. We categorized study designs, similarly to a prior systematic review (Bower et al., 2001), into randomized control trials (RCT; k = 101), controlled before and after (CBA; includes control group; k = 14), and simple before and after (SBA; no control group; k = 28). frontiersin.org How can we classify types of intervention? A wide range of psychosocial interventions provided the basis for over a quarter (29%) of the studies which we further classiﬁed according to the theories forming the basis of the therapies including cognitive behavioral therapy, mindfulness, attention/cognition training, skills training and/or coaching, and alternative therapies. We classiﬁed interventions broadly into three groups depending on the underpinning theories and disciplines in which they were developed. The understanding that ADHD is a neurological imbalance in the brain is dominant in medical disciplines that argue the imbalance can be targeted by speciﬁc drugs and forms the ﬁrst classiﬁcation of pharmacological interventions (Durston, 2003). We based the second classiﬁcation, psychosocial interventions, on theorisations that ADHD can be treated through psychological support (Young and Amarasinghe, 2010), and the ﬁnal classiﬁcation was entitled combination interventions which included studies where a multidisciplinary approach combining both pharmacological and psychosocial interventions is deemed most eﬀective. Table 4 outlines our classiﬁcations and their The remaining 15 studies combined pharmacological and psychosocial interventions. Most of these studies were stimulant treatment combined with cognitive behavioral therapy (k = 9). Other psychosocial therapies combined with medication were mindfulness based cognitive therapy, group psychotherapy, and problem-focused therapy. One study advanced on the traditional two group comparison by comparing individual counseling to group psychotherapy in pharmacologically treated participants Frontiers in Psychology frontiersin.org 06 Lauder et al. 10.3389/fpsyg.2022.893469 TABLE 4 Study classiﬁcations, sub-classiﬁcations, and their representative number of studies and underpinning theory. TABLE 4 Study classiﬁcations, sub-classiﬁcations, and their representative number of studies and underpinning theory. Main classiﬁcation Sub-classiﬁcation Number of studies Underpinning theory (mechanisms) Pharmacological Stimulants 48 Chemical imbalance in neural networks Non-stimulants 27 Anti-depressants 8 Mixture 4 Psychosocial Cognitive behavioral therapy 14 Impact of thought on behavior and emotions Skills training/Coaching 11 Psychoeducation Attention/cognitive training 8 Regulating attention and improving cognition Mindfulness 6 Regulating attention Alternate therapies 2 Combination Stimulant and psychosocial 15 Holistic approach the delivery of the intervention that was not the clinician or individual with ADHD. aiming to explore the most eﬀective psychosocial treatment (Philipsen et al., 2007). Of the ﬁve studies, four included the ADHDer’s signiﬁcant other or family member (Virta et al., 2008; Hirvikoski et al., 2011, 2017; LaLonde et al., 2013). How can we classify types of intervention? The ﬁnal study included a “support person” or “coach” for help with organizational tasks, and if the ADHDer did not have a support person from their own social network, an undergraduate student was allocated to them to adopt the support person role (Stevenson et al., 2002). All studies reported positive ﬁndings with improvements in outcomes beyond reducing symptoms like employment, maintaining relationships, organization skills, self-esteem, and knowledge about ADHD. In short, involving the support network around the ADHDer has marked eﬀects on all outcomes emphasizing that an encouraging and supportive environment can increase the impact of an intervention. Involvement of others Group interventions tended to include individuals from the ADHDers support network as part of the intervention. Including signiﬁcant others during the intervention is arguably eﬀective and linked to psychoeducation whereby the ADHDer and their partners or family is empowered with knowledge about ADHD and how to better support it (Lukens and McFarlane, 2004). So far, research examining the eﬃcacy of including family members in interventions for ADHD has focused on children and adolescents (Kaslow et al., 2012). Only ﬁve interventions in the present review involved someone in In which contexts have the studies been conducted? Contexts are deﬁned as environmental factors that aﬀect behavior change (Denyer et al., 2008). These contextual factors can be separated into four layers: the infrastructural system, the institutional setting, interpersonal relationships and the individual themselves (Pawson and Tilley, 1997). We mapped the 143 included studies onto the four areas with referrals being infrastructural system, location as an institutional setting, clinician-patient relationship as part of the interpersonal relationships and lastly, co-occurring at the individual layer. The group We classiﬁed the interventions classiﬁed by mode of delivery. Of the 143 studies synthesized, 21 were delivered to a group or involved a combination of group and individual delivery. All 21 studies were classiﬁed as psychosocial or a combination of pharmacological and psychosocial. Of the 21 studies, 20 were classiﬁed as eﬀective, 12 were conducted in European countries with 12 being conducted in universities, research centers or university hospitals. Group interventions are argued to be beneﬁcial for sharing lived experiences, coping strategies, increasing feelings of belongingness and knowledge about ADHD (Bramham et al., 2009; Jackson et al., 2014; Fullen et al., 2020). Learning in a group also increases self-eﬃcacy through higher levels of hope and motivation (Bramham et al., 2009; Tian et al., 2018). However, only six studies directly assessed outcomes relating to self-esteem or eﬃcacy with one study additionally measuring social functioning. Therefore, it is diﬃcult to compare the eﬀectiveness of group vs. individual interventions because the theorized beneﬁts are broad and not consistently measured. Referrals and dropout From the 143 studies 90 were outpatient referrals which means that the adults had received a diagnosis and were immediately referred by the psychiatrist for their ﬁrst set of treatment at a specialist center or clinic (Kooij et al., 2010). Another method of recruitment was to advertise the intervention and remunerate participants with a formal Frontiers in Psychology frontiersin.org 07 10.3389/fpsyg.2022.893469 Lauder et al. The clinician-patient relationship The clinician-patient relationship diagnosis (k = 6). These two methods of recruitment attract and include participants who are already aware that they may have ADHD or have recently been diagnosed. Pawson (2013) suggests that with medication it is diﬃcult to pinpoint the exact moment in which the intervention began, which is particularly true with outpatient referrals as basic knowledge or understanding of ADHD may exist prior to the referral for treatment. As a result of going through the diagnostic process, some level of psychoeducation, researching and learning about ADHD, might have already happened prior to medical treatment. Consequently, it is unclear whether the intervention began at the point in which the ADHDer began to learn about ADHD or at the point medication is initiated. This level of self-awareness and knowledge is diﬃcult to measure and is likely to diﬀer greatly between individuals. With an increase in easily available information like self-help and guidance online, it is important to consider how potential misinformation or accurate information may inﬂuence how diﬀerent interventions are perceived before interventions begin. Prior research has drawn attention to the signiﬁcance of the patient and clinician relationship as well as healthcare outcomes (Kelley et al., 2014). As a potential mechanism, it is argued that the better the quality of the relationship, the quicker the recovery and the higher the rate of adherence (Thompson and Mccabe, 2012). The relationship between the clinician-patient is often layered and dynamic making it diﬃcult to directly assess or compare between studies (Street et al., 2009). Key components of an eﬀective relationship are similar in medical and educational settings with research suggesting the following: good management of emotion, high patient or coachee knowledge of their own condition, client/coachee centered approaches and good communication with shared understanding (Street et al., 2009; Jackson et al., 2014; Kelley et al., 2014; Lai and McDowall, 2014). Many of the 143 included studies relied on clinician ratings of symptoms and rarely included the patient. Co-occurrence As a diagnosis, ADHD is rarely present without co- occurrences, clinicians suggest that co-occurrence with depression and anxiety is particularly prevalent due to the experiences of failure, lack of support, and challenges with regulating emotion (Jensen et al., 1997). In experimental study designs, co-occurrence is considered a confounding variable because it is diﬃcult to isolate any beneﬁcial eﬀects of an intervention to a speciﬁc condition (Fortin et al., 2006). Consequently, most studies excluded co-occurrences as part of their criteria (k = 135), whereas others deliberately addressed ADHD with co-occurrences like social anxiety disorder or substance use disorder (k = 8). In contrast, there is an argument as to whether removing or excluding participants with co- occurring conditions lessens the external validity because they provide an unrealistic view of ADHD (Fortin et al., 2006). Referrals and dropout In addition, nearly all the studies had no means of measuring the impact of the clinician/patient relationship with two measuring patient experience directly. In context, as many of the studies were in outpatient clinics, the initial appointment for the treatment was most likely the ﬁrst point of contact after receiving a diagnosis for a large proportion of the ADHDers, enhancing the importance of a positive and meaningful interaction. Although no measures or understandings were assessed in these studies. We therefore identify the clinician-patient relationship as potentially inﬂuential and recommend further investigation. g Dropout rates varied greatly across the studies. Adherence is a central part of assessing the eﬃcacy of an intervention (Horwitz and Horwitz, 1993). In pharmacological studies, adherence is measured through self-report during follow up sessions recording whether the participant has taken the medication or not. In psychosocial interventions, it is typically assessed by attendance. A review of medical adherence in children and adult ADHDers found non-adherence rates that ranged from 13.2 to 64% but concluded that there is minimal research addressing reasons for non-adherence in adults (Adler and Nierenberg, 2015). Qualitative research has identiﬁed forgetfulness, a challenge for ADHDers, and lack of guidance from clinicians as potential barriers to medication treatment adherence (Matheson et al., 2013). Therefore, it important for future research and practice to consider adherence as an inﬂuential factor in the eﬃcacy of pharmacological interventions and identify and remove any potential barriers. Location The most common settings for pharmacological studies were outpatient clinics or multi-center clinics in North America (k = 47). The majority of participants had been referred to the clinic, received a formal diagnosis and then received a treatment. Assessing the eﬀectiveness of pharmaceutical interventions across multiple clinics not only provides researchers and clinicians with information about the impact of the drug in multiple countries, but also provides an indication of the prevalence of ADHD across cultures (Polanczyk et al., 2007; Wang et al., 2017). Research centers and university settings (k = 24) were more likely to study the eﬀectiveness of psychosocial interventions, but unlike common critiques of a student sample and the lack of ecological validity, only two of these studies recruited student samples indicating good generalisability (Ward, 1993). What are the mechanisms in the interventions? Mechanisms can be deﬁned as the processes or underpinning methods in which the intervention operates in a speciﬁc context to produce a speciﬁc outcome that can be triggered in some contexts and not in others (Denyer et al., 2008; Dalkin et al., 2015). We understand mechanisms as the fundamental processes in which interventions are expected to be eﬀective, relating them to the disciplines in which the What are the mechanisms in the interventions? Frontiers in Psychology frontiersin.org 08 Lauder et al. 10.3389/fpsyg.2022.893469 mindfulness was eﬀective for all participants, not just those with ADHD (Bachmann et al., 2018). interventions were developed and theorized, for example, brain chemistry, cognition, and psychoeducation (Denyer et al., 2008). Cognition Cognitive models of ADHD explain a deﬁcit in the prefrontal cortex which is responsible for executive function (Willcutt et al., 2005). Both Barkley (1997) and Brown (2006), leading researchers in ADHD, have developed theoretical models that explain ADHD as a diﬃculty in managing executive function resulting in impulsive and inattentive behavior. These models have been the foundations behind interventions such as cognitive behavioral therapy (CBT), cognitive remediation, and working memory or attention training. CBT was developed to address anxiety and depression by altering thought processes and behavior to avoid the repetitive negative thinking and corresponding behavior (Beck and Beck, 2011). In a Cochrane review, CBT is argued to treat ADHD by tapping into the negative thinking which has been a result of the negative experiences associated with the core symptoms (Lopez et al., 2018). ADHD is also highly co-occurring with anxiety and depression supporting the use of CBT to target co- occurring symptoms. The techniques used in CBT often include psychoeducation followed by the acquisition of techniques to address the individual challenges the person experiences (Huppert, 2009). Goal setting is an integral part of CBT and is useful in assessing eﬀectiveness (Beck and Beck, 2011). Of the 23 studies that assessed the eﬃcacy of CBT alone or with medication, eight were long term and half included measures of depression and/or anxiety ﬁnding positive eﬀects in all studies (k = 11). What outcomes have been addressed? The outcomes assessed in each intervention varied greatly across the 143 studies. Initially, we classiﬁed the primary outcomes according to whether they involved a measurement of the core symptoms. Those outcomes beyond the core symptoms were further classiﬁed into what they assessed: behavior, cognition, physical/functioning, social, and person/emotion. We additionally discuss the outcomes in relation to short-term and long-term and qualitative methods. Brain chemistry Psychoeducation aims to enhance a person’s understanding of mental health by increasing the individual’s knowledge and awareness of their condition and supporting them in sharing their experiences (Getachew et al., 2009). It also oﬀers the opportunity for those supporting the individual such as family members, to help support ADHDers (Anderson et al., 1980). The idea is that self-awareness and knowledge is key in learning strategies to manage any condition or increase functioning rather than simply reduce the symptoms (Lotﬁet al., 2010). In some areas, psychoeducation is argued to adopt a strengths- based approach (Lukens and McFarlane, 2004). Despite the strong evidence base as an intervention for aﬀective disorders and preliminary evidence in interventions for children and adolescents with ADHD, there is limited research applying psychoeducation with ADHDers (Lukens and McFarlane, 2004; Dahl et al., 2020). Seven studies mentioned their use of psychoeducation typically combining it with another therapy or training (Wiggins et al., 1999; Hirvikoski et al., 2017; In de Braek et al., 2017; Bachmann et al., 2018; Hoxhaj et al., 2018; Gaur and Pallanti, 2020; Hartung et al., 2020). These studies indicated improvement in a range of outcomes including executive functioning, time management/organization skills, general functioning, and knowledge, as well as knowledge and coping in signiﬁcant others. Therefore, we consider psychoeducation as a key mechanism that is worth incorporating in future design of interventions because of the variety of beneﬁts for the ADHDer as well as signiﬁcant others. A large proportion of the studies were pharmacological in nature. From our realist synthesis and evidence-based medicine perspective, randomized controlled drug interventions are the gold standard because the mechanisms, contexts and outcomes have been explored years before the randomized control trial is carried out (Pawson, 2018). For interventions involving stimulants, the process prior to testing the stimulant in humans is extensive (Lipsky and Sharp, 2001). Therefore, the theories and mechanisms have been well-established prior to the intervention. Frontiers in Psychology Beyond the core symptoms We categorized a range of outcomes beyond core symptoms further which are displayed with relevant examples in Table 5 alongside the number of studies involving these types of measures. The vast range in outcomes suggests the impact of ADHD to all aspects of life beyond the core symptoms, from social relationships to general self-esteem. Regarding eﬀectiveness, primary outcomes related to cognition were associated with mixed or unclear results. Outcomes relating to social and emotion/person were assessed more often in psychosocial interventions with overall positive eﬀects of the intervention. Aside from cognitive assessments of outcomes which typically involve using technology, and the Clinical Global Impression scale (k = 41), which is purely based on the clinicians rating, many outcomes were assessed using self-report rating scales. In one study, a strength of ADHD, creativity, was measured and improved after the administration of a stimulant which is in line with the expert panel, that strengths are important to consider (Kahn, 2008). Evaluating outcomes associated with strengths supports the neurodiversity conceptualization of ADHD and challenges the pathological approach. Reducing core symptoms Core outcome measures were deﬁned as those assessing the three core symptoms of adult ADHD; impulsivity, inattention, and hyperactivity. Of the 143 studies, 108 assessed a reduction in core symptoms as their primary outcome. There are several validated measures for adult ADHD and in most of the studies a mixture of these measures was used pre and post intervention. The most popular being Conner’s Adult ADHD Rating Scales (CAARS) (k = 44) and the ADHD Rating Scale (ADHD-RS) (k = 38). The authors of the ADHD-RS are prominent authors in the intervention studies and the CAARS was used to validate the measure. It is important to consider the authors as potential Mindfulness formed the intervention in seven of the studies and in many was combined with CBT (Bueno et al., 2015; Edel et al., 2017; Bachmann et al., 2018; Gu et al., 2018; Hoxhaj et al., 2018; Hepark et al., 2019; Nicastro et al., 2021). Similar to CBT, mindfulness aims to tap into cognition. However, CBT is focused on attention rather than other symptoms and aims to focus attention on the present, inner emotions and acceptance (Pirson, 2014). Most of these studies had positive eﬀects on reducing symptoms, one found no diﬀerence in measures of memory (Bueno et al., 2015) and another found that Frontiers in Psychology frontiersin.org 09 Lauder et al. 10.3389/fpsyg.2022.893469 10.3389/fpsyg.2022.893469 sources of bias because of their involvement in developing the measures. theme highlighting the importance of trusting relationships with the clinician delivering the intervention (Björk et al., 2020), and the other emphasizing the sense of belonging and shared experience in group interventions (Nordby et al., 2021). Long-term vs. short-term We classiﬁed studies as short-term if the treatment to follow- up was under 6 months (k = 111) and long-term if they were 6 months or more (k = 32). Combination treatments were more likely to be long-term than short-term (k = 6) indicating a longer follow-up. A total of 78% of pharmacological interventions were short-term with more than half of these (56%) lasting <12 weeks suggesting an immediacy to the expected eﬀectiveness. In contrast, in psychosocial studies, therapeutic eﬀect is assumed to be less immediate as they tend to address a wider range of symptoms and co-occurrences (Biederman et al., 2012). Long- term research into the impact of ADHD across the life span indicates that symptoms beyond the core symptoms such as functionality and anxiety become more prominent over time, yet current research does not reﬂect this because the majority of studies are short term and evaluating pharmacological interventions (Ingram et al., 1999). We then manually searched each study to identify any outcomes relevant to the workplace but not explicitly measuring workplace performance or employment, 35 studies included measurements of outcomes that involved subscales assessing work-relevant outputs. We further grouped these instruments into two categories, those that assess general organization and time management and others that assess general life functioning, including a subscale measuring workplace functioning. General organization and time management scales were present in 13 studies and included scales like “ON-TOP” or “On Time Management Organization and Planning scale” and an adult adapted version of the Child Organisation Skills Measure (COSM). A total of eight studies used the same functioning measure entitled the Sheehan Disability Scale that requires participants to rate on a Likert scale how much they feel their disability impacts their work, family/home life and social/leisure activities. Other measures relating to life satisfaction and functioning use a similar form of including work as a domain that could be impacted. Most of the work-relevant measures lacked suﬃcient reliability or validity estimates or consisted of only one item which further limits the reliability of the study’s ﬁndings and implications. The workplace Our review aimed to include intervention studies that were speciﬁcally related to the workplace, carried out in workplace contexts, or involving workplace outcomes. Unfortunately, no studies were conducted in workplace contexts apart from two studies that were carried out in a simulated workplace environment (Wigal et al., 2010, p. 20). A total of four studies included workplace outcomes as one of their primary outcomes with a further 11 including a including work related outcomes as a secondary outcome. The four studies that primarily assessed work-related outcomes were categorized as pharmacological or combined. The studies that assessed the eﬃcacy of both pharmacological and psychosocial interventions combined had a positive impact on work outcomes improving functioning at work (Dittner et al., 2018) and maintaining employment (LaLonde et al., 2013). The pharmacological studies did not have a positive eﬀect with there being no diﬀerence in work productivity (Adler et al., 2008) and no improvement in occupational status at follow up (Torgersen et al., 2014). Hence, the psychosocial aspect of the intervention might be a key mechanism having a direct impact on work outcomes. Assessment of risk of bias TABLE 7 Adapted risk of bias scoring tool. TABLE 7 Adapted risk of bias scoring tool. Score /24 Risk of bias Interpretation 17–24 Low risk of bias Bias, if present, is unlikely to alter the results seriously 9–16 Unclear risk of bias A risk of bias that raises some doubt about the results 0–8 High risk of bias Bias may alter the results seriously Adapted from Higgins et al. (2011). After the full texts were extracted, we assessed them for risk of bias (Higgins and Green, 2011). We assessed quality using a checklist of 18 questions adapted from three existing quality assessment tools recommended in the Cochrane guidance to calculate a numeric score. We drew on The Newcastle-Ottawa scale (Wells et al., 1999), the Cochrane Collaboration’s tool for assessing risk of bias (Higgins and Green, 2011), and the Qualitative Research Checklist from the Critical Appraisal Skills Programme (Tong et al., 2007) and mapped their criteria on the CIMO framework to reﬂect the purpose and interests of the present review, see Table 6 for domains and example items. We calculated a score for each study based on the checklist which we then compared to the scores in Table 7 as an overall assessment of bias. designed for commercial implications), or little theoretical application which we note upfront as a limitation regarding the generalisability of the ﬁndings. We rated a total of 1% of the studies as high risk of bias and these tended to include poorer ratings across the categories. On the other hand, a large percentage of studies were rated at a low risk of bias in domains of performance (62%) and selection (75%) indicating a strength in research around blinding of the control and the intervention group. A further strength we identiﬁed was the appropriate recruitment strategy to encourage participation from individuals who are generalisable to the ADHD population compared to recruiting student samples which are often critiqued for their lack of ecological validity (Bello et al., 2014). We rated most studies (60%) as unclear regarding risk of bias, which raises some doubt about the results, due to the ambiguity and the lack of detail provided in the studies’ methodology and ﬁndings (see Figure 4). Regarding any psychosocial studies, it was a challenge to comprehend the details of intervention the “skills training” interventions listed the skills they targeted but did not provide examples of how these skills were targeted. Qualitative analysis There were two studies that evaluated the eﬀectiveness of interventions using qualitative methods (Nordby et al., 2021) with one using both qualitative and quantitative data to support their ﬁndings (Björk et al., 2020). Both studies interviewed participants about their experience of completing the interventions and extracted themes using thematic and content analysis. Their analyses support the key mechanisms identiﬁed for eﬀective psychosocial interventions with one Frontiers in Psychology frontiersin.org 10 Lauder et al. 10.3389/fpsyg.2022.893469 TABLE 5 Outcomes by category with examples and total number of studies assessing them. Outcomes Example scales No of studies assessing outcomes Behavioral On Time Management Organization and Planning scale (ON-TOP) Substance/Alcohol use 24 Cognitive Continuous Performance Test (CPT) Verbal memory (WMS-R) 38 Physical/Functioning Clinical Global Impression (CGI) Adult ADHD Quality of Life Scale (AAQoL) Global Assessment of Functioning (GAF) 76 Social Social Adjustment Scale Self-Report (SAS-SR) Family Functioning (FAM-111) 11 Emotion/Person Hamilton Rating Scales for Anxiety/Depression (HAM-A/HAM-D) Beck’s Depression Inventory (BDI) General Self-Eﬃcacy Scale (GSES) 51 No of studies assessing outcomes TABLE 6 Example items from the risk of bias tool. Domain Example items Total items Detection Were participants blind to the outcome assessment? 1 Attrition Did the data suﬃciently support the ﬁndings? 2 Reporting Have ethical issues been considered? 6 Selection Was the recruitment strategy appropriate to the aims of the research? 4 Performance Were participants blind to the intervention rationale? 3 Other What was the length of follow-up in months? 3 Assessment of risk of bias The lack of detail may be due to publication restrictions on word count, privacy (training Frontiers in Psychology 11 frontiersin.org 11 Lauder et al. 10.3389/fpsyg.2022.893469 FIGURE 4 Risk of bias. Discussion eﬃcacy and ultimately the self-awareness and understanding. Student/patient/coachee led interventions also seem to increase the eﬀectiveness by encouraging autonomy of the challenges to address and outcomes relating to self-esteem and self-eﬃcacy. In addition, interventions involving a signiﬁcant other seem to be eﬀective in supporting the person as a whole and increasing the knowledge of those in the individual’s social network. In sum, these theoretical underpinnings can be used to guide further research. Frontiers in Psychology Summary of main ﬁndings Of 143 studies which documented interventions to support ADHDers a large proportion has been conducted in the clinical and medical ﬁelds on the eﬃcacy of pharmacological interventions, subsequently inﬂuencing existing policy and guidelines. Our review did not locate relevant interventions in workplace contexts, designed speciﬁcally for workplaces, based on theory relevant to the workplace, and limited research measured outcomes speciﬁc to employment. This lack of evidence signposts a clear need for robust research investigating psychosocial interventions combined with or compared to pharmacological interventions. Therefore, it is evident from the existing synthesis to build an evidence base that is transferable to the workplace. Key mechanisms to further examine and include in future research are group interventions, inclusion of the support network around the ADHDer, the clinician-patient relationship, psychoeducation, and student/patient/coachee led interventions. Important considerations for future research and practice that are based on the review ﬁndings are listed in Table 8. Our review ﬁndings highlight the necessity for future intervention research aimed at supporting ADHDers to include a workplace component and assess primary work-relevant outcomes using reliable and valid scales. Intervention research should assess the eﬃcacy of the intervention on a range of outcomes including the three core symptoms as well as skills-related outcomes and functioning in life and at work. In addition, studies should examine whether skills-based and cognitive behavioral therapies are applicable across contexts and demonstrate far transfer to the workplace. Based on the NICE guidelines, the ﬁrst step in managing ADHD is to make modiﬁcations to the environment which is in line with the legal requirements for employers to make reasonable adjustments (changes to the environment) for employees with a disability. Future research and practice need to examine what adjustments and modiﬁcations are eﬀective and revisit whether any mechanisms of existing psychosocial interventions can be applied to the workplace More speciﬁcally, our review ﬁndings highlighted that psychosocial interventions, especially training and coaching need to explicitly outline their methods, mechanisms, and theoretical grounding. Psychoeducation is a potential mechanism in interventions that greatly inﬂuences the Frontiers in Psychology 12 frontiersin.org Lauder et al. 10.3389/fpsyg.2022.893469 TABLE 8 Findings from the realist evaluation summarized into important factors. Conclusions From the 143 studies synthesized, none evaluated the eﬃcacy of workplace interventions. Despite the lack of workplace speciﬁc intervention studies, the synthesis identiﬁes eﬀective mechanisms that could be applied to the workplace context such as group-based interventions and those which involve elements of psychoeducation, where the social support network around the ADHDer is included in the intervention. Interventions categorized as psychosocial were applicable to the workplace because they are likely to improve symptoms related to emotional regulation and social interactions that are important for work contexts. Workplace outcomes are often considered as secondary, and any skills-based interventions target general skills rather than speciﬁc work- relevant skills training. As a result, it is unclear whether the recommended support for ADHDers at work is evidence- based and accessible for practitioners who advise employees with ADHD and the employees themselves. There is a clear need to implement theory-driven, rigorously designed and context relevant interventions to facilitate the transfer of Summary of main ﬁndings Important factors in interventions for ADHDers Context Societal Access to diagnosis and support, socioeconomic status, national and international policy and guidelines Settings Applicable to a range including: Medical/educational/workplace Interpersonal Involving others in the intervention, promoting successful clinician-patient/coach-coachee relationships Individual Address co-occurrence Intervention Pharmacological Blinded experimenter and both control and treatment group Psychosocial Autonomy in topics/skills to address, clear methodology and detail of what the intervention involved Group/individual Beneﬁts of the group on shared experiences and meaningfulness Combination Need more studies involving a combination of pharmacological and psychosocial interventions. Mechanisms Pharmacological Psychological Brain chemistry Cognition and psychoeducation Outcomes Core symptoms Measured by the clinician, should include participant response and family/workplace ratings Beyond core symptoms Measure outcomes from all aspects of life and symptomatology e.g., life functioning, emotion, and anxiety. Long-term vs. short-term Long-term eﬀectiveness is imperative Bold is for future considerations for research. Long-term eﬀectiveness is imperative Bold is for future considerations for research. were likely to exhibit symptoms which may have inhibited workplace performance and progression. In other words, we cannot preclude that the samples may have been less likely to comprise ADHDers for example in senior, managerial or professional roles. because this review identiﬁes they are eﬀective for work- relevant outcomes. The expert panel identiﬁed disclosure of ADHD to be a signiﬁcant barrier to accessing workplace support as well as access to coaching. Members of the panel highlighted the importance of psychoeducation and self-awareness of an ADHDers experiences in knowing which support is most eﬀective for them, emphasizing a personalized approach. Therefore, structural barriers to accessing support for ADHDers should also be considered in future research and practice. Frontiers in Psychology Limitations A limitation of the review, and many others in management research, is that it did not include any gray literature despite eﬀorts to ask the expert panel for recommendations (Rojon et al., 2021). Gray literature is important because it can reduce publication bias and inﬂuence the review synthesis (Gough et al., 2012). Hence, there may be existing work-related interventions in practice that are or are not documented in the gray literature and these could include eﬀective mechanisms/designs that inﬂuence the review’s practical recommendations. For example, the expert panel ﬁndings indicated that coaching is often used in practice yet only two studies evaluated its eﬃcacy (Stevenson et al., 2003; Kubik, 2010). Given the typical primary settings, we also surmise that study participants Frontiers in Psychology 13 frontiersin.org Lauder et al. 10.3389/fpsyg.2022.893469 10.3389/fpsyg.2022.893469 Acknowledgments learning to the workplace. Furthermore, additional research is required regarding psychosocial interventions to overcome contemporary workplace challenges such as managing concurrent work tasks and navigating complex teams. Only once we address such important topics can we be conﬁdent that we have a rigorous evidence base to support ADHDers at work. The authors wish to thank the members of the expert panel for their knowledge and advice and Jessica Dark for assisting with the screening of articles. 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https://openalex.org/W4220889736	https://research.rug.nl/files/625551639/08862605221084741.pdf	English	null	The Interplay Between Status and Affection Needs: Testing the Imbalanced Needs Theory of Aggression in Adulthood	CrimRxiv	2,022	cc-by	9,503	University of Groningen University of Groningen The Interplay Between Status and Affection Needs Sijtsema, Jelle J.; Lindenberg, Siegwart M. The Interplay Between Status and Affection Needs Sijtsema, Jelle J.; Lindenberg, Siegwart M. IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Sijtsema, J. J., & Lindenberg, S. M. (2023). The Interplay Between Status and Affection Needs: Testing the Imbalanced Needs Theory of Aggression in Adulthood. 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Download date: 24-10-2024 20916 JIVXXX10.1177/08862605221084741Journal of Interpersonal ViolenceSijtsema and Lindenberg 20916 JIVXXX10.1177/08862605221084741Journal of Interpersonal ViolenceSijtsema and Lindenberg Original Research ttps://doi.org/10.1177/08862605221084741 Journal of Interpersonal Violence 2023, Vol. 38(1-2) 772–795 © The Author(s) 2022 Article reuse guidelines: sagepub.com/journals-permissions DOI: 10.1177/08862605221084741 journals.sagepub.com/home/jiv The Interplay Between Status and Affection Needs: Testing the Imbalanced Needs Theory of Aggression in Adulthood Jelle J. Sijtsema, PhD1and Siegwart M. Lindenberg, PhD2,3 Abstract Status and affection are both goals related to social needs. The imbalanced needs theory of aggression proposes that although aggression can be used to realize status, this strategy is detrimental for realizing affection in the same social context. Thus, to the degree that the social circles overlap in which status and affection needs are realized, it becomes more costly (in terms of affection) to achieve status via aggression. This theory was tested for different forms of aggression, in different contexts, in a sample of adults from the general population (N = 253, M age = 29.95, SD = 2.60, 78% female). Par- ticipants reported on social needs with the Interpersonal Goals Inventory and reported on general measures of physical and social aggression, as well as rule breaking, and aggression at the workplace and in intimate partner relation- ships. As hypothesized, status needs were associated with physical aggression when affection needs were weak. This interaction, though to a lesser degree, also extended to social forms of aggression and rule breaking. At the 1Developmental Psychology, Tilburg University, the Netherlands 2Sociology, University of Groningen, The Netherlands 3Social Psychology, Tilburg University, The Netherlands Corresponding Author: Jelle J. Sijtsema, Department of Developmental Psychology, Tilburg University, 5000 LE Tilburg, The Netherlands. Email: j.j.sijtsema@tilburguniversity.edu Corresponding Author: Jelle J. Sijtsema, Department of Developmental Psychology, Tilburg University, 5000 LE Tilburg, The Netherlands. Email: j.j.sijtsema@tilburguniversity.edu Keywords social goals, aggression, development, status, affection Interpersonal aggressive behavior is often explained by individual charac- teristics related to personality traits, psychopathology, and associated factors (Anderson & Bushman, 2002; Babcock et al., 2014). However, what are the most important social determinants of interpersonal aggression? Decades of research into this question have identiﬁed several social determinants of aggression, such as social rejection, violent environments, and violent media (Warburton & Anderson, 2015). Thus, socially determined aggression is mostly seen as a reaction to a hostile environment. We have no reason to doubt the research in this area. However, much aggression also occurs without exposure to hostile environments, in the pursuit of daily life, and a pressing question is what the determinants of this kind of aggression are, if it is not due to psychopathology or hostile environments. Recently, we suggested that one needs to look at the social function of aggression from the perspective of a need-based goal framework, and we found empirical support for such an approach in adolescence (Sijtsema et al., 2020). The question remains whether we also ﬁnd support for this theory for adults. Abstract Status and affection are both goals related to social needs. The imbalanced needs theory of aggression proposes that although aggression can be used to realize status, this strategy is detrimental for realizing affection in the same social context. Thus, to the degree that the social circles overlap in which status and affection needs are realized, it becomes more costly (in terms of affection) to achieve status via aggression. This theory was tested for different forms of aggression, in different contexts, in a sample of adults from the general population (N = 253, M age = 29.95, SD = 2.60, 78% female). Par- ticipants reported on social needs with the Interpersonal Goals Inventory and reported on general measures of physical and social aggression, as well as rule breaking, and aggression at the workplace and in intimate partner relation- ships. As hypothesized, status needs were associated with physical aggression when affection needs were weak. This interaction, though to a lesser degree, also extended to social forms of aggression and rule breaking. At the 1Developmental Psychology, Tilburg University, the Netherlands 2Sociology, University of Groningen, The Netherlands 3Social Psychology, Tilburg University, The Netherlands 1Developmental Psychology, Tilburg University, the Netherlands 2Sociology, University of Groningen, The Netherlands 3Social Psychology, Tilburg University, The Netherlands 773 0(0) Sijtsema and Lindenberg 2 workplace, aggression was only related to weak affection needs, whereas aggression in intimate partner relationships was, as expected, unrelated to both social needs. Together, these ﬁndings support the results of an earlier test of the imbalanced needs theory of aggression in adolescence, and encourage more research into the link between aggression and the satisfaction of social needs. The Imbalanced Needs Theory of Aggression The background of the “imbalanced needs theory of aggression” is that social goals are important determinants of behavior (Lindenberg, 2013) and have been increasingly explored explicitly in research on the behavior of children and adolescents (Crick & Dodge, 1994; Heidgerken et al., 2004; Ojanen et al., 2005; Renshaw & Asher, 1983; Sijtsema et al., 2009; Volk et al., 2012). The main focus of these studies was on two kinds of goals that are often thought to be negatively correlated, but that turned out to be orthogonal (Ojanen et al., 2005): a communal goal, related to warmth, support, love and intimacy, also referred to as affection goals, and an agentic goal, related to power, domi- nance, and a feeling of superiority, also referred to as status goals (see also Locke, 2003). Both kinds of goals can be linked to universal interpersonal needs (Anderson et al., 2015; Bakan, 1966; Lindenberg, 2013). Most indi- viduals endorse both status and affection goals, to differing degrees (Ormel Journal of Interpersonal Violence 38(1-2)3 774 Sijtse et al., 1997; Steverink et al., 2020). Attaching importance to status goals has been associated with aggression (Caravita & Cillessen, 2012; Ojanen & Findley-Van Nostrand, 2014; Reijntjes et al., 2016; Sijtsema et al., 2009). However, aggression is likely to reduce realizing affection in the relationship with the very same others one would like to be superior to. This very fact led us to formulate and test what we called the imbalanced needs theory of aggression among adolescents (Sijtsema et al., 2020). This theory of social determinants of aggression rests on two main theoretical pillars: ﬁrst, indi- viduals have social needs, for status and for affection, to varying degrees. Second, aggression is an effective means for achieving status as long as it does not diminish the realization of affection. It follows that the strength of the status goal will be positively correlated with showing aggression when status and affection can be realized in separate social circles. To the degree that these circles overlap, two things happen, compared to separate circles: (a) using aggression as a means for achieving status will become costlier in terms of losing affection; and (b) because using aggression for achieving status is detrimental for the satisfaction of social needs, using aggression as a means for achieving status will become more socially disapproved when these circles overlap. Sijtsema and Lindenberg 4 Sijtsema and Lindenberg 4 In our previous study on the imbalanced needs theory of aggression (Sijtsema et al., 2020), we found that the strength of the status goal was associated with direct aggression across adolescence. However, in middle adolescence, this association was only observed when the affection goal was weak. We argued that these ﬁndings were due to more integrated social circles of interaction with age. That is, in comparison to younger adolescents, status and affection for older adolescents were realized more often in the same circle of interaction, namely the peer group. Thus, as the circles of interaction for the achievement of status and affection overlap more, aggression will become more costly and decline. The theory predicts that in adulthood, the circles for the realization of status and affection overlap even more than in later ado- lescence and that the use of aggression for the achievement of status meets with even greater disapproval than in later adolescence. We will apply this theory to different forms of aggression and to different social contexts and test the hypotheses that derive from these applications. Status and different forms of aggression. In line with our previous test of the theory for adolescence regarding physical aggression, our ﬁrst hypothesis for adults to be tested relates to physical aggression as well. Physical ag- gression hypothesis: In adulthood, the strength of the status goal is related to physical aggression, but only when the affection goal is weak. There are also other forms of aggression, especially social aggression and rule breaking, and the question arises how the theory applies to these forms. Social aggression refers to the intention to harm somebody’s social standing and reputation (e.g., “to ridicule someone behind their back”), rather than harming somebody physically. Because social aggression has been shown to be a means for the achievement of status (Adler & Adler, 1998; Prinstein & Cillessen, 2003), just like physical aggression, our second hypothesis to be tested relates to social aggression in analogy to the hypothesis about physical aggression. Social aggression hypothesis: In adulthood, the strength of the status goal is related to social aggression, but only when the affection goal is weak. Theories that aim to explain the mechanism behind a phenomenon should not just explain under what conditions the phenomenon occurs, but also under what conditions it does not occur. The Imbalanced Needs Theory of Aggression As a result, the correlation of the strength of the status goal and aggression will be lower, except for people who happen to be high on the status goal and low on the affection goal. The theory also predicts an age effect: compared to childhood and early adolescence, social circles in later adolescence will increasingly overlap, and thus the correlation between the strength of the status goal and the use of aggression is predicted to decline during later adolescence. In adulthood, this correlation is predicted to decline even further, because the social circles for the realization of status and af- fection are even more integrated (Snijders et al., 2013) and social disapproval of showing aggression becomes even stronger (e.g., Laub & Sampson, 1993), because “being adult” becomes associated with the expectation of being able to restrain one’s aggression (Sweeten et al., 2013). Negative reputational effects of showing aggression (as is observed with gossiping; see Feinberg et al., 2012) will reinforce this expectation. Conversely, self-regulation needed to meet these expectations is likely to become stronger with age, allowing greater restraint on the use of aggression when it interferes with other im- portant goals and external regulation (Williams et al., 1999). The theory also implies context effects in addition to the overlap of social circles: (a) if the context provides good alternatives for achieving status without the use of aggression, the correlation between the strength of the status goal and aggression will be weak, compared to a situation without this al- ternative. (b) If the context makes the achievement of affection more im- portant than the achievement of status, then the correlation between the strength of the status goal and aggression will be also be weak. 775 0(0) Sijtsema and Lindenberg 4 Rule-breaking as a form of aggressive behavior may be used for achieving status only in adolescence. The reason for this is that rule breaking has been linked to the maturity gap—the discrepancy between social and biological maturity—as a form of antisocial behavior, including aggression, that proves status in the form of one’s autonomy vis-a- vis parents and other authorities (Dijkstra et al., 2015; Koepke & Denissen, 2012). The maturity gap closes progressively in later adolescence (Mofﬁtt, 1993), which makes that the circles for the realization of status and affection become even more integrated and that the use of aggression for any goal achievement (including status) becomes socially less acceptable (Weaver & 776 Sijtse Journal of Interpersonal Violence 38(1-2)5 McNeill, 2015). Our third hypothesis to be tested thus concerns rule breaking. Rule-breaking hypothesis: in adulthood, the strengths of the status goal is unrelated to rule breaking, irrespective of the strength of the affection goal. McNeill, 2015). Our third hypothesis to be tested thus concerns rule breaking. Rule-breaking hypothesis: in adulthood, the strengths of the status goal is unrelated to rule breaking, irrespective of the strength of the affection goal. Status and aggression in different contexts. We extend the application of our theory to two speciﬁc contexts that are relevant for virtually everybody: the workplace and intimate relationships. In these speciﬁc contexts, special circumstances might make aggression more or less independent of the strength of the status goal. The imbalanced needs theory of aggression also implies that imbalance may be avoided when aggression becomes unrelated to need fulﬁllment. This is the case (a) when, in particular contexts, status can be better achieved through other means than aggression, or (b) affection is much more relevant in this context than status. We therefore also investigated aggression in the work place (where there are other avenues for status achievement) and aggression in intimate relationships (where affection is presumably much more important than status). With regard to the form of aggression, we opted for behaviors that pertain speciﬁcally to these contexts. For the work place, we focused on “work place aggression”, that is, on more indirect forms pertaining to social aggression, rather than physical forms of aggression; and for intimate partner relation- ships, we focused on the most frequently observed forms of intimate partner aggression, that is, psychological aggression and negotiation strategies that indicate the presence of conﬂict (Feldman & Gowen, 1998; Straus et al., 1996). Sijtsema and Lindenberg 4 ) Work context. In the work context, status is more pronounced than in most other contexts in adulthood. As such, people may adopt different means to achieve status in this context, with aggression being one of them. Previous studies on workplace aggression indicate a relatively high prevalence of workplace aggression and bullying, suggesting that such behaviors are far from uncommon in adulthood (Aquino & Thau, 2009). Several studies also shed light on the motivation for workplace aggression, indicating that ag- gression can be used as a strategy to rise in the ranks in work settings (Neuman & Baron, 2005; Spector et al., 2006). This idea is supported by a recent empirical account showing that also adults who use aggression can be re- warded in terms of social status (Ruschoff et al., 2015). However, even though aggression may still be a way to achieve status at the work place, at work there are other avenues for status that run via competence and promotion, for example via valued knowledge, skills, work effort, and via earning respect and positions (Blau, 1964; Cheng et al., 2013; Maner, 2017; Ng et al., 2005). These alternative ways of achieving status may greatly reduce the use of achieving status via displays of dominance (aggression), thereby also reducing the inﬂuence of the strength of the status goal on aggression. In short, our fourth hypothesis concerns the work context. Work place 777 0(0) Sijtsema and Lindenberg 6 hypothesis: in work settings, the strength of the status goal is only weakly related or unrelated to aggression. hypothesis: in work settings, the strength of the status goal is only weakly related or unrelated to aggression. Romantic contexts. Social relationships, and especially intimate rela- tionships, are likely to be more stable in adulthood than during adolescence (Zimmer-Gembeck, 2002). As romantic relationships become longer lasting, competitive mating contexts are likely to become less numerous (Collins, 2003; Sassler, 2010). Thus, in adult romantic contexts, affection and intimacy, and not status, are likely to be highly salient. In particular, just as in over- lapping social circles, in relationships with a romantic partner, status concerns may impede the realization of affection and thus the quality and stability of the relationship (Sadikaj et al., 2017). Sijtsema and Lindenberg 4 It is thus likely that aggression between romantic partners (intimate partner violence) is not socially induced by the status goal, but is due to individual dispositions, such as personality disorders related to antisocial behavior (e.g., Holtzworth-Munroe, 2000; Ross & Babcock, 2009). Therefore, our ﬁfth hypothesis to be tested is about ro- mantic contexts. Romantic context hypothesis: for intimate partner rela- tionships, the strength of the status goal is unrelated to aggression, irrespective of the strength of the affection goal. In the following, we test these hypotheses in a sample of adults from the general population, while accounting for age and sex. Instruments Aggressive behavior. Physical aggression, social aggression, and rule breaking were assessed with an adapted version of the Subtypes of Antisocial Behavior (STAB) questionnaire (Burt & Donnellan, 2009). Participants were asked how often they felt or behaved aggressively in the past 12 months on a 5-point scale, ranging from 1 (never) to 5 (always). Physical aggression was assessed with a 7-item subscale (α = 0.79), including items such as “got into physical ﬁghts” and “threatened others.” Social aggression was assessed with 10 items (α = 0.78), such as “damaged someone’s reputation on purpose” and “ridiculed someone behind their back.” Rule breaking was assessed with 8 items (α = 0.82), such as “stole things from a shop” and “sold drugs.” Scores were averaged for each subscale across the items with higher scores indicating higher levels of aggression or rule breaking. Workplace aggression. To assess workplace aggression, we adapted the perceived workplace victimization questionnaire (Aquino, 2000). Participants were asked how often they behaved aggressively at the workplace in the past 6 months on a 5-point scale, ranging from 1 (never) to 5 (20 times or more). Workplace aggression perpetration was assessed with ﬁve items (α = 0.61), including items such as “gossiped about a coworker” and “cursed at a co- worker.” Scores were averaged across the items with higher scores indicating higher levels of aggression. g gg Intimate partner violence. To assess aggression in romantic contexts, we opted for the Revised Conﬂict Tactics Scale (CTS2; Straus et al., 1996), which is the most commonly used instrument to assess intimate partner violence in clinical and general population samples (Archer, 2004; Johnson, 2008). Participants ﬁlled out this instrument when they were currently in a romantic relationship or had been in a romantic relationship in the past year. We in- cluded two subscales of this instrument that tapped into psychological ag- gression and conﬂict negotiation strategies. Conﬂicts in intimate relationships are potentially signs of lower affection. The psychological aggression scale consists of eight items and assesses verbal aggression, coercion, and threats. Participants and Procedure Data were collected among 253 Dutch adults from the general population (22% males; M age = 29.95, SD = 2.60; age range 18–67) as part of a data collection on dominance, antisocial personality traits, and life events. Sixty- eight percent of the participants were highly educated or currently followed a university or college education and 70% had a full-time or part-time job. Data collection was administered by three graduate students who approached participants from their personal networks via e-mail and Facebook. Fur- thermore, participants were asked to forward the invitation to their network to increase participation. Participants received a letter including information about the study, with a hyperlink to the digital questionnaire. Participants were fully informed about the subject of the study, the anonymous treatment of data, and the option to withdraw from the study at any time. These research procedures are in line with the ethical standards and guidelines in the Netherlands, following the Declaration of Helsinki (1964). In total, 410 in- dividuals were invited to ﬁll out the online questionnaire. Of these, 157 clicked on the link to the study, but did not complete all measures or only ﬁlled out their sex and age. A chi-square test showed that dropouts were more likely to be male compared to those who participated (Χ2 = 7.46, p < .01), but did not 778 Sijtse Journal of Interpersonal Violence 38(1-2)7 differ in age. Little’s MCAR test indicated that data were missing completely at random (Χ2= 17.24, df = 29, p = .96). differ in age. Little’s MCAR test indicated that data were missing completely at random (Χ2= 17.24, df = 29, p = .96). Instruments This scale includes items such as, “I accused my partner of being a worthless lover” and “I threatened to hit or throw something at my partner.” Conﬂict negotiation strategies were assessed with six items that showed some incli- nation for compromise but were also an indication of having to deal with conﬂicts, such as “I proposed a compromise to end a ﬁght” or “I explained my side of a disagreement to my partner.” Answers were rated on an 8-point scale ranging from 0 (never) to 7 (more than 20 times) in the past 12 months. Cronbach’s alphas were 0.88 for both psychological aggression and conﬂict 779 0(0) Sijtsema and Lindenberg 8 negotiation strategies. Scores were averaged across all items for each subscale, with higher scores indicating more psychological aggression and more use of conﬂict negotiation strategies, respectively. negotiation strategies. Scores were averaged across all items for each subscale, with higher scores indicating more psychological aggression and more use of conﬂict negotiation strategies, respectively. g g , p y Social goals. In the current study, we view agentic and communal goals as trait-like interpersonal motivations that in part resemble universal social needs important for human development (Anderson et al., 2015). The status goal (striving to be dominant and assertive) and the affection goal (seeking re- lational warmth) were assessed with the Interpersonal Goal Inventory in adults (Dryer & Horowitz, 1997; Locke, 2003). Under the frame “When working with someone on a task, it is important to me that…,” participants were asked to rate the subjective importance of 32 interpersonal outcomes on a 7-point scale ranging from 1 (completely disagree) to 7 (completely agree). Items related to agency were averaged to construct the subscale that assessed the strength of the status goal (i.e., a combination of the Agentic and Separate- Agentic scale; 8 items, α = 0.57). Example items are “…Be assertive with the other person” and “… Be ﬁrm when I need to be.” Similarly, items related to communion were averaged to construct the subscale that assessed the strength of the affection goal (i.e., a combination of the Communal and Communal- Separate scale; 8 items, α = 0.68). Instruments Example items are “…Be supportive of the other person’s goals” and “… Share openly my thoughts and ideas.” Following existing literature (Locke, 2003), information represented in the eight goal scales was summarized into overarching status and affection goal vector scores in the circumplex space, via the formulas below: Strength of the status goal = Agentic _ Submissive + [.707 × (Communal and Agentic + Separate and Agentic _ Communal and Submissive _ Separate and Submissive)] Strength of the affection goal = Communal _ Separate + [.707 × (Com- munal and Agentic + Communal and Submissive _ Separate and Agentic _ Separate and Submissive)]. These scores were used to assess status and affection goals respectively. Scores on the four intermediate scales (Communal and Agentic, Communal and Submissive, Separate and Agentic, and Separate and Submissive) were multiplied by .707 because this is the cosine of a 45° angle (the angle of those scales, relative to the status and affection goal vectors). Data Analysis We calculated means, standard deviations, and ranges of all study variables. Because age showed a bimodal distribution, it was recoded into a dummy categorizing participants from 18 up to 30 years and those aged 30 years and over into two different groups. Using independent samples t-tests, we cal- culated mean differences on all variables between these age groups. Next, we computed Pearson correlations between all continuous study variables. 780 Sijtse Journal of Interpersonal Violence 38(1-2)9 Finally, we performed linear regression analyses to test our hypotheses re- garding the interaction between status and affection goals. Examining the distribution of the outcome measures indicated relatively high levels of skewness and kurtosis with values above 2 or below 2 (George & Mallery, 2010) in physical aggression, rule breaking, and psychological aggression between romantic partners. Formal tests of normality indicated that the distributions of all aggression outcome measures were non-linear (Kolmogorov-Smirnov test statistics between 0.097 and 0.250, ps < .05). Therefore, we conducted bootstrapped correlation analyses, creating 1000 random samples to ensure the robustness of our results. This procedure is less sensitive to skewed outcome measures and yields more reliable coefﬁcients and 95% conﬁdence intervals. Next, we tested whether the assumption of a linear relationship between the status goal and the outcome measures was linear. To this end, we produced scatter plots for each outcome measure, which all indicated at least some linearity. We also produced P-P-plots for each regression analysis, which also suggested linearity. Moreover, multi- collinearity indices, such as the Variance Inﬂation Factor (VIF) and the tolerance, indicated no overlap between the independent variable in ex- plaining the various outcome measures, with scores all within the acceptable range (VIFs <1.07; tolerance >0.94). Finally, we calculated scatterplots be- tween the standardized residuals and the standardized predicted values for each regression model, which showed no clear patterns thereby indicating homoscedasticity. To interpret the ﬁndings, signiﬁcant interactions were plotted using simple slope analysis (Aiken & West, 1991). To reduce potential problems with multicollinearity and to ensure that the values plotted in the ﬁgures are accurate representations of the data, independent variables were standardized to a mean of 0 and a standard deviation of 1. All analyses were performed in IBM SPSS 24.0 and hypotheses were tested two-sidedly using a p-value of <.05 to indicate signiﬁcance. Descriptive Analyses In Table 1 and 2, means, standard deviations, and correlations of all study variables are presented for the whole sample (a) and the two age groups separately (b). Independent sample t-tests showed that younger adults reported more physical aggression, social aggression, and rule breaking, as well as more psychological aggression and conﬂict negotiation strategies in intimate partner relationships (ts > 3.22, ps < .05). Age groups did not differ in social goals and workplace aggression. Correlations further indicated that the strength of the affection goal was negatively associated with social aggression in younger adults, suggesting that those with a stronger affection goal reported 781 0(0) Sijtsema and Lindenberg 10 Table 1. Means, Standard Deviations, and Correlations of All Study Variables (Complete Sample; N = 253). 1 2 3 4 5 6 7 8 9 M SD 1. Age (in years) — 0.03 0.05 0.22 0.36 0.21 0.05 0.21 0.22 29.95 12.60 2. Status goals 0.03 0.01 0.03 0.01 0.11 0.04 0.05 0.16 1.12 3. Affection goals 0.03 0.17 0.13* 0.11 0.12 0.01 2.30 1.45 4. Physical aggression 0.56 0.50 0.11 0.41 0.23 1.34 0.38 5. Social aggression 0.66 0.39 0.45 0.27 1.49 0.38 6. Rule-breaking 0.28 .34 0.16* 1.12 0.17 7. Workplace aggression 0.14* 0.18 1.26 0.28 8. Psychological IPVa 0.50 0.94 1.07 9. Negotiation IPVa 3.63 1.64 782 Sijtse Journal of Interpersonal Violence 38(1-2) 11 Table 2. Means, Standard Deviations, and Correlations of All Study Variables (Age 18-30 below and age 31-67 above the diagonal). 1 2 3 4 5 6 7 8 9 Age 18–30 (n = 190) Age 31–67 (n = 63) Independent samples t-tests M SD M SD t-value df 1. Age (in years) — 0.17 0.13 0.07 0.10 0.01 0.14 0.17 0.01 23.31 2.74 50.00 8.87 36.69* 251 2. Status goals 0.03 — 0.01 0.05 0.01 0.09 0.14 0.12 0.02 0.17 1.11 0.16 1.17 0.07 251 3. Affection goals 0.09 0.04 — 0.01 0.05 0.11 0.03 0.05 0.02 2.27 1.51 2.42 1.25 0.74 251 4. Physical aggression 0.02 0.00 0.03 — 0.56 0.18 0.31* 0.16 0.20 1.39 0.41 1.19 0.21 3.75*** 251 5. Social aggression 0.16* 0.05 0.19* 0.53 — 0.64 0.51 0.37 0.42 1.57 0.39 1.27 0.25 5.88* 251 6. Rule-breaking 0.06 0.00 0.13 0.50 0.64 — 0.37 0.11 0.16 1.14 0.18 1.06 0.08 3.48 251 7. Descriptive Analyses Workplace aggression 0.07 0.10 0.13 0.08 0.38 0.26 — 0.16 0.27 1.27 0.29 1.24 0.25 0.74 251 8. Psychological IPV 0.02 0.03 0.01 0.20* 0.19* 0.12 0.17* — 0.35 3.83 1.66 3.01 1.41 3.22 215 9. Negotiation IPV 0.06 0.07 0.14 0.41 0.41 0.34 0.09 0.50 — 1.07 1.06 0.52 1.02 3.31** 215 Sijtsema and Lindenberg 12 783 0(0) less social aggression. In both age groups, status and affection goals were unrelated to the other antisocial outcomes. less social aggression. In both age groups, status and affection goals were unrelated to the other antisocial outcomes. Social Goals and General Measures of Antisocial Behavior In Table 3, main and interaction effects of status and affection goals on physical aggression are presented, while accounting for sex and age group. In line with our Physical aggression hypothesis, this model showed a signiﬁcant interaction effect between status goals and affection goals in the explanation of physical aggression (see Figure 1a). Simple slope analyses indicated that the status goal was positively associated with physical aggression, but indeed only at low levels of the affection goal (b = 0.11, SE = 0.03, p < .001, 95% CI = 0.05 to 0.18). Moreover, a weak status goal was associated with reporting less physical aggression, but only at when the affection goal was strong (b = 0.09, SE = 0.03, p < .01, 95% CI = 0.15 to 0.04). The Johnson-Neyman signiﬁcance region suggested that the interaction between status and affection goals became signiﬁcant at affection goal values of .39 and lower and .54 and higher, which together comprised 57.7% of the sample. Next, we examined the test of our application of the imbalanced needs theory of aggression to other forms of aggression. In line with our Social aggression hypothesis, effects were similar to those of physical aggression (see Figure 1b): a strong status goal was only positively associated with physical aggression at low levels of the affection goal (b = 0.08, SE = 0.03, p < .05, 95% CI = 0.01 to 0.14). The Johnson-Neyman signiﬁcance region suggested that this interaction became signiﬁcant at affection goal values of 0.58 or lower, which comprised 23.3% of the sample. In contrast to our Rule-breaking hypothesis, where we argued that status goals would be unrelated to rule breaking, we found a signiﬁcant interaction between status and affection goals. That is, the strength of the status goal was positively associated with rule breaking, but only at low levels of the affection goal (b = 0.03, SE = 0.01, p < .05, 95% CI = 0.00–0.06) (see Figure 1c). The Johnson-Neyman signiﬁcance region suggested that this interaction became signiﬁcant at affection goal values of .80 and lower, comprising 17.8% of the sample. In this sense, the link between social goals and rule-breaking worked similar to physical and social aggression. Social Goals and Aggression in Different Contexts Next, we tested our hypotheses related to the application of our theory to different contexts, by studying links between the status goal and aggression at the workplace and in romantic relationships. Analogous to the previous analyses, main and interaction effects of status and affection goals are pre- sented, while accounting for sex and age group (see Table 4). Our Work-setting 784 Sijtse Journal of Interpersonal Violence 38(1-2)13 Table 3. Regression Analyses of Status and Affection Goals on Subtypes of Antisocial Behavior. Physical Aggression Social Aggression Rule-breaking B SE LB UB B SE LB UB B SE LB UB Main effects Constant 1.51 0.14 1.23 1.79 1.60 .10 1.40 1.79 1.16 0.05 1.07 1.25 Sex (1 = male; 2 = female) 0.07 0.07 0.21 0.07 0.02 0.05 0.12 0.09 0.01 0.03 0.06 0.04 Age dummy (0 = age 18–30; 1 = age >30) 0.20 0.04 0.27 0.11 0.30 0.05 0.40 0.20 0.08 0.02 0.13 0.03 Status goals 0.00 0.03 0.06 0.06 0.01 0.02 0.04 0.05 0.00 0.01 0.02 0.02 Affection goals 0.01 0.04 0.09 0.07 0.06 0.02 0.10 0.02 0.02 0.01 0.04 -0.00 R2 5.9% 14.6% 6.2% Two-way interactions Constant 1.41 0.10 1.21 1.61 1.54 0.10 1.35 1.74 1.13 .05 1.04 1.22 Sex (1 = male; 2 = female) 0.02 0.06 0.12 0.09 0.01 0.05 0.09 0.12 0.00 0.03 0.05 0.05 Age dummy (0 = age 18–30; 1 = age >30) 0.19 0.05 0.29 0.09 0.30 0.05 0.40 0.20 0.08 0.02 0.12 0.03 Status goals 0.01 0.02 0.04 0.05 0.02 0.02 0.03 0.06 0.00 0.01 0.02 0.02 Affection goals 0.02 0.02 0.07 0.02 0.07 0.02 0.11 0.02 0.03 0.01 0.05 0.01 Status goals x affection goals 0.11 0.02 0.15 0.06 0.06 0.02 0.10 0.02 0.03 0.01 0.05 0.01 R2 14.3% 17.5% 9.6% Note. Figures in bold face are statistically signiﬁcant. Table 3. Regression Analyses of Status and Affection Goals on Subtypes of Antisocial Behavior. Physical Aggression Social Aggression Rule-breaking B SE LB UB B SE LB UB B SE LB UB 785 0(0) Sijtsema and Lindenberg 14 . Plots of simple slopes of the association between the status goa n at high (+1 SD), average (Mean), and low (1 SD) levels of the af hysical aggression (a), social aggression (b), and rule breaking (c). Figure 1. Plots of simple slopes of the association between the status goal and aggression at high (+1 SD), average (Mean), and low (1 SD) levels of the affection goal for physical aggression (a), social aggression (b), and rule breaking (c). 786 Sijtse Journal of Interpersonal Violence 38(1-2)15 Table 4. Regression Analyses of Status and Affection Goals on Workplace Violence and Intimate Partner Violence. Workplace Aggression Negotiation IPV Psychological IPV B SE LB UB B SE LB UB B SE LB UB Main effects Constant 1.35 0.08 1.20 1.50 4.07 0.49 3.11 5.04 0.96 0.32 0.33 1.59 Sex (1 = male; 2 = female) 0.05 0.04 0.13 0.04 0.14 0.27 0.67 0.39 0.06 0.17 0.28 0.40 Age dummy (0 = age 18–30; 1 = age >30) 0.02 0.04 0.10 0.06 0.82 0.26 1.32 .31 0.54 0.17 0.87 0.21 Status goals 0.04 0.02 0.01 0.06 0.08 0.11 0.14 0.30 0.05 0.07 0.10 0.19 Affection goals 0.03 0.02 0.07 0.00 0.03 0.11 0.19 0.25 0.12 0.07 0.26 0.03 R2 2.9% 5.0% 6.2% Two-way interactions Constant 1.32 0.08 1.17 1.48 4.03 0.50 3.04 5.01 0.99 0.32 0.35 1.62 Sex (1 = male; 2 = female) 0.03 0.04 0.12 0.05 0.11 0.27 0.65 0.42 0.05 0.18 0.30 0.40 Age dummy (0 = age 18–30; 1 = age >30) 0.02 0.04 0.10 0.06 0.81 0.26 1.32 0.31 0.54 0.17 0.87 0.22 Status goals 0.03 0.02 0.01 0.07 0.09 0.11 0.14 0.31 0.04 0.07 0.10 0.19 Affection goals 0.04 0.02 0.07 0.00 0.02 0.11 0.20 0.24 0.11 0.07 0.26 0.03 Status goals x affection goals 0.03 0.02 0.06 0.00 0.06 0.10 0.26 0.15 0.03 0.07 0.10 0.16 ΔR2 4.1% 5.2% 6.4% Note. Figures in bold face are statistically signiﬁcant. Note. IPV = Intimate Partner Violence. 787 0(0) Sijtsema and Lindenberg 16 hypothesis stated that in the work place, the strength of status goals is only weakly related or unrelated to aggression. This hypothesis (which is also the null hypothesis) was supported: The status goal was unrelated to aggression in the work context. Table 3. Regression Analyses of Status and Affection Goals on Subtypes of Antisocial Behavior. Physical Aggression Social Aggression Rule-breaking B SE LB UB B SE LB UB B SE LB UB Moreover, the analyses indicated that the status goal did not interact with the affection goal in explaining workplace aggression, but a weaker affection goal was associated with more workplace aggression (b = 0.03, SE = 0.02, p < .05, 95% CI = 0.07 to 0.00). Thus, in line with our expectation and the null hypothesis, we found no support for a link between status goals and aggression in the workplace. Finally, we tested our Romantic context hypothesis suggesting that the strength of the status goal is not associated with intimate partner aggression, irrespective of the affection goal. In Table 4, we present ﬁndings for psy- chological aggression and conﬂict negotiation strategies. In line with the hypothesis, main effects indicate that both status and affection goals were unrelated to psychological aggression as well as to conﬂict negotiation strategies. Moreover, the affection goal did not moderate the association between the status goal and these two types of aggression. Only age group was signiﬁcantly associated with aggression, suggesting that younger participants were more likely to report the use of psychological aggression and conﬂict negotiation strategies in their romantic relationships. Discussion In the current study, we provided a further test of the imbalanced needs theory of aggression. The theory states that to study the social determinants of aggression one has to look at its link to the satisfaction of fundamental needs (status and affection). Aggression can be used to realize status, but at a cost for realizing affection in the same social context, which would create an im- balance in need satisfaction. The theory implies that social contexts in which status need fulﬁllments have to be balanced with affection need fulﬁllment, would be associated with low levels of aggression, except when affection needs are low (that is, when the pursuit of status via aggression cannot create an imbalance). Findings for adolescents (Sijtsema et al., 2020) supported this theory. The question for the present study was whether it would also hold for adults. We conducted ﬁve tests of the theory in adult populations. First, we looked at direct aggression (not context speciﬁc), and found our prediction supported by the data that the strength of the status goal is only related to aggression when the affection goal is weak. Then we looked at different forms of aggression: social aggression and rule-breaking behavior. We hypothesized that we would ﬁnd similar results for social aggression, but not for rule breaking, because rule-breaking for the achievement of status is especially prominent in adolescence, not adulthood. We found that status goals were related to both increased social aggression and rule breaking when affection Journal of Interpersonal Violence 38(1-2) 17 788 Sijtse goals were weak. Seemingly, not just social aggression, but also rule- breaking has a negative inﬂuence on affection, creating an imbalance even in adulthood. The latter ﬁnding goes against our hypothesis. A possible ex- planation is that, contrary to adolescence, rule-breaking behaviors in adult- hood may actually relate to acquiring means for status (such as stealing money or selling drugs), which may negatively affect others within the same social circle (e.g., by being put at risk, or by wishing to dissociate from such be- havior), leading to a loss in affection for the perpetrator. Thus, to the degree that rule-breaking for status occurs in adulthood, it may be more likely in those with a strong status goal and a relatively weak affection goal. Finally, we tested the theory for two different contexts: the workplace and romantic relationships. Discussion There, we tested an implication of the imbalance need theory of aggression with regard to contexts in which the use of aggression for the achievement of status would not occur: contexts in which status can be achieved more easily without aggression (like the workplace), and contexts in which status striving plays a subordinate role in comparison to the striving for affection (as in romantic relationships). These expectations were supported by our results, showing that both in work and romantic settings, the status goal was unrelated to aggression. This also resonates with previous literature showing that at the workplace there are more alternative ways of achieving status (e.g., Cheng et al., 2013; Maner, 2017) and that violence in intimate relationships is often associated with individual traits, such as personality pathology (Holtzworth-Munroe, 2000; Ross & Babcock, 2009). Overall, the important message of our ﬁndings is that for the study of the social determinants of aggression, it is important to consider the possibilities people have to balance need satisfaction of status and affection. When the social circles overlap for the satisfaction of affection and status, the cost of losing affection by achieving status via aggression is high, rendering this social source of aggression less likely. Similarly, there are social contexts that elevate affection needs far above status needs (such as romantic relationships). Scientiﬁc and Societal Implications For the study of aggression, this implies a recommendation for special at- tention to the ways these two social needs are and can be satisﬁed, and the relative social importance of these needs. To the degree that the circles overlap or the context elevates affection needs far above status needs, it becomes more likely that aggression derives not from social circumstances but from per- sonality pathology and related factors, such as negative emotions and poor self-control (Douglas & Martinko, 2001; Sotelo & Babcock, 2013), or par- ticularly low levels of affection needs (e.g., as seen in relation to psychopathy: Moreira et al., 2014). Conversely, social contexts that separate the circles of status and affection (such as strong power differences between social groups 789 0(0) Sijtsema and Lindenberg 18 or the public arena in which the anonymity and lack of past and future social encounters makes status striving with aggression likely), are likely to foster social sources of aggression. Similarly, social contexts that elevate status needs far above affection needs (such as highly competitive contexts) are likely to create social sources of aggression. In such cases, aggression may be triggered by cues from the environment (e.g., work stress or power differ- ences), if aggressive behavior is possible (i.e., low costs to the use of ag- gression) and the outcome could also be beneﬁcial to the aggressor (cf. Salin, 2003). As such, this theory would not only explain variation in proactive forms of aggression, but also in reactive forms of aggression. These rec- ommendations for further scientiﬁc study also offer avenues for policy as it can inﬂuence both the overlap of social circles and the relative importance of status goals in comparison to affection goals, thereby inﬂuencing the social determinants of aggression. Limitations Despite the importance of these ﬁndings, the current study is limited in several respects. First, although it was a great advantage that status and affection goals were measured directly, self-reports also suffer from reporter biases and social desirability. Second, in terms of the diversity of our sample, it should be noted that women were over-represented, which may have affected our ﬁndings. In particular, several studies have shown that women display less physical aggression as compared to men (Hyde, 2005). However, in our study, we mainly included social and psychological aggressive behaviors, which have been found to be equally frequent in males and females (Little et al., 2003; Heilbron & Prinstein, 2008). Moreover, we showed that the pattern of ﬁndings for general indices of aggression is similar for both direct and more covert forms of aggression. Finally, a convenience sample was collected using a snowball-technique, which may have led to a more homogeneous sample as participants may have come from the same social group or the same area. We should thus be careful in generalizing the current ﬁndings to other populations that may show more variation in gender, socio-economic background, or culture. Also, some caution is warranted because the effects that we found were by and large modest, thus suggesting that additional factors, such as personality and social inﬂuences not considered by us, are important to in- clude when explaining variations in self-reported aggression. Notwithstanding these shortcomings, we provided an extensive test of the imbalanced needs theory of aggression, testing its merits in different contexts and pertaining to different forms of antisocial behavior. It stands to reason that replication and further extension of the current study is needed. Avenues that are worth exploring include a more thorough analysis of the conditions under which the satisfaction of status and affection needs becomes imbalanced. For 790 Sijtse Journal of Interpersonal Violence 38(1-2) 19 example, under what conditions do power differences in organizations create such an imbalance, and when does competition in organizations create an imbalance between the importance of status and affection goals? Also, neighborhood studies could proﬁt from looking into the degree to which ethnic heterogeneity creates separate social circles for status and affection need sat- isfaction. There is potentially a wide application of the imbalanced needs theory of aggression in research on the social determinants of aggression. Funding The author(s) received no ﬁnancial support for the research, authorship, and/or publication of this article. Jelle J. Sijtsema https://orcid.org/0000-0001-5822-7783 Jelle J. Sijtsema https://orcid.org/0000-0001-5822-7783 Declaration of Conﬂicting Interests The author(s) declared no potential conﬂicts of interest with respect to the research, authorship, and/or publication of this article. 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Developmental Psy- chology, 35(1), 205–213. https://doi.org/10.1037//0012-1649.35.1.205 795 0(0) Sijtsema and Lindenberg 24 Author biographies Jelle J. Sijtsema, PhD, is Associate Professor at the department of Devel- opmental Psychology at Tilburg University. His research focuses on the links between social relationships, social cognitions, and violence. He is interested in connecting insights into social dynamics to programs aimed at reducing violence and improving offender treatment. Siegwart M. Lindenberg, PhD, is Professor of Cognitive Sociology at the University of Groningen, and at the Tilburg Institute for Behavioral Eco- nomics Research, the Netherlands. He is one of the founders of the Inter- university Center for Social science theory and methodology (ICS). His main interests lie in the areas of micro-foundations for theories on collective phenomena, self-regulation and pro- and antisocial behavior, and groups and relationships.
https://openalex.org/W3179467597	https://biblio.ugent.be/publication/8743631/file/8745428.pdf	English	null	The MicroRNA Landscape of Acute Beta Cell Destruction in Type 1 Diabetic Recipients of Intraportal Islet Grafts	Cells	2,021	cc-by	14,562	Article The MicroRNA Landscape of Acute Beta Cell Destruction in Type 1 Diabetic Recipients of Intraportal Islet Grafts Gorus 1, Pieter Mestdagh 3,5, Dieter De Smet 2, Jo Vandesompele 3,5 , Bart Keymeulen 1 and Sarah Roels 1 1 Diabetes Research Center, Brussels Free University (VUB), Laarbeeklaan 103, 1090 Brussels, Belgium; geert.stange@vub.ac.be (G.S.); zhidong.ling@vub.ac.be (Z.L.); daniel.pipeleers@vub.ac.be (D.G.P.); frans.gorus@vub.ac.be (F.K.G.); bart.keymeulen@uzbrussel.be (B.K.); sarah.roels@colruytgroup.com (S.R.) 2 Department of Laboratory Medicine, Molecular Diagnostics Unit, AZ Delta General Hospital, 8800 Roeselare, Belgium; dieter.desmet@azdelta.be 1 Diabetes Research Center, Brussels Free University (VUB), Laarbeeklaan 103, 1090 Brussels, Belgium; geert.stange@vub.ac.be (G.S.); zhidong.ling@vub.ac.be (Z.L.); daniel.pipeleers@vub.ac.be (D.G.P.); frans.gorus@vub.ac.be (F.K.G.); bart.keymeulen@uzbrussel.be (B.K.); sarah.roels@colruytgroup.com (S.R.) 1 Diabetes Research Center, Brussels Free University (VUB), Laarbeeklaan 103, 1090 Brussels, Belgium; geert.stange@vub.ac.be (G.S.); zhidong.ling@vub.ac.be (Z.L.); daniel.pipeleers@vub.ac.be (D.G.P.); frans.gorus@vub.ac.be (F.K.G.); bart.keymeulen@uzbrussel.be (B.K.); sarah.roels@colruytgroup.com (S.R.) 2 Department of Laboratory Medicine, Molecular Diagnostics Unit, AZ Delta General Hospital, 8800 Roeselare, Belgium; dieter.desmet@azdelta.be , g ; 3 Department of Biomolecular Medicine, Ghent University, 9000 Gent, Belgium; Jasper.Anckaert@UGent.be (J.A.); Pieter.Mestdagh@UGent.be (P.M.); jo.vandesompele@ugent.be (J.V.) 4 Di b R h I i U i i à Vi S l S R ff l 20132 Mil I l i i l @h i g 3 Department of Biomolecular Medicine, Ghent University, 9000 Gent, Belgium; Jasper.Anckaert@UGent.be (J.A.); Pieter.Mestdagh@UGent.be (P.M.); jo.vandesompele@ugent.be (J.V.) 4 Diabetes Research Institute, Università Vita-Salute San Raffaele, 20132 Milan, Italy; piemonti.lorenzo@hsr.it 5 C t f M di l G ti U i it H it l Gh t (UZ G t) D Pi t l 185 9000 Gh t B l i g 3 Department of Biomolecular Medicine, Ghent University, 9000 Gent, Belgium; Jasper.Anckaert@UGent.be (J.A.); Pieter.Mestdagh@UGent.be (P.M.); jo.vandesom Department of Biomolecular Medicine, Ghent University, 9000 Gent, Belgium; Jasper.Anckaert@UGent.be (J.A.); Pieter.Mestdagh@UGent.be (P.M.); jo.vandesompele@ugent.be (J.V.) 4 Diabetes Research Institute, Università Vita-Salute San Raffaele, 20132 Milan, Italy; piemonti.lorenzo@hsr.it 5 Center for Medical Genetics, University Hospital Ghent (UZ Gent), De Pintelaan 185, 9000 Ghent, Belgium * Correspondence: geert.martens@azdelta.be; Tel.: +32-2-477-45-48; Fax: +32-2-477-50-60 Jasper.Anckaert@UGent.be (J.A.); Pieter.Mestdagh@UGent.be (P.M.); jo.vandesompele@ugent.be (J.V.) 4 Diabetes Research Institute, Università Vita-Salute San Raffaele, 20132 Milan, Italy; piemonti.lorenzo@hsr.it 5 Center for Medical Genetics, University Hospital Ghent (UZ Gent), De Pintelaan 185, 9000 Ghent, Belgium 4 Diabetes Research Institute, Università Vita-Salute San Raffaele, 20132 Milan, Italy; piemonti.lorenzo@hsr.it 5 Center for Medical Genetics, University Hospital Ghent (UZ Gent), De Pintelaan 185, 9000 Ghent, Belgium Diabetes Research Institute, Università Vita Salute San Raffaele, 20132 Milan, Italy; piemonti.lorenzo@h 5 Center for Medical Genetics, University Hospital Ghent (UZ Gent), De Pintelaan 185, 9000 Ghent, Belgiu 5 Center for Medical Genetics, University Hospital Ghent (UZ Gent), De Pintelaan 185, 9000 Ghent, Belgium * Correspondence: geert.martens@azdelta.be; Tel.: +32-2-477-45-48; Fax: +32-2-477-50-60 , y p ( ), , , g * Correspondence: geert.martens@azdelta.be; Tel.: +32-2-477-45-48; Fax: +32-2-477-50-60 Abstract: Ongoing beta cell death in type 1 diabetes (T1D) can be detected using biomarkers selec- tively discharged by dying beta cells into plasma. Citation: Martens, G.A.; Stangé, G.; Piemonti, L.; Anckaert, J.; Ling, Z.; Pipeleers, D.G.; Gorus, F.K.; Mestdagh, P.; De Smet, D.; Vandesompele, J.; et al. The MicroRNA Landscape of Acute Beta Cell Destruction in Type 1 Diabetic Recipients of Intraportal Islet Grafts. Cells 2021, 10, 1693. https://doi.org/ 10.3390/cells10071693 Academic Editors: Md Shahidul Islam and Michael E. Boulton Received: 4 May 2021 Accepted: 24 June 2021 Published: 4 July 2021 Keywords: beta cell; type 1 diabetes; islet transplantation; biomarkers; microRNA 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. Article The MicroRNA Landscape of Acute Beta Cell Destruction in Type 1 Diabetic Recipients of Intraportal Islet Grafts microRNA-375 (miR-375) ranks among the top biomarkers based on studies in animal models and human islet transplantation. Our objective was to identify additional microRNAs that are co-released with miR-375 proportionate to the amount of beta cell destruction. RT-PCR proﬁling of 733 microRNAs in a discovery cohort of T1D patients 1 h before/after islet transplantation indicated increased plasma levels of 22 microRNAs. Sub-selection for beta cell selectivity resulted in 15 microRNAs that were subjected to double-blinded multicenter analysis. This led to the identiﬁcation of eight microRNAs that were consistently increased during early graft destruction: besides miR-375, these included miR-132/204/410/200a/429/125b, microR- NAs with known function and enrichment in beta cells. Their potential clinical translation was investigated in a third independent cohort of 46 transplant patients by correlating post-transplant microRNA levels to C-peptide levels 2 months later. Only miR-375 and miR-132 had prognostic potential for graft outcome, and none of the newly identiﬁed microRNAs outperformed miR-375 in multiple regression. In conclusion, this study reveals multiple beta cell-enriched microRNAs that are co-released with miR-375 and can be used as complementary biomarkers of beta cell death. Citation: Martens, G.A.; Stangé, G.; Piemonti, L.; Anckaert, J.; Ling, Z.; Pipeleers, D.G.; Gorus, F.K.; Mestdagh, P.; De Smet, D.; Vandesompele, J.; et al. The MicroRNA Landscape of Acute Beta Cell Destruction in Type 1 Diabetic Recipients of Intraportal Islet Grafts. Cells 2021, 10, 1693. https://doi.org/ 10.3390/cells10071693 Academic Editors: Md Shahidul Islam and Michael E. Boulton Received: 4 May 2021 Accepted: 24 June 2021 Published: 4 July 2021 cells cells Citation: Martens, G.A.; Stangé, G.; Piemonti, L.; Anckaert, J.; Ling, Z.; Pipeleers, D.G.; Gorus, F.K.; Mestdagh, P.; De Smet, D.; Vandesompele, J.; et al. The MicroRNA Landscape of Acute Beta Cell Destruction in Type 1 Diabetic Recipients of Intraportal Islet Grafts. Cells 2021, 10, 1693. https://doi.org/ 10.3390/cells10071693 Article The MicroRNA Landscape of Acute Beta Cell Destruction in Type 1 Diabetic Recipients of Intraportal Islet Grafts Geert A. Martens 1,2,3,, Geert Stangé 1 , Lorenzo Piemonti 4 , Jasper Anckaert 3,5 , Zhidong Ling 1, Daniel G. Pipeleers 1, Frans K. 1. Introduction The triggers and kinetics of pancreatic beta cell destruction in type 1 diabetes (T1D) are largely enigmatic. It is thought that insidious episodes of beta cell death induced by in- fectious, nutritional or environmental stressors trigger progressive bouts of T-cell-mediated destruction in individuals with unfavorable HLA backgrounds, with a more aggressive course in younger subjects. Real-time monitoring of cell death in rare cell types such as the beta cells requires highly selective biomarkers with a sufﬁciently long half-life in plasma, measured by extremely sensitive analytical techniques. To date, only three chemically distinct biomarkers were independently conﬁrmed in models of synchronous necrotic beta cell destruction such as streptozotocin-induced diabetes in rodents or islet transplantation in human T1D: GAD65 protein, unmethylated insulin DNA and microRNA-375 (miR-375). GAD65, the prototypic T1D autoantigen, can be used to predict the outcome of islet trans- plantation [1], but its use is limited due to interference by GAD65-autoantibodies and Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/cells Cells 2021, 10, 1693. https://doi.org/10.3390/cells10071693 Cells 2021, 10, 1693 2 of 19 technical limitations of analytical sensitivity of sandwich immunoassay technology [2]. Analytically, nucleic acid ampliﬁcation assays potentially offer superior sensitivity. Beta cell-selective patterns of DNA methylation provide an acceptable level of speciﬁcity, al- lowing the use of unmethylated insulin DNA to detect acute beta cell destruction [3,4]. However, liquid biopsies for DNA methylation patterns are analytically complex, and inter-laboratory agreement of assays is to date disappointing [5]. Furthermore, it is still unknown whether insulin gene DNA methylation patterns are uniformly present in all beta cells or show variations related to functional and developmental heterogeneity or pathological variations in stressed cells. Multiplexing various DNA loci is likely needed to attain the required speciﬁcity [6,7]. miR-375 shows an excellent level of islet enrichment [8], is expressed at very high levels by beta cells [9,10] and can be used to detect beta cell death in animal models and islet graft recipients [11]. We previously reported that miR-375 and GAD65 proteins are expressed at near equimolar levels in human beta cells and are co- released at near equimolar levels with roughly comparable half-lives in the circulation. 1. Introduction The plasma levels of GAD65 and miR-375 measured 1 h after intraportal islet transplantation are both good indicators of the amount of early graft destruction and can clinically be used to predict the insulin secretory graft function 2 months later [9]. Using optimized assays with analytical sensitivities in the femtomolar range, incidental surges above the limits of detection (LoD) of GAD65 or miR-375 assays were also observed in the follow-up period between day 2 and day 60 after transplantation [9]. A remaining challenge was the conﬁdent attribution of such incidental surges above the LoD to actual events of insidious beta cell death rather than analytical noise around the LoD. We reasoned that one way to circumvent this uncertainty was a multiplexing approach: the simultaneous detection above LoD of multiple beta cell-selective intracellular molecules could thus minimize false discovery rate and enable a more speciﬁc screening for occult beta cell death in vivo. y p g The aim of this study was to identify other beta-cell-enriched microRNAs that are co-released with reference marker miR-375 in the well-controlled model of intraportal islet transplantation in T1D subjects. A stepwise approach was followed to prioritize novel candidate biomarkers (graphically summarized in Figure 1). In a discovery cohort, plasma samples before and 1 h after islet transplantation were proﬁled for the presence and level of 733 human microRNAs. Second, likely beta cell-derived microRNAs were sub-selected by recovery experiments and co-linearity with GAD65 or miR-375. This resulted in a panel of 15 microRNAs that were then subjected to central blinded veriﬁcation on independent validation cohorts obtained from two different transplant centers. Finally, a core panel of eight microRNAs including miR-375 was then clinically validated in a third independent cohort of 46 islet transplant events for their potential to quantify acute graft destruction and predict long-term graft function (C-peptide secretion). In sum, our study aims to provide a detailed view of the plasma microRNA landscape in the model of intraportal islet transplantation and serve as a resource of novel candidate biomarkers for beta cell death in vivo. 3 of 19 6 of 21 Cells 2021, 10, 1693 Cells 2021, 10, x Figure 1. Study design. (a). Plasma samples before and 1 h after intraportal islet transplantation (Tx) were arrayed by hydrolysis probe PCR for presence and relative level of 733 human microRNAs in a discovery cohort of 8 subjects trans- planted in Brussels. 1. Introduction After stringent data filtering, 220 microRNAs were confidently detected and analyzed for differential levels before and after transplantation. A further selection of likely beta cell-enriched microRNAs was done by identifying microRNAs that showed NRQ levels that correlated with plasma GAD65 and/or miR-375 as measured by calibrated quan- titative assays; and/or that show a linear response to the number of beta cells spiked into a control plasma pooled sample in a recovery experiment. This led to the composition of a panel of 15 candidate biomarker microRNAs that was subjected to blinded verification by targeted hydrolysis-probe PCR in two independent validation cohorts of 8 patients transplanted in Brussels (validation cohort 1) and 7 patients transplanted in Milan (validation cohort 2). (b). The Venn diagram shows the 22 microRNAs that were significantly increased after islet transplantation in the discovery cohort, the 16 microRNAs that increased proportionately to the number of beta cells spiked in the recovery experiment and the 36 microRNAs of which post-transplant NRQ correlated positively with miR-375 and/or GAD65. The 15 microRNAs selected as candidate biomarkers for blinded validation are listed left of the Venn diagram. Finally, microRNAs confirmed in the validation cohorts were additionally analyzed for possible beta cell selectivity by tissue-comparative PCR analysis. Table 1. Panel of 15 candidate microRNA biomarkers of beta cell destruction identified in the discovery cohort and sub- Figure 1. Study design. (a). Plasma samples before and 1 h after intraportal islet transplantation (Tx) were arrayed by hydrolysis probe PCR for presence and relative level of 733 human microRNAs in a discovery cohort of 8 subjects transplanted in Brussels. After stringent data ﬁltering, 220 microRNAs were conﬁdently detected and analyzed for differential levels before and after transplantation. A further selection of likely beta cell-enriched microRNAs was done by identifying microRNAs that showed NRQ levels that correlated with plasma GAD65 and/or miR-375 as measured by calibrated quantitative assays; and/or that show a linear response to the number of beta cells spiked into a control plasma pooled sample in a recovery experiment. This led to the composition of a panel of 15 candidate biomarker microRNAs that was subjected to blinded veriﬁcation by targeted hydrolysis-probe PCR in two independent validation cohorts of 8 patients transplanted in Brussels (validation cohort 1) and 7 patients transplanted in Milan (validation cohort 2). (b). 1. Introduction The Venn diagram shows the 22 microRNAs that were signiﬁcantly increased after islet transplantation in the discovery cohort, the 16 microRNAs that increased proportionately to the number of beta cells spiked in the recovery experiment and the 36 microRNAs of which post-transplant NRQ correlated positively with miR-375 and/or GAD65. The 15 microRNAs selected as candidate biomarkers for blinded validation are listed left of the Venn diagram. Finally, microRNAs conﬁrmed in the validation cohorts were additionally analyzed for possible beta cell selectivity by tissue-comparative PCR analysis. Figure 1. Study design. (a). Plasma samples before and 1 h after intraportal islet transplantation (Tx) were arrayed by hydrolysis probe PCR for presence and relative level of 733 human microRNAs in a discovery cohort of 8 subjects trans- planted in Brussels. After stringent data filtering, 220 microRNAs were confidently detected and analyzed for differential levels before and after transplantation. A further selection of likely beta cell-enriched microRNAs was done by identifying microRNAs that showed NRQ levels that correlated with plasma GAD65 and/or miR-375 as measured by calibrated quan- titative assays; and/or that show a linear response to the number of beta cells spiked into a control plasma pooled sample in a recovery experiment. This led to the composition of a panel of 15 candidate biomarker microRNAs that was subjected to blinded verification by targeted hydrolysis-probe PCR in two independent validation cohorts of 8 patients transplanted in Brussels (validation cohort 1) and 7 patients transplanted in Milan (validation cohort 2). (b). The Venn diagram shows the 22 microRNAs that were significantly increased after islet transplantation in the discovery cohort, the 16 microRNAs that increased proportionately to the number of beta cells spiked in the recovery experiment and the 36 microRNAs of which post-transplant NRQ correlated positively with miR-375 and/or GAD65. The 15 microRNAs selected as candidate biomarkers for blinded validation are listed left of the Venn diagram. Finally, microRNAs confirmed in the validation cohorts were additionally analyzed for possible beta cell selectivity by tissue-comparative PCR analysis. T bl 1 P l f 15 did t i RNA bi k f b t ll d t ti id tifi d i th di h t d b Figure 1. Study design. (a). 1. Introduction Plasma samples before and 1 h after intraportal islet transplantation (Tx) were arrayed by hydrolysis probe PCR for presence and relative level of 733 human microRNAs in a discovery cohort of 8 subjects transplanted in Brussels. After stringent data ﬁltering, 220 microRNAs were conﬁdently detected and analyzed for differential levels before and after transplantation. A further selection of likely beta cell-enriched microRNAs was done by identifying microRNAs that showed NRQ levels that correlated with plasma GAD65 and/or miR-375 as measured by calibrated quantitative assays; and/or that show a linear response to the number of beta cells spiked into a control plasma pooled sample in a recovery experiment. This led to the composition of a panel of 15 candidate biomarker microRNAs that was subjected to blinded veriﬁcation by targeted hydrolysis-probe PCR in two independent validation cohorts of 8 patients transplanted in Brussels (validation cohort 1) and 7 patients transplanted in Milan (validation cohort 2). (b). The Venn diagram shows the 22 microRNAs that were signiﬁcantly increased after islet transplantation in the discovery cohort, the 16 microRNAs that increased proportionately to the number of beta cells spiked in the recovery experiment and the 36 microRNAs of which post-transplant NRQ correlated positively with miR-375 and/or GAD65. The 15 microRNAs selected as candidate biomarkers for blinded validation are listed left of the Venn diagram. Finally, microRNAs conﬁrmed in the validation cohorts were additionally analyzed for possible beta cell selectivity by tissue-comparative PCR analysis. ded validation. The table lists, f d f d f 2. Material and Methods used for targeted confirmation, the average Normalized Relative Quantity (NRQ) after g he Spearman Rank correlation coefficient to plasma GAD65 and miR 375 of the 15 cand 2.1. Intrahepatic Transplantation of Human Beta Cells in Type 1 Diabetic Recipients used for targeted confirmation, the average Normalized Relative Quantity (NRQ) after he Spearman Rank correlation coefficient to plasma GAD65 and miR 375 of the 15 can 2.1. Intrahepatic Transplantation of Human Beta Cells in Type 1 Diabetic Recipients he Spearman Rank correlation coefficient to plasma GAD65 and miR-375 of the 15 candidate ected, as shown in Figure 1 in the discovery cohort (n = 8). Right columns show the average fter versus before transplantation and the corresponding p-value (paired student t-test) in the hort 1 (Brussels, n = 8) and validation cohort 2 (Milan, n = 7). ND: not detected. Tx: islet trans- Discovery Cohort (n = 8) Validation Cohort 1 (n = 8) Validation Cohort 2 (n = 7) miR Level Post/Pre- Tx Correlation GAD65 Correlation miR- 375 miR Level Post/Pre-Tx miR Level Post/Pre-Tx NRQ P rs P rs P RQ P RQ P 118.1 0.007 0.88 <0.0001 - - 249.5 0.000 178.0 0.002 210.9 0.005 0.50 0.051 0.87 <0.0001 2.0 0.047 4.2 0.004 172.0 0.002 0.71 0.002 0.93 <0.0001 315.4 0.003 33.0 0.016 49.1 0.034 0.46 0.072 0.57 0.022 3.7 0.002 12.6 0.014 6249.4 0.044 0.49 0.054 0.82 <0.0001 397.0 0.000 70.4 0.000 36.5 0.016 0.64 0.008 0.92 <0.0001 287.0 0.007 53.5 0.002 52.1 0.001 0.83 0.000 0.79 0.000 6.8 0.039 6.5 0.282 Plasma was sampled 1 h before and 1 h after intrahepatic infusion of human islet preparations in the University Hospital Brussels, Brussels, Belgium (n = 16, Brussels cohorts, Diabetes Research Center, B. Keymeulen) and Ospedale San Raffaele, Milan, Italy (n = 8, Milan cohort, Diabetes Research Institute, L. Piemonti). The Brussels cohort was divided into a discovery cohort (n = 8) and an independent validation cohort 1 (n = 8). MicroRNA proﬁling by Low-Density Arrays was ﬁrst done on a discovery cohort (n = 8) of GAD65 autoantibody-negative (GADA < 23WHO Units/mL) T1D individuals who received at least 2 × 10 [6] beta cells/kg bodyweight with islet graft compositions detailed in Supplementary Table S1. Selected microRNAs with biomarker potential were then integrated into a targeted LDA RT-PCR panel, which was veriﬁed on an independent validation cohort 1 (n = 8) in Brussels and validation cohort 2 in Milan (n = 8). For blinded analysis, all pre-and post-transplant patient samples and healthy controls received a random numerical code within each cohort. All samples were then centrally analyzed by PCR and only after ﬁnalization of data processing with normalization were samples unblinded. 2.3. GAD65 Cytometric Bead Array and Calibrated miR-375 Assay 2.3. GAD65 Cytometric Bead Array and Calibrated miR-375 Assay GAD65 was measured by an in-house developed Cytometric Bead Array (CBA) [2,15] with Limit of Detection (LoD) and Limit of Quantiﬁcation (LoQ) of 0.121 pmol/L and 0.288 pmol/L respectively. miR-375 was quantiﬁed using a calibrated hydrolysis probe- PCR after sequence-speciﬁc capture by hybridization using the Taqman miRNA ABC Puriﬁcation Kit Human Panel A [9]. LoD and LoQ of this miR-375 assay are, respectively, 0.049 pmol/L and 0.102 pmol/L. 2.4. microRNA Array Proﬁling MicroRNAs were extracted from human plasma using the Taqman miRNA ABC Puriﬁcation Kit Human Panel A and Panel B (Applied Biosystems, Waltham, MA, USA). microRNAs were measured by hydrolysis-probe PCR with Taqman Megaplex Pools consist- ing of matching primer pools and Taqman MicroRNA Arrays for 733 human microRNAs according to manufacturer’s protocol (Applied Biosystems, Waltham, MA, USA). mi- croRNA was converted to cDNA using Megaplex RT Primers Human Pool Set v3.0 and pre-ampliﬁed using Megaplex PreAmp Primers Human Pool set v3.0. Taqman MicroRNA Arrays (TaqMan Array Human MicroRNA Card Set v3.0) were run on a 7900HT Fast Real- Time PCR System using the 384-well Taqman Array Block with default thermal-cycling conditions (Applied Biosystems, Waltham, MA, USA). used for targeted confirmation, the average Normalized Relative Quantity (NRQ) after he Spearman Rank correlation coefficient to plasma GAD65 and miR 375 of the 15 can 2.1. Intrahepatic Transplantation of Human Beta Cells in Type 1 Diabetic Recipients Transplantations in the Brussels cohorts were done using cultured human islets pooled from one to six donors, characterized for beta cell number, endocrine purity and viability by immunohistochemistry (insulin, glucagon), electron microscopy and Cells 2021, 10, 1693 4 of 19 propidium iodide counting [12]: an average of 9.7 (95% CI: 8.4–11.0) million cells/kg bodyweight, of which 2.9 (95% CI: 2.5–3.3) million/kg bodyweight insulin-positive cells were implanted. Grafts were composed of 40% (95% CI: 34–47) endocrine cells with 32% (95% CI: 27–36) beta cells and 12% (95% CI: 9–16) alpha cells. The rest of the graft consisted of 47% (95% CI: 41–54) non-granulated (mainly ductal) cells, 2% (range 0–8%) acinar exocrine cells and 10% (range 7–14) dead cells (Supplementary Table S1). Patients received immune suppression using Anti-Thymocyte Globulin, mycophenolate mofetil and tacrolimus (protocol NCT00623610, ethical approval 98/059D) [12,13]. Islet isolation and intrahepatic transplantation in the Milan cohort were done according to similar protocols albeit with shorter pre-transplant culture times [14]. Plasma samples were collected in K3-EDTA Monovette tubes (Sarstedt, Nümbrecht, Germany) supplemented with 1% of a 0.12 mg/mL solution of aprotinin (Stago, Asnières sur Seine, France) in 0.9% NaCl. Samples were stored at −80 ◦C after centrifugation for 15 min at 1600× g. 2.2. Recovery—Linearity Experiments with Human Beta Cell Lysate Endocrine-enriched islet preparations (52% insulin positive, pool of 3 donors) were washed 3 times with PBS to remove albumin and resuspended in RIPA buffer supplemented with proteinase inhibitor cocktail PIC 1 and PIC 2 (Roche, Basel, Switzerland) at a ﬁnal cell density of 104 cells/µL. Lysis was done by freezing at −80 ◦C, thawing on ice, sonication and collection of supernatants after centrifugation for 10 min at 4 ◦C at 10,000× g. This lysate was then spiked to K3-EDTA human plasma to obtain the equivalent of 5, 50 and 250 × 103 cells/mL (K cells/mL). 2.6. Targeted PCR in Validation Cohorts Selected (n = 15) candidate biomarker microRNAs (Figure 1) were integrated as single- plex Taqman™miRNA expression assays in the TaqMan® low-density array format (LDA, Applied Biosystems, ThermoFisher, Waltham, MA, USA) based on sequences provided in Supplementary Table S2. The LDA additionally included candidate reference microR- NAs (n = 4) for data normalization selected from the microRNA proﬁling data as follows: standard deviation of all NRQ values (for all samples) per microRNA in the microarray analysis of the discovery cohort were calculated. The microRNAs with the lowest NRQ standard deviation and that were detected in at least 15 of 16 samples (n = 8, pre- and post- transplant) were imported in qbase+ Software v3.0 (10,11) to determine the 15 best-ranked normalizers (geNorm M value, in Supplementary Document S1). Selection of the optimal number of stable normalizers (geNorm V value) was based on geNorm’s pairwise variation analysis between subsequent normalization factors using a cut-off value of 0.15 for the inclusion of additional normalizers [17]. The genomic location of the four microRNAs (miR-524-3p, miR-30e-3p, miR-208a-3p and miR-1291) candidate reference microRNAs was veriﬁed to avoid including microRNAs that are putatively co-regulated [21]. All four candidate reference microRNAs failed in LDA in both subsequent validation cohorts, with inconsistent or no detection. As a consequence, PCR data in the validation cohorts were expressed as RQ instead of reference-gene geometrically normalized values and calculated on paired pre- and post-transplant samples as 2 ∆Ct with ∆Ct = (mean of duplicate Ct of microRNAi pre-transplant–mean of duplicate Ct of microRNAi post-transplant). In the clinical validation cohort of 46 islet transplantations, RQ values were calculated on the post-transplant samples and calculated as 2 ∆Ct where Ct = (mean Ct of microRNAi across all 46 samples—mean of duplicate Ct of microRNAi in sample X). Tissue selectivity of microRNA expression was analyzed on in-house prepared pancreatic cell fractions obtained by COBE 2991 Cell Processor and enriched in exocrine amylase-expressing acinar cells (1.5% contaminating beta cells), CK19-expressing duct cells (0.4% contaminating beta cells) and endocrine fractions with low (8.3%) or high-percentage (61.0%) insulin-positive cells. Body-wide tissue selectivity was addressed using public repositories of microRNA expression in 61 human tissue types [22] and next-generation sequencing of human islets versus other human tissues [10]. 2.5. microRNA Array Data Analysis 7900HT Fast Real-Time PCR System output ﬁles (.sds) of the discovery cohort samples were analyzed using ExpressionSuite Software v1.0.3 (Applied Biosystems, Waltham, MA, USA). Raw Cq values (quantiﬁcation cycle, the standard name for Ct according to Real-time PCR Data Markup Language, MIQE and ISO 20395:2019 guidelines [16]) were calculated applying automatic baseline settings, and threshold settings were adjusted for each individual microRNA with equal threshold settings across all samples within this study. Only Cq values equal to or below 35 were considered detectable and taken into account for downstream quantitative analysis. The raw Cq data were further analyzed using qbase+ Software v3.0 [17,18], where raw Cq values were transformed to linear scale (RQ values) and normalization factors were calculated following global mean normalization Cells 2021, 10, 1693 5 of 19 strategy [19] to obtain one NRQ (normalized relative quantity) value for each microRNA- sample combination. NRQ data were then log10 transformed and uploaded in RStudio (R package version 2.17.0), where an additional data ﬁltering was performed: microRNAs that were detected with low conﬁdence (Cq > 35) in at least 4 of 8, either in the pre-transplant or post-transplant sample group, were excluded. For lysate spiking, a maximum of one missing value was allowed per microRNA. For calculation of fold increase or decrease at group level, missing values of individual microRNAs (Cq > 35) were replaced by the minimum log10(NRQ) minus 1. These ﬁnal data were converted into a heat map using R package gplots [20] with default clustering settings (complete linkage method for Ward clustering and Manhattan distance measure). For the proﬁling of in beta cell recovery experiments, microRNAs were included when the slope of Cq/log (K beta cells/mL plasma) < −1.5; R2 > 0.9 and when at least 4/6 criteria (Cq(0 K/mL) > Cq(5 K/mL); Cq(5 K/mL) > Cq(50 K/mL); Cq(50 K/mL) > Cq(250 K/mL); NRQ(0 K/mL) < NRQ(5 K/mL); NRQ(5 K/mL) < NRQ(50 K/mL); NRQ(50 K/mL) < NRQ(250 K/mL)) were fulﬁlled. Furthermore, the Cq values should progressively decline, and the ∆Cq between subsequent spiked samples ≥1. 3.1. Study Design 3.1. Study Design Intrahepatic infusion of islets is associated with an acute destruction of 2–10% of implanted beta cells, resulting in a synchronous discharge of beta cell-selective biomarkers such as GAD65 and miR-375 [9]. To identify other microRNAs discharged from necrotic graft cells, a step-wise approach was followed as summarized in Figure 1. First, blood samples just before and 1 h after transplantation were proﬁled by hydrolysis-probe PCR array for the presence of 733 human microRNAs in a discovery cohort of eight patients. After stringent data ﬁltering, with exclusion at sample level (all microRNAs with Cq > 35) and at group level (all microRNAs that were non-detected in at least 4 of 8 samples in either the pre-or post-transplant samples), 220 microRNAs were considered conﬁdently detected and used for further quantitative analysis by global mean normalization and calculation of normalized relative quantities (NRQ) (all data in Supplementary Table S3). Of these 220 microRNAs, 29 microRNAs showed signiﬁcantly different NRQ levels between the pre- and post-transplant phase: seven microRNAs showed lower post-transplant levels, and 22 microRNAs showed a least four times higher post-transplant levels (Figure 1a). Next, these 22 increased microRNA were then further manually down-selected to a panel of 15 candidate microRNAs, guided by following criteria: (i) the magnitude of post-transplant surge (NRQ levels); (ii) a correlation with plasma concentrations of GAD65 and/or miR-375 measured by calibrated assays; and/or (iii) a co-linearity with the amount of spiked-in beta cell lysate in a separate recovery experiment (Figure 1b). The resulting panel of 15 microRNAs (shown in Figure 1b and Table 1) was then subjected to blinded veriﬁcation by targeted PCR in two independent validation cohorts: eight transplant events in Brussels (validation cohort 1) and seven transplant events in Milan (validation cohort 2) (Figure 1a), resulting in a core panel of eight microRNAs that were subsequently clinically validated. Table 1. Panel of 15 candidate microRNA biomarkers of beta cell destruction identiﬁed in the discovery cohort and subjected to independent blinded validation. The table lists, from left to right, the microRNA name and corresponding TaqMan® hydrolysis probe used for targeted conﬁrmation, the average Normalized Relative Quantity (NRQ) after global mean normalization, and the Spearman Rank correlation coefﬁcient to plasma GAD65 and miR-375 of the 15 candidate biomarker microRNAs selected, as shown in Figure 1 in the discovery cohort (n = 8). 2.7. Statistical Analyses Correlation and linear regression analyses were done using Prism 5 (GraphPad, San Diego, CA, USA). Statistical differences in NRQ or RQ of microRNA levels before and after transplantation were done using two-tailed paired Student’s t-test in R without correction for multiple comparison. The area under the Receiver Operating Characteristic Cells 2021, 10, 1693 6 of 19 (ROC) curve (AUC) (non-parametric model by DeLong et al. [23]), correlogram (Spearman rank) and multiple regression analysis of microRNAs versus graft outcome were analyzed using MedCalc (version 19.2.1, Mariakerke, Belgium). (ROC) curve (AUC) (non-parametric model by DeLong et al. [23]), correlogram (Spearman rank) and multiple regression analysis of microRNAs versus graft outcome were analyzed using MedCalc (version 19.2.1, Mariakerke, Belgium). 3.2. microRNA Proﬁling in the Discovery Cohort 3.2. microRNA Proﬁling in the Discovery Cohort Microarray proﬁling conﬁrmed miR-375 as excellent biomarker of beta cell destruction: in pre-transplant samples, levels were consistently low, and its post-transplant NRQ values (118-times up, p = 0.007) correlated well with plasma concentrations of GAD65 quantiﬁed by a cytometric bead array [15] (rS = 0.88; p < 0.0001, Figure 1B, Table 1). Besides miR- 375, several other microRNAs with a previously reported [8,24] islet endocrine-enriched expression pattern were increased: Let-7g-5p and three members of the miR-376 cluster. The levels of miR-376a-5p and miR-376a-3p correlated positively with GAD65 (rs = 0.52; p = 0.0396) and with the numbers of implanted beta cells (rS = 0.81; p = 0.0218), respectively. Some of these increased microRNAs might also originate from other cell types contaminat- ing the graft: miR-197-3p correlated positively with the number of duct cells (rS = 0.9048; p = 0.0046), and three microRNAs correlated positively with the number of alpha cells (Figure 2a): miR-125b-5p (rS = 0.8264; p = 0.0154), miR-204-5p (rS = 0.8623; p = 0.0107) and miR-132-3p (rS = 0.7545; p = 0.0368). The miR-375 levels showed a tight hierarchical clustering with miR-200a-3p, miR-369-5p, miR-125b-5p, miR-204-5p, miR-429 and miR- 216b-5p (Figure 2a): all showed consistently low baseline levels and marked increases (NRQ range 36- to 6249-fold) 1 h after islet infusion. Of note, intraportal transplantation was not associated with signiﬁcantly increased levels of hepatocyte-speciﬁc microRNA-122 (NRQ = 18, p = 0.294) nor muscle-speciﬁc miR-133a (NRQ = 7.59, p = 0.0619) potentially originating from surgical procedure-associated tissue injury (Supplementary Table S3). 8 of 21 Figure 2. microRNA profiling after islet cell transplantation and beta cell recovery experiment. A total of 733 human mi- croRNAs were measured before and 1 h after intraportal islet cell transplantation in a discovery cohort of 8 transplant events. (a). Twnety-two microRNAs showed at least 4-fold increase after islet cell transplantation. Log(NRQ) values were hierarchically clustered and for every microRNA the row z-score is shown. Sc/S indicates the paired samples before and after islet infusion. (b). Bar graphs indicate the GAD65 (pM) and miR-375 (NRQ) levels before and one hour after islet cell transplantation and their linear correlation (Inset, rS = 0.88; p < 0.0001). The full line represents a regression line and the dashed lines their 95% confidence interval. (c). Sixteen microRNAs increased linearly after spiking of ascending amounts Figure 2. 3.1. Study Design Right columns show the average Relative Quantitity (RQ) after versus before transplantation and the corresponding p-value (paired student t-test) in the independent validation cohort 1 (Brussels, n = 8) and validation cohort 2 (Milan, n = 7). ND: not detected. Tx: islet transplantation. Discovery Cohort (n = 8) Validation Cohort 1 (n = 8) Validation Cohort 2 (n = 7) miR Level Post/Pre-Tx Correlation GAD65 Correlation miR-375 miR Level Post/Pre-Tx miR Level Post/Pre-Tx MicroRNA Assay ID NRQ P rs P rs P RQ P RQ P miR-375 hsa-miR-375-000564 118.1 0.007 0.88 <0.0001 - - 249.5 0.000 178.0 0.002 miR-132-3p hsa-miR-132-000457 210.9 0.005 0.50 0.051 0.87 <0.0001 2.0 0.047 4.2 0.004 miR-204-5p hsa-miR-204-000508 172.0 0.002 0.71 0.002 0.93 <0.0001 315.4 0.003 33.0 0.016 miR-410-3p hsa-miR-410-001274 49.1 0.034 0.46 0.072 0.57 0.022 3.7 0.002 12.6 0.014 miR-200a-3p hsa-miR-200a-000502 6249.4 0.044 0.49 0.054 0.82 <0.0001 397.0 0.000 70.4 0.000 miR-429 hsa-miR-429-001024 36.5 0.016 0.64 0.008 0.92 <0.0001 287.0 0.007 53.5 0.002 miR-125b-5p hsa-miR-125b-000449 52.1 0.001 0.83 0.000 0.79 0.000 6.8 0.039 6.5 0.282 miR-216b-5p hsa-miR-216b-002326 85.9 0.019 0.66 0.005 0.94 <0.0001 21.6 0.275 191.4 0.084 let-7g-5p hsa-let-7g-002282 90.5 0.036 0.29 0.274 0.69 0.003 0.6 0.109 1.3 0.650 miR-103a-3p hsa-miR-103-000439 130.3 0.020 0.18 0.494 0.49 0.054 1.1 0.497 1.2 0.274 miR-130b-3p hsa-miR-130b-000456 97.1 0.021 0.26 0.336 0.62 0.010 0.8 0.337 1.5 0.086 miR-210-3p hsa-miR-210-000512 156.7 0.010 0.35 0.188 0.76 0.001 0.0 0.766 0.2 0.317 miR-376a-5p hsa-miR-376a-5p-002127 9707.1 0.014 0.52 0.040 0.26 0.322 0.0 0.351 2.7 0.115 miR-369-5p hsa-miR-369-5p-001021 1705.0 0.000 0.93 <0.0001 0.78 0.000 ND ND miR-520e hsa-miR-520e-001119 167.6 0.027 0.45 0.079 0.75 0.001 ND ND ND: not detected; NRQ: normalized relative quantity; RQ; relative quantiy; Tx: islet transplantation. Cells 2021, 10, 1693 7 of 19 3.2. microRNA Proﬁling in the Discovery Cohort microRNA proﬁling after islet cell transplantation and beta cell recovery experiment. A total of 733 human microRNAs were measured before and 1 h after intraportal islet cell transplantation in a discovery cohort of 8 transplant events. (a). Twnety-two microRNAs showed at least 4-fold increase after islet cell transplantation. Log(NRQ) values were hierarchically clustered and for every microRNA the row z-score is shown. Sc/S indicates the paired samples before and after islet infusion. (b). Bar graphs indicate the GAD65 (pM) and miR-375 (NRQ) levels before and one hour after islet cell Figure 2. microRNA profiling after islet cell transplantation and beta cell recovery experiment. A total of 733 human mi- croRNAs were measured before and 1 h after intraportal islet cell transplantation in a discovery cohort of 8 transplant events. (a). Twnety-two microRNAs showed at least 4-fold increase after islet cell transplantation. Log(NRQ) values were hierarchically clustered and for every microRNA the row z-score is shown. Sc/S indicates the paired samples before and after islet infusion. (b). Bar graphs indicate the GAD65 (pM) and miR-375 (NRQ) levels before and one hour after islet cell transplantation and their linear correlation (Inset, rS = 0.88; p < 0.0001). The full line represents a regression line and the Figure 2. microRNA proﬁling after islet cell transplantation and beta cell recovery experiment. A total of 733 human microRNAs were measured before and 1 h after intraportal islet cell transplantation in a discovery cohort of 8 transplant events. (a). Twnety-two microRNAs showed at least 4-fold increase after islet cell transplantation. Log(NRQ) values were hierarchically clustered and for every microRNA the row z-score is shown. Sc/S indicates the paired samples before and after islet infusion. (b). Bar graphs indicate the GAD65 (pM) and miR-375 (NRQ) levels before and one hour after islet cell 8 of 19 Cells 2021, 10, 1693 transplantation and their linear correlation (Inset, rS = 0.88; p < 0.0001). The full line represents a regression line and the dashed lines their 95% conﬁdence interval. (c). Sixteen microRNAs increased linearly after spiking of ascending amounts of human beta cell lysate to a healthy control plasma pool (0 K beta cells/mL; 5 K beta cells/mL; 50 K beta cells/mL and 250 K beta cells/mL, 1 K = 10 [3] beta cells). Log (NRQ) values were hierarchically clustered (color key according to z score). (d). 3.2. microRNA Proﬁling in the Discovery Cohort Fold change with control levels (2∆Ct) are shown versus the number of spiked beta cells (log-scale). 3.3. microRNAs Correlating Linearly to Beta Cell Number in Recovery Experiment A restricted set of 16 microRNAs showed a dose-dependent increase proportionate to the number of spiked-in beta cells (Figure 2c). These included miR-375, which showed the strongest correlation with both the number of spiked beta cells and the associated GAD65 concentration (Figure 2d), but also several other microRNAs that clustered tightly with miR- 375 in the post-transplant samples, such as miR-125b-5pmiR-429, miR-204-5p and miR-200a- 3p (Figure 2a). Overall, 27% (6/22) microRNAs that increased after transplantation showed dose-dependency in the recovery experiment, suggesting their predominant provenance from the grafts (Figure 1b, Figure 2d). 3.4. Selection of Candidate Biomarker MicroRNAs for Validation Studies 3.4. Selection of Candidate Biomarker MicroRNAs for Validation Studies A panel of 15 microRNAs was sub-selected for blinded validation on two independent validation cohorts. The criteria for selection (Figure 1a) were (i) signiﬁcant correlation with plasma GAD65 and/or miR-375 levels after transplantation, (ii) increased levels in the re- covery experiment proportionate to the spiked-in beta cell number (miR-375, miR-125b-5p, miR-204-5p, miR-429, miR-132-3p and miR-200a-3p), (iii) absence of correlation with duct cell marker miR-197-3p and (iv) one microRNA (miR-103a-3p), because of its consistently low pre-transplant baseline plasma levels and strong correlation to several of the selected microRNAs mentioned above. The 15 candidate biomarker microRNAs selected for valida- tion studies are shown in Table 1, along with their average fold increase (average NRQ ratio in discovery cohort) and correlation with GAD65 and miR-375 concentrations. These 15 microRNAs were integrated into a TaqMan® low-density array (LDA) PCR, along with four reference microRNAs for geometric normalization. Reference microRNAs were selected for their stable expression and conﬁdent detection in all pre/post-transplant samples in the discovery cohort as described in Supplement 1. However, these four reference microRNAs were not consistently detected in all samples of the validation cohorts precluding their use for geometric normalization and restricting statistical analysis to RQ instead of NRQ values (Table 1). Samples from two independent validation cohorts were subjected to blinded central analysis. Validation cohorts additionally contained eight healthy control blood samples to assess speciﬁcity. Only 8 of 15 (53%) candidate biomarkers showed clear post-transplant increases in both validation cohorts (RQ values in Table 1): signiﬁcant (p < 0.05) in case of miR-375, miR-132-3p, miR-204-5p, miR-410-3p, miR-200a-3p, miR-429 and miR-125b-5p and a trend for miR-216b-5p. 3.5. Veriﬁcation of Pancreatic Cell Type and Tissue Tropism of the Eight Micrornas Consistently Increased after Islet Transplantation 3.5. Veriﬁcation of Pancreatic Cell Type and Tissue Tropism of the Eight Micrornas Consistently Increased after Islet Transplantation The pancreatic cell type tropism of these eight microRNAs conﬁrmed in both valida- tion cohorts was additionally analyzed by measuring their relative expression in pancreatic cell fractions enriched in CK19-positive duct cells, amylase-expressing acinar cells and islet endocrine-enriched fractions with lower or higher beta cell number (insulin posi- tivity) and by correlating their post-transplant level to cellular composition of the graft. Whole-body tissue tropism of these microRNAs was additionally analyzed using public data repositories: a comparative analysis of microRNA expression in human islets versus other tissues by next-generation sequencing [10] and a human microRNA tissue atlas, comparing microRNA expression in 61 human tissues to obtain a tissue selectivity index (Supplementary Document S1) [22]; the latter data set did not contain islets of Langerhans Cells 2021, 10, 1693 9 of 19 but did contain pituitary endocrine cells with known similarity to beta cells in terms of selective gene expression [25]. but did contain pituitary endocrine cells with known similarity to beta cells in terms of selective gene expression [25]. A ﬁrst set of four microRNAs including miR-375, miR-132-3p, miR-204-5p and miR- 410 showed an islet-enriched expression at the whole-body level (Figure 3a upper panel) and a relative beta cell-selective expression within the pancreas (Figure 3a, lower panel). All but miR-410 also showed a statistical correlation to C-peptide content or alpha cell number in the graft (Table 2). 10 of 21 Figure 3. Relative expression levels in human pancreatic cell fractions. (a). set of 4 microRNA with a relative islet endo- crine-enriched expression and (b) set of 4 microRNA with a relative pancreatic exocrine cell-enriched expression. For each set, the upper panel indicates a tissue-comparative enrichment score obtained by next-generation sequencing in human pancreatic islets (n = 3 isolates from 6 organs) versus the other indicated tissues as derived from a public data set [10] and the lower panel the RQ measured in this study on cell preparations of human pancreas (all from n = 3 independent organ preparations), enriched in duct cells, exocrine acinar cells and islet endocrine-enriched fractions with lower (INS + low) or higher (INS + high) percentage insulin-positive cells. These fractions respectively contained 0.4%, 1.5%, 8.3% and 61.0% insulin-positive cells Figure 3. Relative expression levels in human pancreatic cell fractions. (a). set of 4 microRNA with a relative islet endocrine- enriched expression and (b) set of 4 microRNA with a relative pancreatic exocrine cell-enriched expression. 3.5. Veriﬁcation of Pancreatic Cell Type and Tissue Tropism of the Eight Micrornas Consistently Increased after Islet Transplantation For each set, the upper panel indicates a tissue-comparative enrichment score obtained by next-generation sequencing in human pancreatic islets (n = 3 isolates from 6 organs) versus the other indicated tissues as derived from a public data set [10] and the lower panel the RQ measured in this study on cell preparations of human pancreas (all from n = 3 independent organ preparations), enriched in duct cells, exocrine acinar cells and islet endocrine-enriched fractions with lower (INS + low) or higher (INS + high) percentage insulin-positive cells. These fractions respectively contained 0.4%, 1.5%, 8.3% and 61.0% insulin-positive cells. Figure 3. Relative expression levels in human pancreatic cell fractions. (a). set of 4 microRNA with a relative islet endo- crine-enriched expression and (b) set of 4 microRNA with a relative pancreatic exocrine cell-enriched expression. For each set, the upper panel indicates a tissue-comparative enrichment score obtained by next-generation sequencing in human pancreatic islets (n = 3 isolates from 6 organs) versus the other indicated tissues as derived from a public data set [10] and the lower panel the RQ measured in this study on cell preparations of human pancreas (all from n = 3 independent organ preparations), enriched in duct cells, exocrine acinar cells and islet endocrine-enriched fractions with lower (INS + low) or higher (INS + high) percentage insulin-positive cells. These fractions respectively contained 0.4%, 1.5%, 8.3% and 61.0% insulin-positive cells. Figure 3. Relative expression levels in human pancreatic cell fractions. (a). set of 4 microRNA with a relative islet endocrine- enriched expression and (b) set of 4 microRNA with a relative pancreatic exocrine cell-enriched expression. For each set, the upper panel indicates a tissue-comparative enrichment score obtained by next-generation sequencing in human pancreatic islets (n = 3 isolates from 6 organs) versus the other indicated tissues as derived from a public data set [10] and the lower panel the RQ measured in this study on cell preparations of human pancreas (all from n = 3 independent organ preparations), enriched in duct cells, exocrine acinar cells and islet endocrine-enriched fractions with lower (INS + low) or higher (INS + high) percentage insulin-positive cells. These fractions respectively contained 0.4%, 1.5%, 8.3% and 61.0% insulin-positive cells. 10 of 19 10 of 19 Cells 2021, 10, 1693 Table 2. Correlation between post-transplant microRNA levels and cellular composition of the islet graft and with secretory graft function 2 months after transplantation. 3.5. Veriﬁcation of Pancreatic Cell Type and Tissue Tropism of the Eight Micrornas Consistently Increased after Islet Transplantation Correlations were analyzed in an independent validation cohort 3 of patients (n = 46) receiving at least 2 million beta cells/kg BW, on RQ levels for the indicated microRNAs calculated on paired 1 h post-transplant versus pre-transplant samples for each individual patient. The left panel lists the statistical signiﬁcance (p-value) of Spearman’s rank correlation of RQ to the composition of the grafts in terms of number of insulin-positive cells, C-peptide content, number of glucagon-positive cells, exocrine cells and dead cells measured just prior to implantation. The right panel indicates the correlation of RQ values to the magnitude of the C-peptide increment two months after transplantation [1,9,12] as an indicator of late-stage insulin secretory function of the graft. For correlation with C-peptide and functional graft outcome, multivariate analysis with multiple linear regression was additionally done for parameters retained as signiﬁcant in univariable analysis. Correlation with Graft Characteristics Functional Outcome Beta Cells C-Peptide Content Alpha Cells Exocrine Cells Dead Cells C-Peptide Increment after 2 Months (ng/mL) Spearman (P) Spearman (P) Linear Regression (P) Spearman (P) Spearman (P) Spearman (P) Spearman (P) Linear Regression (P) miR-375 0.0541 p < 0.0001 0.0001 0.0441 0.0023 0.0424 0.0059 0.0535 miR-132-3p 0.1418 0.0198 0.0037 0.0782 0.0271 0.0262 0.0102 0.0432 miR-204-5p 0.2528 0.0230 0.0074 0.0384 0.5109 0.4291 0.1371 / miR-410 0.3239 0.1175 / 0.9689 0.0491 0.6401 0.0432 / miR-200a-3p 0.2320 0.0245 0.0040 0.0859 0.0221 0.0839 0.0828 / miR-429 0.6479 0.0158 0.0006 0.0778 0.0603 0.6225 0.1249 / miR-125b-5p 0.5322 0.0328 / 0.0043 0.0900 0.8033 0.1644 / miR-216b-5p 0.7800 0.1152 / 0.6951 0.3092 0.4530 0.9714 / A second set of four microRNAs including miR-200a-3p, miR-429, miR-125b-5p and miR-216b-5p (Figure 3b, lower panel) showed a more predominant expression in pancreatic exocrine cell fractions. Only miR-200a-3p and miR-429 showed relative enriched expression in islets (Figure 3b, upper panel) and correlated to graft C-peptide content (Table 2). miR- 216b-5p showed a highly restricted expression in the exocrine pancreas (Figure 3b), clearly indicating acinar cell expression. In order to be clinically useful as a biomarker of acute beta cell loss in conditions with a higher probability of such a rare event, consistently low to undetectable baseline levels in conditions with a low probability of beta cell loss, are a prerequisite. 3.5. Veriﬁcation of Pancreatic Cell Type and Tissue Tropism of the Eight Micrornas Consistently Increased after Islet Transplantation From top to bottom, panels show the NRQ microRNA levels in individual sam- ples the discovery cohort (n = 8 transplants, Brussels) and RQ microRNA levels in validation cohort 1 (n = 8 transplants and n = 6 healthy controls h1-h6, Brussels) and validation cohort 2 (n = 7 transplants and n = 2 healthy controls, H5-H6, Milan). Average ± SD and p value ( p < 0.05; ** p < 0.001, paired student-t test) in inset. Bar colors indicate pre-transplant ( e T blue) 1 h o t t a la t ( o t T ed) o healthy o t ol ( ee ) Figure 4. microRNA levels before and 1 h after islet transplantation in three independent cohorts of 4 microRNAs with the strongest islet endocrine enrichment. From top to bottom, panels show the NRQ microRNA levels in individual samples the discovery cohort (n = 8 transplants, Brussels) and RQ microRNA levels in validation cohort 1 (n = 8 transplants and n = 6 healthy controls h1-h6, Brussels) and validation cohort 2 (n = 7 transplants and n = 2 healthy controls, H5-H6, Milan). Average ± SD and p value (* p < 0.05; ** p < 0.001, paired student-t test) in inset. Bar colors indicate pre-transplant (pre-Tx, blue) 1 h post-transplant (post-Tx red) or healthy control (green) Figure 4. microRNA levels before and 1 h after islet transplantation in three independent cohorts of 4 microRNAs with the strongest islet endocrine enrichment. From top to bottom, panels show the NRQ microRNA levels in individual sam- ples the discovery cohort (n = 8 transplants, Brussels) and RQ microRNA levels in validation cohort 1 (n = 8 transplants and n = 6 healthy controls h1-h6, Brussels) and validation cohort 2 (n = 7 transplants and n = 2 healthy controls, H5-H6, Milan). Average ± SD and p value (* p < 0.05; ** p < 0.001, paired student-t test) in inset. Bar colors indicate pre-transplant (pre-Tx, blue), 1 h post-transplant (post-Tx, red) or healthy control (green). Figure 4. microRNA levels before and 1 h after islet transplantation in three independent cohorts of 4 microRNAs with the strongest islet endocrine enrichment. 3.5. Veriﬁcation of Pancreatic Cell Type and Tissue Tropism of the Eight Micrornas Consistently Increased after Islet Transplantation As shown in Figure 4, miR-375 perfectly meets this criterion, with low to undetectable NRQ/RQ values in all pre-transplant samples (n = 23) and all healthy control samples (n = 8) of the three study cohorts. The three other islet endocrine-enriched microRNAs, however, show variably detectable levels in pre-transplant samples (in 78% of samples for miR-132-3p, 30% for miR-410) or in healthy control samples (75% of samples for miR-132-3p, 38% for miR-204-5p). The set of four microRNAs with a more predominant pancreatic exocrine expression showed generally low baseline levels both in pre-transplant and healthy control samples, with the exception of miR-125b (Figure 5). 11 of 19 12 of 21 11 of 19 12 of 21 Cells 2021, 10, 1693 Cells 2021, 10, x Figure 4. microRNA levels before and 1 h after islet transplantation in three independent cohorts of 4 microRNAs with the strongest islet endocrine enrichment. From top to bottom, panels show the NRQ microRNA levels in individual sam- ples the discovery cohort (n = 8 transplants, Brussels) and RQ microRNA levels in validation cohort 1 (n = 8 transplants and n = 6 healthy controls h1-h6, Brussels) and validation cohort 2 (n = 7 transplants and n = 2 healthy controls, H5-H6, Milan). Average ± SD and p value (* p < 0.05; ** p < 0.001, paired student-t test) in inset. Bar colors indicate pre-transplant (pre-Tx, blue), 1 h post-transplant (post-Tx, red) or healthy control (green). Figure 4. microRNA levels before and 1 h after islet transplantation in three independent cohorts of 4 microRNAs with the strongest islet endocrine enrichment. From top to bottom, panels show the NRQ microRNA levels in individual samples the discovery cohort (n = 8 transplants, Brussels) and RQ microRNA levels in validation cohort 1 (n = 8 transplants and n = 6 healthy controls h1-h6, Brussels) and validation cohort 2 (n = 7 transplants and n = 2 healthy controls, H5-H6, Milan). Average ± SD and p value (* p < 0.05; ** p < 0.001, paired student-t test) in inset. Bar colors indicate pre-transplant (pre-Tx, blue), 1 h post-transplant (post-Tx, red) or healthy control (green). Figure 4. microRNA levels before and 1 h after islet transplantation in three independent cohorts of 4 microRNAs with the strongest islet endocrine enrichment. 3.5. Veriﬁcation of Pancreatic Cell Type and Tissue Tropism of the Eight Micrornas Consistently Increased after Islet Transplantation From top to bottom, panels show the NRQ microRNA levels in individual samples the discovery cohort (n = 8 transplants, Brussels) and RQ microRNA levels in validation cohort 1 (n = 8 transplants and n = 6 healthy controls h1-h6, Brussels) and validation cohort 2 (n = 7 transplants and n = 2 healthy controls, H5-H6, Milan). Average ± SD and p value (* p < 0.05; ** p < 0.001, paired student-t test) in inset. Bar colors indicate pre-transplant (pre-Tx, blue), 1 h post-transplant (post-Tx, red) or healthy control (green). 12 of 19 13 of 21 12 of 19 13 of 21 Cells 2021, 10, 1693 Cells 2021, 10, x Figure 5. microRNA levels before and 1 h after islet transplantation in three independent cohorts of 4 microRNAs with lower islet endocrine enrichment. From top to bottom, panels show the NRQ microRNA levels in individual samples in the discovery cohort (n = 8 transplants, Brussels) and RQ microRNA levels in the validation cohort 1 (n = 8 transplants and n = 6 healthy controls h1-h6, Brussels) and validation cohort 2 (n = 7 transplants and n = 2 healthy controls, H5-H6, Milan). Average ± SD and p-value (* p < 0.05; p < 0.005; * p < 0.001, paired student t-test) in inset. Bar colors indicate pre- transplant (pre-Tx, blue), 1 h post-transplant (post-Tx, red) or healthy control (green). Cli i l lid i C l i f l l i l S Figure 5. microRNA levels before and 1 h after islet transplantation in three independent cohorts of 4 microRNAs with lower islet endocrine enrichment. From top to bottom, panels show the NRQ microRNA levels in individual samples in the discovery cohort (n = 8 transplants, Brussels) and RQ microRNA levels in the validation cohort 1 (n = 8 transplants and n = 6 healthy controls h1-h6, Brussels) and validation cohort 2 (n = 7 transplants and n = 2 healthy controls, H5-H6, Milan). Average ± SD and p-value (* p < 0.05; p < 0.005; * p < 0.001, paired student t-test) in inset. Bar colors indicate pre-transplant (pre-Tx, blue), 1 h post-transplant (post-Tx, red) or healthy control (green). Figure 5. microRNA levels before and 1 h after islet transplantation in three independent cohorts of 4 microRNAs with lower islet endocrine enrichment. 3.5. Veriﬁcation of Pancreatic Cell Type and Tissue Tropism of the Eight Micrornas Consistently Increased after Islet Transplantation From top to bottom, panels show the NRQ microRNA levels in individual samples in the discovery cohort (n = 8 transplants, Brussels) and RQ microRNA levels in the validation cohort 1 (n = 8 transplants and n = 6 healthy controls h1-h6, Brussels) and validation cohort 2 (n = 7 transplants and n = 2 healthy controls, H5-H6, Milan). Average ± SD and p-value (* p < 0.05; p < 0.005; * p < 0.001, paired student t-test) in inset. Bar colors indicate pre- transplant (pre-Tx, blue), 1 h post-transplant (post-Tx, red) or healthy control (green). Figure 5. microRNA levels before and 1 h after islet transplantation in three independent cohorts of 4 microRNAs with lower islet endocrine enrichment. From top to bottom, panels show the NRQ microRNA levels in individual samples in the discovery cohort (n = 8 transplants, Brussels) and RQ microRNA levels in the validation cohort 1 (n = 8 transplants and n = 6 healthy controls h1-h6, Brussels) and validation cohort 2 (n = 7 transplants and n = 2 healthy controls, H5-H6, Milan). Average ± SD and p-value (* p < 0.05; p < 0.005; * p < 0.001, paired student t-test) in inset. Bar colors indicate pre-transplant (pre-Tx, blue), 1 h post-transplant (post-Tx, red) or healthy control (green). 3.6. Clinical Validation: Correlation of early Post-Transplant Microrna Levels to Late-Stage Graft Function 3.6. Clinical Validation: Correlation of early Post-Transplant Microrna Levels to Late-Stage Graft Function Finally, a clinical verification of these eight microRNAs was performed in a third previously reported cohort of 46 T1D patients undergoing islet transplantation [1,9]. In these graft recipients, an elevated GAD65 level (> 12.2 pmol/L) and/or miR-375 level (> 7.6 pmol/L) 1h after graft infusion, predicted a poor secretory function in these patients 2 months later, defined as a C-peptide increment below 0.5 ng/mL as compared to the fast- ing C-peptide levels before transplantation [9,12]. Generally, post-transplant GAD65 and miR-375 levels linearly correlate to the C-peptide content of the graft as a proxy of the Finally, a clinical veriﬁcation of these eight microRNAs was performed in a third previously reported cohort of 46 T1D patients undergoing islet transplantation [1,9]. In these graft recipients, an elevated GAD65 level (>12.2 pmol/L) and/or miR-375 level (>7.6 pmol/L) 1h after graft infusion, predicted a poor secretory function in these patients 2 months later, deﬁned as a C-peptide increment below 0.5 ng/mL as compared to the fast- ing C-peptide levels before transplantation [9,12]. Generally, post-transplant GAD65 and miR-375 levels linearly correlate to the C-peptide content of the graft as a proxy of the num- ber of infused beta cells [1,9]. Outliers on these linear regressions—with disproportionately Cells 2021, 10, 1693 13 of 19 13 of 19 elevated GAD65 or miR-375, as expected from the amount of infused beta cells—generally indicate excessive early graft destruction and poor late-stage outcomes [1]. Figure 6a shows the molar amounts of miR-375 1 h post-transplant, plotted as a function of graft function at 2 months. Patients with outliers for GAD65 (blue dots), miR-375 (green dots) or both (red dots) are indicated in color. Molar amounts of miR-375 correlate well with its RQ values (Figure 6b), thus allowing a similar outlier analysis of the eight candidate biomarker microRNAs at RQ level. As shown in Figure 6c–j, 6 of 7 transplant events with outliers for GAD65 and/or miR-375 also showed outliers for one or more of the other microRNAs. However, none of the other microRNAs was individually superior to miR-375, and their inclusion did not result in the identiﬁcation of additional poor outcomes, using a C-peptide increment < 0.5 ng/mL as a dichotomous cut-off. Linear regression analysis indicated that besides miR-375, only miR-410 and miR-132 were correlated to graft outcome (Table 2). In multivariate analysis, only miR-132 and miR-375 were retained. 3.6. Clinical Validation: Correlation of early Post-Transplant Microrna Levels to Late-Stage Graft Function 3.6. Clinical Validation: Correlation of early Post-Transplant Microrna Levels to Late-Stage Graft Function ROC analysis conﬁrmed that only miR-375 (AUC = 0.783, 95% CI 0.637–0.891) and miR-132 (AUC = 0.703, 95% CI 0.550–0.828) had signiﬁcant diagnostic power to predict poor graft outcome at 2 months (Figure 7a). The various microRNAs showed a high degree of redundancy as shown by the correlogram in Figure 7b. In multiple regression, only miR-375 (p = 0.0028) was retained as an independent predictor of poor outcome. 15 of 21 Figure 6. Cont. Figure 6. Cont. 14 of 19 Cells 2021, 10, 1693 Cells 2021, Figure 6. Diagnostic performance of microRNAs to predict poor secretory graft outcome 2 months post-transplantation. (a). Molar miR-375 levels (pmol/L) in plasma as measured by calibrated RT-PCR assay 1 h after islet graft infusion in n = 46 T1D recipients (X-axis) as a function of the graft-induced gain in endogenous C-peptide production (C-peptide incre- ment). Dotted lines indicate operator-chosen thresholds for acceptable graft function at C-peptide increment of 0.5 ng/mL and 1 ng/mL. Further analyses were done taking C-peptide < 0.5 ng/mL as a dichotomous indicator of poor outcome. Generally, 1h post-transplant levels of miR-375 and GAD65 (not shown, [9]) correlate linearly with the number of im- planted beta cells and C-peptide content of the graft. Individual post-transplant patient samples that are outliers on these correlations, indicating excessive beta cell destruction, are numbered and marked as colored dots throughout all panels: outliers for GAD65 (blue dots), miR-375 (green dots) or both (red dots). (b). Linear correlation (95% confidence interval as dotted lines) of post-transplant miR-375 values expressed as RQ values (see methods) and molar levels measured by cali- brated PCR assay. Panels (c–j): RQ values of the indicated microRNAs 1 h post-transplant for all n = 46 transplant events as a function of C-peptide increment 2 months post-transplant. In case the distribution of RQ values of any microRNA shows statistical outliers, then this threshold is marked with a dotted line on the X-axis. Figure 6. Diagnostic performance of microRNAs to predict poor secretory graft outcome 2 months post-transplantation. (a). Molar miR-375 levels (pmol/L) in plasma as measured by calibrated RT-PCR assay 1 h after islet graft infusion in n = 46 T1D recipients (X-axis) as a function of the graft-induced gain in endogenous C-peptide production (C-peptide increment). Dotted lines indicate operator-chosen thresholds for acceptable graft function at C-peptide increment of 0.5 ng/mL and 1 ng/mL. 3.6. Clinical Validation: Correlation of early Post-Transplant Microrna Levels to Late-Stage Graft Function 3.6. Clinical Validation: Correlation of early Post-Transplant Microrna Levels to Late-Stage Graft Function Figure 6. Diagnostic performance of microRNAs to predict poor secretory graft outcome 2 months post-transplantation. (a). Molar miR-375 levels (pmol/L) in plasma as measured by calibrated RT-PCR assay 1 h after islet graft infusion in n = 46 T1D recipients (X-axis) as a function of the graft-induced gain in endogenous C-peptide production (C-peptide increment). Dotted lines indicate operator-chosen thresholds for acceptable graft function at C-peptide increment of 0.5 ng/mL and 1 ng/mL. Further analyses were done taking C-peptide < 0.5 ng/mL as a dichotomous indicator of poor outcome. Generally, 1 h post-transplant levels of miR-375 and GAD65 (not shown, [9]) correlate linearly with the number of implanted beta cells and C-peptide content of the graft. Individual post-transplant patient samples that are outliers on these correlations, indicating excessive beta cell destruction, are numbered and marked as colored dots throughout all panels: outliers for GAD65 (blue dots), miR-375 (green dots) or both (red dots). (b). Linear correlation (95% conﬁdence interval as dotted lines) of post-transplant miR-375 values expressed as RQ values (see methods) and molar levels measured by calibrated PCR assay. Panels (c–j): RQ values of the indicated microRNAs 1 h post-transplant for all n = 46 transplant events as a function of C-peptide increment 2 months post-transplant. In case the distribution of RQ values of any microRNA shows statistical outliers, then this threshold is marked with a dotted line on the X-axis. ells 2021, 10, x 17 of 21 Figure 7. ROC analysis of endocrine-enriched microRNAs 1 h after transplantation to predict secretory graft function 2 months later. (a). Area under the receiver operating characteristics (AUC) of the indicated microRNAs in n = 46 intraportal islet transplantations, measured 1 h post-transplant, to predict a poor secretory graft function 23 months later (<0.5 ng/mL increment of C-peptide as compared to fasting C-peptide before transplantation). (b). Spearman rank correlation coeffi- cients of the indicated microRNAs with a color grading ranging from low (blue) to high (red) correlation. 4 Discussion Figure 7. ROC analysis of endocrine-enriched microRNAs 1 h after transplantation to predict secretory graft function 2 months later. (a). Area under the receiver operating characteristics (AUC) of the indicated microRNAs in n = 46 intraportal islet transplantations, measured 1 h post-transplant, to predict a poor secretory graft function 23 months later (<0.5 ng/mL increment of C-peptide as compared to fasting C-peptide before transplantation). (b). 3.6. Clinical Validation: Correlation of early Post-Transplant Microrna Levels to Late-Stage Graft Function 3.6. Clinical Validation: Correlation of early Post-Transplant Microrna Levels to Late-Stage Graft Function Further analyses were done taking C-peptide < 0.5 ng/mL as a dichotomous indicator of poor outcome. Generally, 1 h post-transplant levels of miR-375 and GAD65 (not shown, [9]) correlate linearly with the number of implanted beta cells and C-peptide content of the graft. Individual post-transplant patient samples that are outliers on these correlations, indicating excessive beta cell destruction, are numbered and marked as colored dots throughout all panels: outliers for GAD65 (blue dots), miR-375 (green dots) or both (red dots). (b). Linear correlation (95% conﬁdence interval as dotted lines) of post-transplant miR-375 values expressed as RQ values (see methods) and molar levels measured by calibrated PCR assay. Panels (c–j): RQ values of the indicated microRNAs 1 h post-transplant for all n = 46 transplant events as a function of C-peptide increment 2 months post-transplant. In case the distribution of RQ values of any microRNA shows statistical outliers, then this threshold is marked with a dotted line on the X-axis. Cells 2021, 10, x 17 of 21 Figure 6. Diagnostic performance of microRNAs to predict poor secretory graft outcome 2 months post-transplantation. (a). Molar miR-375 levels (pmol/L) in plasma as measured by calibrated RT-PCR assay 1 h after islet graft infusion in n = 46 T1D recipients (X-axis) as a function of the graft-induced gain in endogenous C-peptide production (C-peptide incre- ment). Dotted lines indicate operator-chosen thresholds for acceptable graft function at C-peptide increment of 0.5 ng/mL and 1 ng/mL. Further analyses were done taking C-peptide < 0.5 ng/mL as a dichotomous indicator of poor outcome. Generally, 1h post-transplant levels of miR-375 and GAD65 (not shown, [9]) correlate linearly with the number of im- planted beta cells and C-peptide content of the graft. Individual post-transplant patient samples that are outliers on these correlations, indicating excessive beta cell destruction, are numbered and marked as colored dots throughout all panels: outliers for GAD65 (blue dots), miR-375 (green dots) or both (red dots). (b). Linear correlation (95% confidence interval as dotted lines) of post-transplant miR-375 values expressed as RQ values (see methods) and molar levels measured by cali- brated PCR assay. Panels (c–j): RQ values of the indicated microRNAs 1 h post-transplant for all n = 46 transplant events as a function of C-peptide increment 2 months post-transplant. In case the distribution of RQ values of any microRNA shows statistical outliers, then this threshold is marked with a dotted line on the X-axis. 4. Discussion Here, we conducted a comprehensive proﬁling of the microRNA landscape in plasma in a model of acute synchronous necrotic islet cell destruction. A remarkably high number of microRNAs (>200) could be detected in pre-transplant control samples. Islet infusion was, however, associated with prominent surges of a highly restricted set of microRNAs. These expectedly included miR-375, but also several other microRNAs with a previously described beta cell-selective expression and/or function. Four microRNAs (miR-132, miR- 204, miR-410 and miR-375) showed a clearly beta cell-enriched expression within the cell populations in our graft preparations and a relevant neuroendocrine/islet-enriched expression at whole-body level [10,22,26] as measured by PCR and sequencing. All were previously shown to be functionally important for beta cells. Reference marker miR-375 was the ﬁrst microRNA reported to speciﬁcally regulate glucose-stimulated insulin secretion and beta cell mass [8,27,28]. It is selectively expressed by pituitary cells and alpha and beta cells with only a 2-fold higher expression in beta versus alpha cells. Its biomarker potential additionally derives from its extremely high molar expression levels in the beta cell, with sequencing read counts 10 times higher than the second most abundant microRNAs, miR-7 and let-7a [10,26] corresponding to an estimated 600,000 miR-375 molecules per beta cell as measured by a calibrated assay [9]. miR-132-3p was recently implied as a positive regulator of alpha cell mass and resistance to stress-induced apoptosis in both alpha [29] and beta [30] cell apoptosis in mouse models and also showed a moderate 3-fold enrichment in beta versus alpha cells [31]. miR-410 is upregulated in glucose-responsive murine insulinoma cell clones [32] and belongs to the top-beta cell enriched microRNAs with 17-fold higher levels in beta versus alpha cells [31]. However, the most exciting microRNA resulting from our selection is miR-204. miR-204 was identiﬁed as an important regulator of beta cell function and resistance to endoplasmic reticulum stress: its upregulation by TXNIP- STAT3 signaling leads to suppression of insulin gene transcription via downregulation of MAFA transcription factor [33], and it regulates PERK and ATF4 signaling in beta cells [34] and mediates protection to cytokine-induced ER stress in human beta cells [35]. From a biomarker perspective, miR-204 was the most beta-cell-enriched microRNA with 108- times higher level in human beta versus alpha cells and a relatively abundant read count, albeit 177 times lower than miR-375 [10,26]. Our ﬁndings conﬁrm the recent ﬁndings by Xu et al. 3.6. Clinical Validation: Correlation of early Post-Transplant Microrna Levels to Late-Stage Graft Function 3.6. Clinical Validation: Correlation of early Post-Transplant Microrna Levels to Late-Stage Graft Function Spearman rank correlation coefﬁcients of the indicated microRNAs with a color grading ranging from low (blue) to high (red) correlation. Figure 7. ROC analysis of endocrine-enriched microRNAs 1 h after transplantation to predict secretory graft function 2 months later. (a). Area under the receiver operating characteristics (AUC) of the indicated microRNAs in n = 46 intraportal islet transplantations, measured 1 h post-transplant, to predict a poor secretory graft function 23 months later (<0.5 ng/mL increment of C-peptide as compared to fasting C-peptide before transplantation). (b). Spearman rank correlation coeffi- cients of the indicated microRNAs with a color grading ranging from low (blue) to high (red) correlation. Figure 7. ROC analysis of endocrine-enriched microRNAs 1 h after transplantation to predict secretory graft function 2 months later. (a). Area under the receiver operating characteristics (AUC) of the indicated microRNAs in n = 46 intraportal islet transplantations, measured 1 h post-transplant, to predict a poor secretory graft function 23 months later (<0.5 ng/mL increment of C-peptide as compared to fasting C-peptide before transplantation). (b). Spearman rank correlation coefﬁcients of the indicated microRNAs with a color grading ranging from low (blue) to high (red) correlation. Cells 2021, 10, 1693 15 of 19 15 of 19 4. Discussion g gg y The strengths of our study are its rigorous design with (i) a stepwise selection of putatively beta cell-enriched microRNAs in well-characterized islet transplant models, allowing the study of correlation between biomarker proﬁles and cellular graft composition, size and outcome; (ii) the use of several independent validation cohorts originating from two different clinical centers, with centralized but blinded analysis, thus increasing the robustness of our biomarker prioritization; and (iii) the use of a sufﬁciently sized cohort for clinical validation of their prognostic potential of graft outcome. Cross-comparison with recent literature indicates that this crystallized into a highly relevant panel with siven islet endocrine-selective microRNAs and one exocrine-selective microRNA that requires further study for its potential for early diagnosis of pancreatitis. Our study also had limitations. Technically we failed to identify proper housekeeping microRNAs for data normalization in the targeted RT-PCRs. Despite their vast biomarker potential, microRNAs have not yet made it into the routine clinical diagnostic lab and independent reproduction of study ﬁndings remains challenging. Bench-to-bed translation of microRNAs will require the development of calibrated assays for absolute quantiﬁcation of circulating microRNA levels or absolute quantiﬁcation via digital PCR methods. A second limitation for biomarker discovery might be the choice of the model: acute early islet graft destruction likely involves necrotic cell death of endocrine, acinar and ductal cells alike and thus only serves as a rough proxy for the pure beta cell death in autoimmune T1D. Furthermore, it is not clear if gene expression patterns in isolated, cultured beta cells are a reliable approximation of the phenotype of pro-apoptotic, stressed beta cells during the preclinical phase of T1D. It is thus possible that stressed beta cells, around the clinical onset of T1D, upregulate microRNAs involved in endoplasmic reticulum stress responses such as miR-204, and this might boost their biomarker potential by increasing the clinical sensitivity. This could explain why miR-204 could be detected around the onset of T1D [36] but was clearly inferior to miR-375 in our transplant cohorts. More research is needed to evaluate if combinatorial proﬁling of miR-375 with miR- 132/204/410/200a/429/125b has potential for sensitive detection of occult beta cell death in the preclinical phase and around the onset of T1D. In our opinion, microRNAs have signiﬁcant potential as compared to both protein-type or (un)methylated DNA markers. 4. Discussion [36] that miR-204 is increased after islet transplantation. More importantly, the latter study also observed increases in plasma miR-204 in the recent-onset phase of T1D, indicating restricted beta cell provenance and highlighting the potential of this microRNA to screen for autoimmune-mediated beta cell destruction. A second set of four microRNA (miR-200a, miR-429, miR-125b and miR-216b) showed in our analysis a higher relative expression in pancreatic acinar-enriched fractions. One of those, miR-216b, is undoubtedly pancreatic acinar cell-selective: at whole-body level, it is highly enriched in total pancreas tissue, and in rodent models, it was recently reported to be an excellent marker for pancreatitis, superior even to amylase and lipase [37]. For the other three, our observed enrichment in exocrine pancreatic cell fractions conﬂicts with several reports suggesting endocrine-selective expression and function. miR-200a and miR-429 belong to the co-regulated miR-200a/b/c/142/429 cluster with endocrine- selective expression at whole-body level [38], a relative 2-fold enrichment in beta versus alpha cells [31]. This cluster is upregulated in glucose-responsive murine insulinoma clones [32] and protects beta cells from stress-induced apoptosis in mouse models [39]. Finally, miR-125b was identiﬁed as one of the most beta cell-enriched microRNAs, superior even to miR-410 and miR-132. It is possible that miR-125b and miR-200a/429 are indeed endocrine-expressed but that their expression is upregulated ex vivo, in the small subset (1.5%) of beta cells that is co-puriﬁed with exocrine cell fractions, resulting in cell stress by the isolation or culture procedures. In a third independent cohort of 46 transplant events, we then evaluated the diagnos- tic performance of these eight microRNAs to predict late-stage secretory graft outcome 2 months later. These ﬁndings were disappointing: though we observed a clear co-discharge Cells 2021, 10, 1693 16 of 19 16 of 19 of several newly identiﬁed microRNAs with miR-375, only miR-132 showed signiﬁcant prognostic power (95% conﬁdence interval around AUC excluding AUC = 0.5) but in regression analysis, only miR-375 was retained. This indicates that the newly discovered microRNAs have no added value over miR-375 to predict late-stage graft outcome and are likely inferior to miR-375 as a biomarker of beta cell loss. However, this does not completely erode their diagnostic potential: the addition of the sixendocrine-enriched microRNA (miR-132/204/410/200a/429/125b) to miR-375 in a multiplex- or panel-based PCR in future studies could still be useful to increase the diagnostic speciﬁcity in studies screening for possible triggers of nutrients- or cytokine-induced beta cell death. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. References 1. Ling, Z.; De Pauw, P.; Jacobs-Tulleneers-Thevissen, D.; Mao, R.; Gillard, P.; Hampe, C.S.; Martens, G.A.; In’t Veld, P.; Lernmark, A.; Keymeulen, B.; et al. Plasma GAD65, a marker for early beta-Cell Loss After Intraportal Islet Cell Transplantation in Diabetic Patients. J. Clin. Endocrinol. Metab. 2015, 100, 2314–2321. [CrossRef] 2. 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Multiplexing DNA methylation markers to detect circulating cell-free DNA derived from human pancreatic beta cells. JCI Insight 2020, 5, e136579. [CrossRef] 7 Sklenarova J ; Petruzelkova L ; Kolouskova S ; Lebl J ; Sumnik Z ; Cinek O Glucokinase gene may be a more suitable target 7. Sklenarova, J.; Petruzelkova, L.; Kolouskova, S.; Lebl, J.; Sumnik, Z.; Cinek, O. 4. Discussion Though relatively low throughput to execute, PCR assay development is easy as compared to the selection of high afﬁnity-antibody couples for sandwich immunoassays. Their half- life in plasma (ranging from 1.5 to 13 h [40]) is also relatively favorable with extended circulation likely due to microRNA binding to the 97kDa Argonaute-2 protein or association to extracellular vesicles. As compared to beta cell-selective patterns of (un)methylated DNA, microRNAs have the beneﬁt of their vastly higher molar abundance per cell, and also their longer half-life, with short (170 bp) circulating cell-free DNA fragments being typically cleared within 15 min after discharge. y y g In conclusion, our study shows that at least six endocrine-enriched microRNAs are co-released with miR-375 from damaged islet grafts. Individually, these newly identiﬁed microRNAs are inferior to miR-375 to detect acute synchronous beta cell destruction, quantify early graft destruction and predict late-stage graft function. Cells 2021, 10, 1693 17 of 19 17 of 19 Supplementary Materials: The following ﬁles available online at https://www.mdpi.com/article/ 10.3390/cells10071693/s1, Supplementary Document S1 Supplementary methods selection reference genes and analysis of beta cell-selectivity. Supplementary Table S1 Graft characteristics discovery cohort and validation cohort 1 (Brussels). Supplementary Table S2 Details of targeted PCR assays used for indicated miRbase accessions and sequence. Supplementary Table S3 NRQ data of microarray proﬁling in discovery cohort. Author Contributions: Conceptualization: G.A.M. and D.G.P.; Data curation: G.S., J.A., Z.L., D.D.S. and S.R.; Formal analysis: G.A.M., Z.L., F.K.G., D.D.S. and S.R.; Funding acquisition: G.A.M.; Investi- gation: G.A.M. and S.R.; Methodology: G.A.M., G.S., L.P., Z.L., J.V. and S.R.; Project administration: G.A.M. and F.K.G.; Resources, L.P., D.G.P., F.K.G. and B.K.; Software: J.V. and S.R.; Supervision: G.S., Z.L. and F.K.G.; Validation: G.S. and S.R.; Visualization: J.A. and P.M.; Writing—original draft, G.A.M. and S.R.; Writing—review and editing, G.S., D.D.S. and J.V. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Juvenile Diabetes Research Foundation (JDRF) gran (JDRF project 1-PNF-2012-615 to G.A.M. and F.G and by the Willy Gepts Foundation (Universitair Ziekenhuis Brussel). Funding agencies had no inﬂuence on data interpretation and manuscript preparation. Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: All source data presented in this study are available in Supplementary Tables S1–S3. 4. Discussion Data Availability Statement: All source data presented in this study are available in Supplementary Tables S1–S3. References Comparing the areas under two or more c characteristic curves: A nonparametric approach. Biometrics 1988, 44, 837–845. [CrossRef] p pp 24. Joglekar, M.V.; Joglekar, V.M.; Hardikar, A.A. Expression of islet-speciﬁc microRNAs during human Gene Expr. 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Edinburgh Research Explorer Making Virtual Learning Environments Accessible to People with Disabilities in Universities in Uganda Edinburgh Research Explorer Citation for published version: Baguma, R & Wolters, M 2021, 'Making Virtual Learning Environments Accessible to People with Disabilities in Universities in Uganda', Frontiers in Computer Science, vol. 3, 638275. https://doi.org/10.3389/fcomp.2021.638275 Published In: Frontiers in Computer Science Take down policy Take down policy The University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorer content complies with UK legislation. If you believe that the public display of this file breaches copyright please contact openaccess@ed.ac.uk providing details, and we will remove access to the work immediately and investigate your claim. Download date: 24. Oct. 2024 ORIGINAL RESEARCH published: 16 June 2021 doi: 10.3389/fcomp.2021.638275 Keywords: virtual learning environment, learning management system, elearning, accessibility, people with disabilities, higher education institutions, Uganda Edited by: Carlos Duarte, University of Lisbon, Portugal Making Virtual Learning Environments Accessible to People with Disabilities in Universities in Uganda Rehema Baguma 1* and Maria K. Wolters 2 1School of Computing and IT, College of Computing and IT, Makerere University, Kampala, Uganda, 2School of Informatics, Institute for Design Informatics, University of Edinburgh, Edinburgh, United Kigdom Public and private universities in Uganda have been using Virtual Learning Environments (VLEs) since early 2000s to support delivery of blended learning owing to the increased uptake of technology in many aspects of life, and the beneﬁts of blended learning/ eLearning. eLearning is of particular beneﬁt to people with disabilities, since they may ﬁnd it difﬁcult to attend classes on a university campus. Accessibility of a VLE has a strong impact on user engagement and adoption and consequently on students’ learning outcomes. Current research on use of VLEs and eLearning in general in Ugandan universities focuses on sensitization and training, the potential of social media like WhatsApp and Facebook, and required resources like Internet connectivity, and change management. In stark contrast, there is no investigation of accessibility to people with disabilities, even though about 12.4% of the population have some form of disability. This paper examines the extent to which Uganda’s policy environment promotes making eLearning accessible, reviews the accessibility of a sample of VLEs of public and private universities in Uganda, and suggests recommendations on addressing the existing accessibility gaps in policy and implementation of VLEs. Keywords: virtual learning environment, learning management system, elearning, accessibility, people with disabilities, higher education institutions, Uganda INTRODUCTION Reviewed by: Claire Kearney-Volpe, New York University, United States Mike Kent, Reviewed by: Claire Kearney-Volpe, New York University, United States Mike Kent, Curtin University, Australia Correspondence: Rehema Baguma rehema.baguma@gmail.com Reviewed by: Claire Kearney-Volpe, New York University, United States Mike Kent, Curtin University, Australia Correspondence: Rehema Baguma rehema.baguma@gmail.com As part of the ongoing Covid-19 lockdown, all tertiary institutions across Uganda were required to shift to Open Distance and eLearning (ODeL). Before an institution can be cleared to offer remote teaching and learning, they need to show that they adhere to guidelines that were issued in July 2020 by the National Council for Higher Education (NCHE). One of the requirements is an interactive learning management system (LMS) that effectively supports eLearning, which should provide for student-to-student interactions; student and instructor interactions, and evaluation of interaction. However, institutions are not required to ensure that all remote learning activities, and in particular the LMS and its content, are accessible to students with disabilities, even though 12.4% of the population have a disability (National Population and Housing Census, 2014). Curtin University, Australia *Correspondence: Rehema Baguma rehema.baguma@gmail.com Received: 05 December 2020 Accepted: 20 April 2021 Published: 16 June 2021 Citation: Following the UN (Convention on the Rights of Persons with Disabilities (CRPD), 2020), we deﬁne people with disabilities as those who have long-term impairments that make it difﬁcult for them to fully and effectively participate in society on an equal footing with others (Article 1). These impairments can be sensory, physical, mental, or cognitive. Our use of the term “people with disabilities” follows the recommendations in Hanson et al. (2015) and echoes the particular terminology and person-ﬁrst language used in many relevant legal and policy documents. We Baguma R and Wolters MK (2021) Making Virtual Learning Environments Accessible to People with Disabilities in Universities in Uganda. Front. Comput. Sci. 3:638275. doi: 10.3389/fcomp.2021.638275 Baguma R and Wolters MK (2021) Making Virtual Learning Environments Accessible to People with Disabilities in Universities in Uganda. June 2021 \| Volume 3 \| Article 638275 1 Frontiers in Computer Science \| www.frontiersin.org Making Virtual Learning Environments Accessible Baguma and Wolters recognize, however, that terms such as disability and impairment are inherently problematic and will return to this in Section Discussion. For VLEs to effectively facilitate learning for all categories of learners including those with disabilities, all learners must be able to ﬁnd course content, participate, collaborate, communicate with the facilitator and peers, and complete required tasks. Accessibility of a VLE has a strong impact on user engagement and adoption and consequently on students’ learning outcomes. Students may fail to execute or participate in certain activities, they may fail to access or use learning resources, and they may not come back for more learning if they have not had a great experience. However, if the VLE was built with accessibility in mind, and content authors adhere to principles of accessibility, it has the potential to make higher education more accessible to everyone, particularly those with visual, hearing, and cognitive impairments (Hersh, 2008). In an accessible ODeL environment, people with disabilities have a high chance of learning better given the advantages ODeL brings to learning such as self-paced learning, ubiquitous access to learning resources, and remote collaboration, among others (Baguma, 2017). eLearning allows learners with disabilities to be more self-reliant, and to dispel the alleged misconceptions by some educators that such learners are incompetent and means that such learners no longer need to rely on other students to do their assignments (Beyene et al., 2020). eLearning in Uganda Makerere University and other public universities in Uganda have been using Virtual Learning Environments (VLEs) since early 2000s. Makerere University started with Blackboard in 2003 and later moved to an instance of Moodle called Makerere University eLearning Environment (MUELE). Other universities like Makerere University Business School, and Gulu University followed with Moodle (Mayoka and Kyeyune, 2012). To date, private universities have also set up VLEs to support delivery of blended learning owing to the increased uptake of technology in many aspects of life, the beneﬁts of blended/eLearning and most recently the Covid-19 lockdown. 1https://moodle.org/(accessed on 15th November 2020). Citation: The 2014 Uganda National Population and Housing Census deﬁned disability prevalence as the proportion of the population aged two years and above who had difﬁculty in seeing, walking, hearing or remembering (National Population and Housing Census, 2014). In order to ensure that those with disabilities can access higher education, strong laws and policies that promote inclusive education need to be in place. Article 24 of the UN (Convention on the Rights of Persons with Disabilities (CRPD), 2020) deﬁnes inclusion as a process of systemic reform embodying changes and modiﬁcations in content, teaching methods, approaches, structures and strategies in education to overcome barriers with a vision serving to provide all students of the relevant age range with an equitable and participatory learning experience and environment that best corresponds to their requirements and preferences.” This paper examines the extent to which Uganda’s policy environment promotes inclusive ODeL, reviews the extent VLEs of selected public and private universities in Uganda are currently accessible to people with disabilities, and suggests recommendations on how existing gaps in policy and implementation of VLEs can be addressed to improve the accessibility of VLEs of universities in Uganda. Accessible eLearning g Guglielman (2010) categorized eLearning accessibility into technological and pedagogical accessibility. Technological accessibility includes the accessibility of hardware and software, adaptive and assistive technology, websites, and eLearning platforms covering both the static and conﬁgurable design. On the other hand, pedagogical accessibility involves access to content, resources, and documents, interaction and collaboration tools such as chats, forums, Wikis; and access to learning activities like labs, group work, peer practices, quizzes, projects, debates, etc. Making content accessible involves being able to read text, convert content into another format and download content in different formats such as Doc, PDF, audio and video. Moodle is designed to provide equal functionality and information to all people regardless of disabilities, assistive technologies used, different screen sizes and different input devices such as mouse, keyboard and touchscreen1. According to Rogers, the Moodle LMS incorporates best practices such as “Alt tag” text descriptions for images and ﬁgures. Students can be designated as the user of a screen reader so that page content adapts to the read-out-loud format, the interface is simpliﬁed to remove clutter, and the long lists of links can be skipped by the screen reader. Moodle is also zoom-enabled, allowing users with low vision to increase the size of content for better readability. It supports keyboard navigation which is important for users of screen readers and those with mobility limitations that prevent them from using a mouse. However, even with an accessible eLearning platform, content must still be created, organized, and formatted following accessibility best practices for an eLearning platform and its contents to be accessible to people with disabilities. On 9th November 2020, Moodle released version Virtual Learning Environments (VLEs) are software packages through which learning activities are delivered online in the context of eLearning or blended learning. To-date, there are close to 200 VLEs on the market and more than half of these are open source (Hersh, 2008). Presently, Moodle is the most widely used open source VLE around the world (Medevel.com). VLEs should be accessible to all learners, including those with disabilities, to support an inclusive educational experience. Accessibility of a VLE should cover all student, administrator, and teacher functions, including editing and content authoring, with prompts for features such as alternative text descriptions of ﬁgures/images, content and formatting of documents posted on the system, and system modiﬁcation in the case of open source software (Hersh, 2008). Rights of People with Disabilities Rights of People with Disabilities The 2030 Sustainable Development Goals In September 2015, while Uganda was holding the UN presidency, the 192 member states of the UN adopted a resolution committing themselves to the 2030 Agenda for Sustainable Development. The 2030 agenda and associated 17 SDGs informs and guides global and national development. The 17 SDGs are centered on the principle of leaving no one behind, a holistic approach to achieving sustainable development for all. Throughout the 17 SDGs, disability is referenced in multiple parts, speciﬁcally related to education, growth and employment, inequality, accessibility of human settlements, as well as data collection and monitoring the SDGs (UN.org). SDG 4 states: “To ensure equitable and inclusive quality education and promote lifelong learning opportunities for all.” Speciﬁcally, target 4.5 states: “By 2030, eliminate gender disparities in education and ensure equal access to all levels of education and vocational The Constitution of Uganda, adopted in 1995, enshrines relevant disability rights in three articles: Article 16 recognizes the right of persons with disabilities to respect and human dignity, Article 32 outlaws discrimination on the basis of disability, and Article 34 recognizes the right of all children to beneﬁt from education. In order to address violations of these articles, the National Council for Disability Act was passed in 2003. The rights of people with disabilities were further spelled out in the 2006 Disability Act, which promotes equal opportunities, empowerment, and participation and protects disability rights regardless of age, gender, or type of disability. The Disability Act was accompanied by a human rights-based policy framework, the 2006 National Policy on Disability. Article 24b of the 2001 Universities and Other Tertiary Institutions Act speciﬁcally notes that all people, including those with disabilities, should have the opportunity of acquiring higher education. The Special Needs and Inclusive Education Policy (still in its 2011 draft form) promotes provision of specialized instructional materials, equipment and supportive services to all categories of learners at all levels of The Constitution of Uganda, adopted in 1995, enshrines relevant disability rights in three articles: Article 16 recognizes the right of persons with disabilities to respect and human dignity, Article 32 outlaws discrimination on the basis of disability, and Article 34 recognizes the right of all children to beneﬁt from education. In order to address violations of these articles, the National Council for Disability Act was passed in 2003. Accessible eLearning June 2021 \| Volume 3 \| Article 638275 Frontiers in Computer Science \| www.frontiersin.org 2 Making Virtual Learning Environments Accessible Baguma and Wolters training for the vulnerable, including Persons with Disabilities (PWDs). . ..” 3.10 and Moodle 3.9.3 in which it addressed WCAG 2.1 Level A and Level AA issues raised from an external audit that had been sanctioned earlier1. Most accessibility improvements were also back-ported to versions 3.7.5+ and 3.8.2+. UN Convention on the Rights of Persons with Disabilities In 2018, Uganda ratiﬁed the CRPD which mandates all states to protect, respect and fulﬁll the right to education without discrimination. However, in a 2016 dialogue with the CRPD Committee and the Ugandan government regarding conditions for people with disabilities in Uganda, delegates from Disabled Persons Organizations noted the need to translate the CRPD into amendments to national law, including the 2006 Persons with Disabilities Act, and the need for budgetary allocations to disability programs particularly in education, health and employment (Disability Rights Fund, 2016; Uganda DPOs Present Critical Rights Issues to CRPD Committee, 2016). Research on use of VLEs and eLearning in general in Ugandan universities is still limited. The focus has been more on sensitization and training, required resources like Internet connectivity, and change management (Mayoka and Kyeyune, 2012). Other studies have examined the potential of emerging technologies like WhatsApp (Baguma et al., 2019) and Facebook (Bagarukayo, et al., 2016). To the best of our knowledge, this paper is the ﬁrst to assess the accessibility of the VLEs of universities in Uganda, which form a core part of Ugandan students’ ODeL experience, for people with disabilities. 1https://moodle.org/(accessed on 15th November 2020). g y Commitment to the Global Agenda g y Commitment to the Global Agenda At the global level, Uganda has committed herself to the global disability agenda. We focus on three policy documents that cover the rights of adults with disabilities: the 2030 Sustainable Development Goals (SDGs), the UN Convention on the Rights of Persons with Disabilities (CRPD), and the Marrakesh Treaty. The Marrakesh Treaty The Marrakesh Treaty, adopted on June 27, 2013 in Marrakesh forms part of the body of international copyright treaties administered by The World Intellectual Property Organization (WIPO). Its main goal is to create a set of mandatory limitations and exceptions for the beneﬁt of the blind, visually impaired, and otherwise print disabled (VIPs) through facilitating access to Published Works for the visually impaired learners (Access to Information for the Visually Impaired has been Made Possible, 2020). Uganda ratiﬁed the Treaty in 2018, and in the same year, it entered into force in the country (Afri-can.org). Both international and national policy instruments demand for access, equity and quality as regards educational services for persons with special learning needs. Uganda is a signatory to International Agreements on making education accessible to people with disabilities. At the national level, a number of laws and policies that promote provision of education to those with disabilities, and accessibility of ICTs and ICT enabled services, have also been developed. This section reviews key international and national policies that promote making education accessible. Existing Policy Environment At the National level, a number of laws and polices promote provision of education to people with disabilities, and accessibility of ICTs and ICT enabled services. In addition, the Education and Sports Sector Strategic Plan 2017/18 - 2019/20 earmarks promotion of eLearning and computer literacy in secondary and tertiary education in order to enhance learning outcomes. Here, we focus on those relevant to Higher Education. Gaps in the Policy Environment Partial Coverage of Access for People with Disabilities by ICT Policies Uganda has many enabling policies and laws aimed at protecting the interests of children with disabilities and creating equal opportunities for people with disabilities (Abimanyi-Ochom and Mannan, 2014). However, there are still considerable policy gaps, and key policies such as the Policy on Special Needs and Inclusive Education and the ICT for Disability Policy, are still in draft form. Relevant ofﬁcials in Government of Uganda attribute the delay to pass the policies to lack of ﬁnancial resources to implement the policies once they are passed. The delay to pass and implement inclusion policies shows that Government of Uganda is yet to give inclusion issues in general, and inclusive education in particular, the priority they deserve. Lack of substantive policy instruments limits the demand for access, equity and quality to educational services. This can explain why the ODeL guidelines released by the National Council for Higher Education (NCHE) in July 2020 to guide remote teaching and learning activities during the Covid- 19 lockdown are silent on ensuring that all remote learning activities, and in particular the LMS and its content, are accessible to students with disabilities, despite the fact that 12.4% of the population have a disability (National Population and Housing Census, 2014). The global and national anti- discrimination laws and treaties on education and ICT must be accompanied by country-speciﬁc statutes that specify the steps that should be taken to facilitate inclusion in education and ODeL (Singal et al. 2017). It has also been noted that there is a substantial lack of data to inform policy making Whereas Uganda boasts of an ICT regulatory framework that covers the needs of people with disabilities, the relevancy of existing ICT policies to the promotion of ICTs and ICT enabled services such as eLearning that are accessible to those with disabilities is low. In the NITA-U Act 2009, The Uganda Communications Act 2000 and the National ICT Policy 2014–2019, people with disabilities have been categorized with youth and women as a special interest group. Due to this generalization, key issues that are speciﬁc to those with disabilities, such as the use of ICT as a means to remove barriers to learning, are not highlighted. This limits the relevancy of such policy instruments to the demand for access, equity and quality to educational services. Where speciﬁc provisions are made, they are limited to speciﬁc types of disability. Gaps in the Policy Environment Partial Coverage of Access for People with Disabilities by ICT Policies For example, the Uganda Communications Act 2000 only refers to promotion of accessibility of communication services to the hearing impaired. But, given the current technological and communications convergence, people with other disabilities particularly visual and learning disabilities are also affected by the telecommunication and broadcasting services regulated by the Communications Act 2000 (ICT for Disability Policy, 2017). ICT for People with Disabilities The Uganda Communications Act 2000 (UCC) promotes research into the development and use of new communications techniques and technologies, including those which promote accessibility of hearing-impaired people to communication services. The National IT Authority–Uganda (NITA-U) Act (2009) provided for the establishment of the National IT Authority-Uganda (NITA-U). One of the goals of NITA-U (section Evaluation of University Virtual Learning Environments for Accessibility, sub-section (f)) is to promote access to and use of ICT for youth, women, and people with disabilities. These three “special interest groups” are also highlighted in Section 4.6.1.1 of the National ICT Policy (2014–2019). A draft ICT for Disability Policy was published in 2017. It promotes sector-wide interventions to improve the lives of all Ugandans through ICT. The policy seeks to augment government efforts to promote the social economic development of people with disabilities mainly through provision of health services and special needs/inclusive education, which is to be achieved by developing and supplying accessible ICTs and ICT enabled services in Uganda. The 2011 draft Policy on Special Needs and Inclusive Education does not specify ICT or education technology as one of the instructional materials, equipment and supportive service. On the other hand, the 2017 second draft Policy on ICT for Disability proposes comprehensive sector-wide interventions to improve the lives of all Ugandans by facilitating and promoting the use of ICTs, but does not make reference to making eLearning accessible to people with disabilities, or to the 2011 draft Policy on Special Needs and Inclusive Education, although it came into existence when the latter had been in place albeit in draft form, for six years. Rights of People with Disabilities The rights of people with disabilities were further spelled out in the 2006 Disability Act, which promotes equal opportunities, empowerment, and participation and protects disability rights regardless of age, gender, or type of disability. The Disability Act was accompanied by a human rights-based policy framework, the 2006 National Policy on Disability. Article 24b of the 2001 Universities and Other Tertiary Institutions Act speciﬁcally notes that all people, including those with disabilities, should have the opportunity of acquiring higher education. The Special Needs and Inclusive Education Policy (still in its 2011 draft form) promotes provision of specialized instructional materials, equipment and supportive services to all categories of learners at all levels of June 2021 \| Volume 3 \| Article 638275 Frontiers in Computer Science \| www.frontiersin.org 3 Making Virtual Learning Environments Accessible Baguma and Wolters standalone policy. The Special Needs and Inclusive Education policy that was drafted in 2011 has remained in draft till today, a situation the Government of Uganda attributes to lack of ﬁnancial resources for its implementation (The Independent, 2019). But even this draft does not mention eLearning, much less accessible eLearning. In the absence of such policy guidance, consideration of disability-speciﬁc needs in the implementation of eLearning in higher education institutions is limited, and in most cases non- existent. Educators and the education environment are not supported in providing eLearning that is accessible to people with disabilities. This greatly disadvantages those living with disabilities, especially given the potential beneﬁts of eLearning to them. Similarly, the ICT and Disability Policy has been in draft form since 2017 for the same reason of inadequate resources, and makes no mention of accessible eLearning/education technology. education. It also provides for training of special needs and inclusive education personnel, and guides on access to physical environment in schools, on the curriculum, and on assessment and information. However, there are no special higher education institutions for people with disabilities. Hence, they rely on support available in mainstream universities. Automatic Accessibility Evaluation To assess the accessibility of the eLearning platforms under study, WAVE, a suite of evaluation tools that can identify Web Content Accessibility Guideline (WCAG) errors, but also facilitates human evaluation of web content was used. WAVE can be used online by entering the address of the website/system under evaluation at: https://wave.webaim.org/, or installing browser extensions (Firefox and Chrome) for evaluating local, dynamic, or password-protected pages and site-wide WAVE tools for easily evaluating numerous pages2. For this study, the online option was used. WAVE reports the number and category of errors (issues that need to be ﬁxed), alerts (potential problems that need to be checked), and good accessibility practices. In this study, we focused on errors and alerts. Table 3 provides an overview of the errors and alerts we observed, and outlines ways to address them. Universities Sampled Universities Sampled Uganda has 11 public and 39 private universities. We selected 3 institutions from each category. The 3 public universities covered include: • Makerere University (MUK), founded in 1922, the oldest and biggest university in the country located in Kampala, the capital city, which has an Eastern Campus in Jinja. The web address of each VLE studied was entered in the WAVE site at: https://wave.webaim.org/, and all errors and alerts returned were noted. p y p j • Kyambogo University (KYU), founded in 2003, which has a Faculty of Special Needs and Rehabilitation, also located in Kampala. 2https://webaim.org/(accessed on 20th October 2020). Lack of Policies Focused on Making eLearning Accessible to People with Disabilities To date, there is no policy on eLearning accessibility to people with disabilities either as part of other related policies or as a June 2021 \| Volume 3 \| Article 638275 Frontiers in Computer Science \| www.frontiersin.org 4 Making Virtual Learning Environments Accessible Baguma and Wolters (Abimanyi-Ochom and Mannan, 2014; Wozniak et al., 2020). The implementation of the existing policy framework is weak, and resources made available to translate policies into services for people with disabilities are always insufﬁcient (Abimanyi-Ochom and Mannan, 2014). Thus, in spite of government efforts, people with disabilities continue to face difﬁculties accessing quality education. summarized in Table 1. For each University, we chose six courses from a variety of Schools, including Health, Engineering, Education, Science, and Agriculture. The selected courses are summarized in Table 2. Types of Disabilities Covered Our evaluation focused mostly on accessibility to people with visual, and to some extent hearing impairments, and those with motor impairments who ﬁnd it difﬁcult to use a mouse. This was due to the fact that use of digital video and audio materials in teaching and learning in Uganda is still limited, and the ﬁeld of automated and manual assessment of visual accessibility has been widely studied and provides more mature tools and guidelines to use. Finally, we note that the use of the term “people with disabilities” and the emphasis on impairments does not reﬂect the complex lived experience of those who face digital exclusion because of the way in which they perceive and interact with the world around them. In Disability Studies, this tension has been framed as a contrast between a medical and a social model of disability (see e.g., Haegele and Hodge, 2016 for a summary). The legal and policy documents we cite are strongly inﬂuenced by the medical model of disability, which sees impairments as defects to be cured. In the medical model, people with impairments are provided with assistance to function in a society designed for able-bodied people. In the social model, on the other hand, society itself should change to remove barriers to participation. Methodology Automatic Accessibility Evaluation VLE website TABLE 2 \| Courses evaluated. University School Courses in the VLE evaluated Public Makerere university (MUK) School of computing and IT 1. Audit and security assurance principles 2. Web-based information systems and web mining technologies School of open distance and eLearning 1. Multimedia design for instruction 2. e-tutoring and training School of public health 1. Introduction to health care and health systems 2. Principles of public health Kyambogo university (KYU) Faculty of education 1. Research methodology 2. Critical discourses in education and training School of management and entrepreneurship 1. Business ﬁnance 2. Principles of accounting Faculty of special needs and rehabilitation 1. Community psychology 2. Computer applications in research Busitema university (BUS) Faculty of engineering 1. Engineering mathematics 1 2. Engineering geology Faculty of health sciences 1. Principles of health communication 2. Principles of public health and disease control Faculty of agriculture and animal sciences 1. Soil science for engineers 2. Thermodynamics Private Uganda Technology and management university (UTAMU) School of computing and engineering 1. IT project planning and management 2. System analysis and design 3. Computer networks School of business and management 1. Business ﬁnance 2. Introduction to monitoring and evaluation 3. Management information systems Uganda Martyrs university (UMU) Faculty of agriculture 1.Climatology and Field Engineering 2. Agricultural Extension Education Practical Skills Faculty of science in education 1. Discrete Mathematics 2. Entomology and Parasitology Faculty of science 1. Cell Biology 2. Circuit Theory Kampala international university (KIU) Faculty of education 1.Selection and utilization of instructional resources 2. Principles and practices of open, distance and e-learning School of mathematics and computing 1. Calculus 2. Structured programming School of applied and natural science 1. Natural resources and landscape processes 2. Aquaculture production systems TABLE 2 \| Courses evaluated. Private Uganda Technology and management university (UTAMU) Uganda Martyrs university (UMU) Uganda Martyrs university (UMU) Kampala international university (KIU) Hence, graphical material should be accessible to both the sighted and the blind or visually impaired people. VLEs that support accessibility should prompt for alternative text descriptions for ﬁgures/images. But these descriptions should provide the same type of information as that perceived by sighted users, in order to support learning for non-sighted users in the same way as the ﬁgures themselves. Cases where only ﬁgure captions instead of meaningful descriptions of the ﬁgures are provided just to meet the technical accessibility requirements are common. Manual Accessibility Evaluation Using Heuristics From Accessibility Standards and Guidelines • Busitema University (BUS), founded in 2007, located in the eastern part of the country, which also has campuses in Mbale, Tororo, Soroti, and Kamuli Districts Based on international accessibility standards and guidelines such as The Web Content Accessibility Guidelines 1.0 and 2.0 (WCAG 1.0 and 2.0), eLearning accessibility principles, and VLE accessibility evaluation metrics, a set of nine accessibility principles and best practices (heuristics) were identiﬁed and subsequently used in the evaluation. Using the heuristics, the ﬁrst author, who has extensive experience in web accessibility and usability, walked through the user interfaces of the six sample VLEs to identify existing accessibility problems. The nine accessibility principles and best practices (heuristics) include: The 3 private universities include: • Uganda Technology and Management University (UTAMU), founded in 2012 and located in Kampala. • Uganda Martyrs University (UMU), founded in 1993 and afﬁliated with the Roman Catholic church in Uganda, which has 10 campuses across the country with the main campus located along the Equator at Nkozi, 80 kms west of Kampala. Around 75% of all students are enrolled in distance learning programmes. 1) Appropriate alt text descriptions for graphics and rich media: Graphics play an important role in teaching, particularly in the sciences and engineering, by providing important supplementary or complementary information to the main text. Sometimes, graphical representation may be the main or sole representation of a particular content. • Kampala International University (KIU), founded in 2001, which has a main campus in Kampala, a Western campus in Ishaka, and a campus in Dar es Salaam, Tanzania. All six universities use VLEs based on Moodle. Information about their institutional websites and websites of their VLEs is June 2021 \| Volume 3 \| Article 638275 Frontiers in Computer Science \| www.frontiersin.org 5 Making Virtual Learning Environments Accessible Baguma and Wolters TABLE 1 \| Universities whose VLEs were studied and courses evaluated. University Main website VLE website Public Makerere university (MUK) https://www.mak.ac.ug/ https://muele.mak.ac.ug/login/index.php Kyambogo university (KYU) https://kyu.ac.ug/ https://kelms.kyu.ac.ug/ Busitema university (BUS) https://www.busitema.ac.ug/ https://lms.busitema.ac.ug/login/index.php Private Uganda Technology and management university (UTAMU) https://utamu.ac.ug/ https://elearning.utamu.ac.ug/ Uganda Martyrs university (UMU) https://www.umu.ac.ug/ https://elearning.umu.ac.ug/ Kampala international university (KIU) https://kiu.ac.ug/ https://lms.kiu.ac.ug/login/index.php TABLE 2 \| Courses evaluated. University School Courses in the VLE evaluated Public Makerere university (MUK) School of computing and IT 1. Audit and security assurance principles 2. Web-based information systems and web mining technologies School of open distance and eLearning 1. Multimedia design for instruction 2. Manual Accessibility Evaluation Using Heuristics From Accessibility Standards and Guidelines e-tutoring and training School of public health 1. Introduction to health care and health systems 2. Principles of public health Kyambogo university (KYU) Faculty of education 1. Research methodology 2. Critical discourses in education and training School of management and entrepreneurship 1. Business ﬁnance 2. Principles of accounting Faculty of special needs and rehabilitation 1. Community psychology 2. Computer applications in research Busitema university (BUS) Faculty of engineering 1. Engineering mathematics 1 2. Engineering geology Faculty of health sciences 1. Principles of health communication 2. Principles of public health and disease control Faculty of agriculture and animal sciences 1. Soil science for engineers 2. Thermodynamics Private Uganda Technology and management university (UTAMU) School of computing and engineering 1. IT project planning and management 2. System analysis and design 3. Computer networks School of business and management 1. Business ﬁnance 2. Introduction to monitoring and evaluation 3. Management information systems Uganda Martyrs university (UMU) Faculty of agriculture 1.Climatology and Field Engineering 2. Agricultural Extension Education Practical Skills Faculty of science in education 1. Discrete Mathematics 2. Entomology and Parasitology Faculty of science 1. Cell Biology 2. Circuit Theory Kampala international university (KIU) Faculty of education 1.Selection and utilization of instructional resources 2. Principles and practices of open, distance and e-learning School of mathematics and computing 1. Calculus 2. Structured programming School of applied and natural science 1. Natural resources and landscape processes 2. Aquaculture production systems TABLE 1 \| Universities whose VLEs were studied and courses evaluated. VLE website For example, if a product image and product name are in the same link, the image can usually be given alt "" for alternative text Place the top heading of the page within a <h1> element Ensure headings are used as appropriate, and without skipping heading levels If the content of the image is already conveyed elsewhere (through text or the alternative text of a nearby image), determine whether the additional image is necessary. It is also possible to assign empty alternative text (alt "") Suspicious link text Link text should clearly describe the destination or function of the link Link to PDF PDF documents often have accessibility issues or require a separate plug in/application Link to video Videos are often not accessible Reword link text to clearly describe destination or function Convey information in native HTML. If that is not possible, ensure that the PDF is accessible Ensure all video content is accessible (subtitling/audio description) 2) Support for text-only navigation including link shortcuts, hidden links and descriptive link texts: In Moodle, this can be achieved if the student is designated as the user of a screen reader so that page content adapts to the read-out-loud format and the interface is simpliﬁed to remove clutter (Hersh, 2008). This also makes the screen reader skip long lists of links when relaying the system interface to the user. 5) Organize, structure, and make content clear: Screen readers read the markup not the page presented visually in the web browser. Hence, content authors should use markup correctly to organize and identify different levels of headings: H1-H6, use the bulleted or numbered list styles to bullet content instead of hyphens or other characters; only use table markup if presenting an actual table of data instead of other purposes like styling content; use bold for importance, italic for emphasis, and blockquote for call- outs or quotes instead of coloring or highlighting text to make it stand out. 2) Support for text-only navigation including link shortcuts, hidden links and descriptive link texts: In Moodle, this can be achieved if the student is designated as the user of a screen reader so that page content adapts to the read-out-loud format and the interface is simpliﬁed to remove clutter (Hersh, 2008). This also makes the screen reader skip long lists of links when relaying the system interface to the user. VLE website June 2021 \| Volume 3 \| Article 638275 Frontiers in Computer Science \| www.frontiersin.org 6 Making Virtual Learning Environments Accessible Baguma and Wolters TABLE 3 \| Accessibility issues and suggestions for addressing them. Name Description Suggested solution Errors Very low color contrast Text difﬁcult to read for all users with low vision, especially if text is small Use clear default contrasts between foreground (text) and background colors Empty link Function of the link cannot be presented to user of screen reader Providing text within link that describes functionality or target of the link Empty button Button without descriptive text that can be read out by screen reader Place text content within the <button > element or give the <input > element a value attribute Broken ARIA menu Accessible rich internet applications menu points to something that does not exist Check all ARIA menus for broken links Alerts Redundant title text The value of the title attribute should provide additional information to the user when the mouse hovers over an element, not repeat the text of the title element The title attribute should be either removed or edited to be more informative Very small text Text is too small to read for people with vision impairment Increase font size so that it can be read by people with mild to moderate vision problems Orphaned form label Label that is not associated with a corresponding form control Associate lable with the correct form control or remove it if unneccessary Suspicious alternative text The alternative text for an image does not summarize the content or information conveyed by the image Make alternative text informative; avoid text such as “image of” Redundant links Adjacent links that go to the same location result in additional navigation for keyboard and screen reader users Merge links. For example, if a product image and product name are in the same link, the image can usually be given alt "" for alternative text Missing ﬁrst level heading Nearly all pages should contain a ﬁrst level heading with the most important heading of the page for ease of navigation Place the top heading of the page within a <h1> element Skipped heading level Headings provide document structure and facilitate keyboard navigation by users of assistive technology. VLE website Skipping heading levels will confuse users Ensure headings are used as appropriate, and without skipping heading levels Duplicate alt text Two images near each other have the same alternative text If the content of the image is already conveyed elsewhere (through text or the alternative text of a nearby image), determine whether the additional image is necessary. It is also possible to assign empty alternative text (alt "") Suspicious link text Link text should clearly describe the destination or function of the link Reword link text to clearly describe destination or function Link to PDF PDF documents often have accessibility issues or require a separate plug in/application Convey information in native HTML. If that is not possible, ensure that the PDF is accessible Link to video Videos are often not accessible Ensure all video content is accessible (subtitling/audio description) TABLE 3 \| Accessibility issues and suggestions for addressing them. Name Description Errors Very low color contrast Text difﬁcult to read for all users with low vision, especially if text is small Empty link Function of the link cannot be presented to user of screen reader Empty button Button without descriptive text that can be read out by screen reader Broken ARIA menu Accessible rich internet applications menu points to something that does not exist Alerts Redundant title text The value of the title attribute should provide additional information to the user when the mouse hovers over an element, not repeat the text of the title element Very small text Text is too small to read for people with vision impairment Orphaned form label Label that is not associated with a corresponding form control Suspicious alternative text The alternative text for an image does not summarize the content or information conveyed by the image Redundant links Adjacent links that go to the same location result in additional navigation for keyboard and screen reader users Missing ﬁrst level heading Nearly all pages should contain a ﬁrst level heading with the most important heading of the page for ease of navigation Skipped heading level Headings provide document structure and facilitate keyboard navigation by users of assistive technology. Skipping heading levels will confuse users Duplicate alt text Two images near each other have the same alternative text Suggested solution The title attribute should be either removed or edited to be more informative Merge links. VLE website 3) Scalable fonts (text) and graphics: Designation of a student as a user of a screen reader will enable the zoom functionality which allows users to increase or decrease the size of content for better readability (Hersh, 2008). To test this functionality, the home pages of VLEs should be zoomed to 200%, and the content, and layout checked to see if it has automatically adjusted to ﬁt on the screen or if there are overlaps or disappearance of some of the content/layout elements. 3) Scalable fonts (text) and graphics: Designation of a student as a user of a screen reader will enable the zoom functionality which allows users to increase or decrease the size of content for better readability (Hersh, 2008). To test this functionality, the home pages of VLEs should be zoomed to 200%, and the content, and layout checked to see if it has automatically adjusted to ﬁt on the screen or if there are overlaps or disappearance of some of the content/layout elements. 6) Make content clear and discernible for users with limited vision or learning disabilities: Avoid using color on its own to distinguish or organize content, as color-blind users might have difﬁculty understanding it. It is also advisable to ensure sufﬁcient contrast between visual elements. 4) Keyboard access to all system components: Designation of a student as a user of a screen reader will also enable keyboard navigation for the whole system which is important for users of screen readers and those with mobility limitations (Hersh, 2008). This can be tested using the Tab and Shift- Tab keys to establish whether all elements of the VLE can be reached through the keyboard. 7) Make learning content accessible: Provide learning content such as word documents, pdf documents and presentations in accessible formats. 8) Make learning activities accessible: There is need to ensure that learning activities like labs, group work, peer practices, quizzes, projects, debates among others are accessible to users of screen readers. 8) Make learning activities accessible: There is need to ensure that learning activities like labs, group work, peer practices, quizzes, projects, debates among others are accessible to users of screen readers. VLE website June 2021 \| Volume 3 \| Article 638275 Frontiers in Computer Science \| www.frontiersin.org Frontiers in Computer Science \| www.frontiersin.org 7 Making Virtual Learning Environments Accessible Baguma and Wolters TABLE 4 \| Summary accessibility evaluation results for university VLEs from WAVE accessibility evaluation tool. University Errors Alerts Details Makerere university (mak) 26 22 Errors • 5 empty buttons • 6 empty links • 15 very low color contrast Alerts • 15 orphaned form labels • 1 skipped heading level • 6 redundant title text Kyambogo university (KyU) 11 10 Errors • 5 empty links • 6 very low color contrast Alerts • 1 missing ﬁrst level heading • 1 skipped heading level • 8 redundant title text Busitema university (BUSU) 20 27 Error • 1 empty heading • 9 empty links • 10 very low color contrast Alerts • 10 suspicious alternative text • 7 duplicate alternative text • 4 skipped heading level • 3 suspicious link text • 3 link to PDF document Uganda technology and management university (UTAMU) 15 13 Errors • 2 empty links • 13 very low color contrast Alerts • 2 suspicious alternative text • 7 redundant title text • 2 redundant links • 1 duplicate alternative text • 1 missing ﬁrst level heading Uganda martyrs university (UMU) 25 22 Errors • 10 empty buttons • 6 empty links • 9 very low color contrast Alerts • 8 redundant title text • 12 orphaned form labels • 2 skipped heading level Kampala international university (KIU) 23 59 Errors • 7 very low color contrast • 1 empty link 3 b k ARIA uation tool. Frontiers in Computer Science \| www.frontiersin.org Uganda martyrs university (UMU) Makerere university (mak) Busitema university (BUSU) Kampala international university (KIU) June 2021 \| Volume 3 \| Article 638275 9) Ensure that interaction and collaboration tools such as chats, forums, and wikis are accessible to users of screen readers. commonly used widgets can be passed to assistive technologies. For example, for forms, sometimes there is additional information beyond the <label> tag that the user needs to understand, such as a password requirement or some other requirement that is not standard. Visually, this is usually presented in additional helper text under the label. But for users of assistive technologies who navigate by form controls, adding “aria-describedby” will make the screen reader read both the label and the element that aria-describedby is pointing to (Pope, 2020). This is a good example of a case where technology to improve accessibility is provided within the system, but not used correctly. 3https://www.mozilla.org/en-US/(accessed on 20th October 2020). Alerts (N 157) The alerts fall into three main categories, issues that occur as part of the process of making content more accessible, issues that can be addressed by providing better templates to course creators, and issues that can be addressed by providing appropriate content design guidance. VLE website Alerts Details 22 Errors • 5 empty buttons • 6 empty links • 15 very low color contrast Alerts • 15 orphaned form labels • 1 skipped heading level • 6 redundant title text 10 Errors • 5 empty links • 6 very low color contrast Alerts • 1 missing ﬁrst level heading • 1 skipped heading level • 8 redundant title text 27 Error • 1 empty heading • 9 empty links • 10 very low color contrast Alerts • 10 suspicious alternative text • 7 duplicate alternative text • 4 skipped heading level • 3 suspicious link text • 3 link to PDF document 13 Errors • 2 empty links • 13 very low color contrast Alerts • 2 suspicious alternative text • 7 redundant title text • 2 redundant links • 1 duplicate alternative text • 1 missing ﬁrst level heading 22 Errors • 10 empty buttons • 6 empty links • 9 very low color contrast Alerts • 8 redundant title text • 12 orphaned form labels • 2 skipped heading level 59 Errors • 7 very low color contrast • 1 empty link • 3 broken ARIA menu Alerts • 28 very small text • 11 redundant title text • 10 redundant links • 2 insufﬁcient alternative text • 2 suspicious link text • 1 missing ﬁrst level heading • 1 embedded or linked video (Continued on following page) Kyambogo university (KyU) Busitema university (BUSU) Busitema university (BUSU) Uganda technology and management university (UTAMU) Uganda technology and management university (UTAMU) Uganda martyrs university (UMU) Kampala international university (KIU) 23 59 June 2021 \| Volume 3 \| Article 638275 Frontiers in Computer Science \| www.frontiersin.org 8 Making Virtual Learning Environments Accessible Baguma and Wolters Baguma and Wolters TABLE 4 \| (Continued) Summary accessibility evaluation results for university VLEs from WAVE accessibility evaluation tool. Automatic Evaluation Detailed results are presented in Table 4 in the Supplementary Materials section. Overall, there were more alerts (N 157) reported than errors (N 110). The low number of outright accessibility problems (errors) could be due to the fact that all the studied VLEs use Moodle which has inbuilt accessibility features such as zoom which enables users to increase the size of content for better readability, and support for keyboard navigation. Nevertheless, the alerts also need to be analyzed and those found a potential problem ﬁxed to improve the accessibility of VLEs to people with disabilities. VLE website University Errors Alerts Details Total 110 157 Errors • 55 very low color contrast (60%) • 28 empty link (25%) • 15 empty button (14%) • 3 broken ARIA menu (3%) Alerts • 49 redundant title text (31%) • 28 very small text (18%) • 27 orphaned form label (17%) • 15 suspicious alternative text (10%) • 13 redundant links (8%) • 12 missing ﬁrst level heading (8%) • 8 duplicate alt text (5%) • 7 suspicious link text (4%) • 3 link to PDF document (2%) • 1 embedded or linked video (<1%) TABLE 4 \| (Continued) Summary accessibility evaluation results for university VLEs from WAVE accessibility evaluation tool. University Errors Alerts Details Total 110 157 Errors • 55 very low color contrast (60%) • 28 empty link (25%) • 15 empty button (14%) • 3 broken ARIA menu (3%) Alerts • 49 redundant title text (31%) • 28 very small text (18%) • 27 orphaned form label (17%) • 15 suspicious alternative text (10%) • 13 redundant links (8%) • 12 missing ﬁrst level heading (8%) • 8 duplicate alt text (5%) • 7 suspicious link text (4%) • 3 link to PDF document (2%) • 1 embedded or linked video (<1%) University Total 9) Ensure that interaction and collaboration tools such as chats, forums, and wikis are accessible to users of screen readers. Baguma and Wolters Errors (N 110) The most frequent error, low contrast between foreground and background colours (n 60, 55%), affects both users with low vision and users with normal vision who are reading the screen under adverse conditions, such as glare from the Sun. The remaining errors (empty link, n 28, 25%; empty button (n 15, 14%); broken ARIA menu (n 3, 3%)) mainly affect screen reader users, because information that screen readers require to help users navigate is not provided. Low contrast issues can be addressed by providing course content creators with well- designed course templates, but the other three types of issues require course content creators to perform checks on the material they create. Three of the four most frequent issues, redundant title text (n 49, 31%), orphaned form labels (n 27, 17%), and suspicious alternative text that does not properly describe the image it refers to (n 15, 10%), can be viewed as issues that occur during the part of the process where accessibility information is added. If two images near each other have the same alternative text (Duplicate alt text, n 8, 5%), this is a sign that either one of them should be eliminated, or the textual descriuptions need to be reﬁned. In addition, all linked videos (n 1, <1%) and PDF documents (n 3, 2%) should be checked for accessibility. The second most frequent alert, very small text (n 28, 18%), is closely linked to the most frequent error, low color contrast. The easiest remedy for this issue is to provide templates that specify an acceptable minimum text size, such as 14 pt. Of particular interest are the broken ARIA menus. Accessible Rich Internet Applications (ARIA) is a set of attributes that deﬁne ways to make web content and web applications, especially those developed with JavaScript, more accessible to those with disabilities3. It supplements HTML to ensure interactions and The majority of the remaining issues regard content design, and will beneﬁt all students, regardless of whether they have a disability. Errors (N 110) Text should be clearly structured, with a top level heading (missing ﬁrst level heading, n 12, 8%), and a logical sequence of subheadings, where no heading levels are skipped June 2021 \| Volume 3 \| Article 638275 Frontiers in Computer Science \| www.frontiersin.org 9 Making Virtual Learning Environments Accessible Baguma and Wolters TABLE 5 \| Summary Results from Expert Evaluation using Heuristics. Heuristics VLEs Makerere Kyambogo Busitema UTAMU UMU KIU Appropriate alt text descriptions for graphics and rich media No No No Yes No No Support for text-only navigation, including link shortcuts, hidden links and descriptive link texts No No No No No No Scalable fonts (text) and graphics Yes Yes No Yes Yes Yes Keyboard access to all system components Yes No No No No No Organize, structure, and make content clear for screen reader users No No No No No No Make content clear and discernible for users with limited vision or learning disabilities Yes Yes Yes Yes Yes Yes Provide learning content in accessible formats No No No No No No Interaction and collaboration tools such as chats, forums, and wikis that are accessible to users of screen readers Yes Yes Yes Yes Yes Yes Learning activities like labs, group work, quizzes, projects, debates that are accessible to users of screen readers Yes No No No No No TABLE 5 \| Summary Results from Expert Evaluation using Heuristics. There were inaccessible pdfs and presentation ﬁles, and videos without synchronized captions. (skipped heading level, n 8, 5%). Where adjacent links go to the same location (Redundant links, n 13, 8%), for example a linked product image and an adjacent linked product name, content authors should choose one anchor, preferably the text, which should be automatically highlighted as a link by the style sheet. In general, a link should be associated with text that describes clearly he function or destination of the link (suspicious link text, n 7, 4%). Discussion We conducted automatic and manual evaluations for the VLEs of six Ugandan universities, three public, and three private. Most of these universities have their main campus in the capital, Kampala, but four out of the six have satellite campuses across the country. All universities adopted Moodle as their VLE. Frontiers in Computer Science \| www.frontiersin.org Manual Evaluation p The automatic evaluation found a substantial number of errors and alerts mostly related to content while the potential accessibility barriers established from the manual evaluation were mainly platform related. Out of the four categories of errors established from automatic evaluation, three were content related namely: very low contrast, empty links, and empty buttons. The three were also the most frequent errors. The fourth type of error, i.e. broken aria menu, was associated to both platform and content accessibility and had the lowest frequency. The eLearning platform needs the “aria-described by” attribute to make the screen reader read both the label and the element that “aria-describedby” is pointing to (additional helper text). But also content authors should ensure that the additional helper text under the label passes on the same message to visual interfaces and users of screen readers. The platform should include a prompt for provision of functionality/information about each form control to the user, while the content author should provide functionality/information about each form control that communicates the same message to visual and screen reader users. The results of the Manual Evaluation are summarized in Table 5 in the Supplementary Materials section. All the 6 VLEs had clear and discernible content for users with limited vision or learning disabilities. The interaction and collaboration tools such as chats, forums, and wikis for all the VLEs evaluated were also accessible to users of screen readers due to Moodle’s accessible interface. All the six VLEs had scalable fonts (text) and graphics. Five of the six VLEs did not have appropriate alt text descriptions for graphics and rich media. Only the VLE of UTAMU had appropriate alt text descriptions for graphics and rich media, and only the VLE of Makerere University had keyboard access to all system components. Keyboard access was a problem for the Kyambogo University VLE due to use of frames. Although keyboard navigation could reach every function in the VLE of UTAMU, the order of presentation was not chronological. From the top, the tab key jumped to the middle section of the page, then to the right section, next was the left side, and ﬁnally the bottom part. Such a presentation of the interface is confusing to a non- visual user relying on a screen reader. Keyboard access was not possible for the VLEs of Busitema, UMU and KIU. eLearning Accessibility in Context g y Much of the literature on the accessibility of eLearning reports similar levels of problems. For example, in their evaluation of higher education websites for 20 universities across the globe, Acosta-Vargas et al. found that the majority of websites did not comply with WCAG 2.0 guidelines to an acceptable level (Acosta- Vargas, Luján-Mora and Salvador-Ullauri, 2016). Qualitative studies have illustrated the impact that lack of accessibility has on the experience of LMIC students (Ro’fah et al., 2020) and lecturers (Zongozzi, 2020). Ro’fah et al. note that in addition to the types of accessibility problems discussed above, students with disabilities also reported problems with contacting and obtaining support from the Disability ofﬁce, which can potentially result in a vicious circle of exclusion. In interviews with lecturers from a South African ODeL institution, Zongozzi (2020) found that lecturers are well aware of the negative impact that lack of accessibility has on students with disabilities, but ﬁnd it difﬁcult to identify students who have disabilities and to provide appropriate support. While most of our ﬁndings related to presentation of visual information, the manual evaluation also uncovered issues with content structuring, which supports people with cognitive difﬁculties. The accessibility gaps established in the studied VLEs pose a big barrier to meaningful eLearning for people with disabilities. Our analysis methodology focused mostly on accessibility for those with visual impairments, screen reader users, those with hearing impairment, and those with motor impairments that make it difﬁcult to use a mouse. Adequate contrast between text and background color is necessary for all users but in particular for users with low vision especially for content presented in small text (less than 14 point), Lack of or inappropriate text description for links, buttons, form controls, aria menus, images or ﬁgures introduces confusion for screen reader users because the function or purpose of those elements will not be known. If the alternative text for an image does not provide the same information conveyed by the image, that content will not be available to screen reader users. Adjacent links that go to the same location like a linked product image and a linked product name for the same product results into additional navigation and repetition for keyboard and screen reader users. Similarly, two images with the same alternative text, causes redundancy or indicates incorrect alternative text which confuses users of screen readers. Manual Evaluation From the manual evaluation, two of the ﬁve potential accessibility barriers detected were platform related namely: lack of support for keyboard only access, and lack of support for text-only navigation while the other three were content related namely: lack of appropriate alt text descriptions for graphics and rich media, content that is not organized, structured, and clear for screen reader users, and learning content in inaccessible formats. There were four main sources of problems in the parts of the VLEs we investigated–perceptual difﬁculties, lack of sufﬁcient alternative text for users of screen readers, challenges navigating From the manual evaluation, two of the ﬁve potential accessibility barriers detected were platform related namely: lack of support for keyboard only access, and lack of support for text-only navigation while the other three were content related namely: lack of appropriate alt text descriptions for graphics and rich media, content that is not organized, structured, and clear for screen reader users, and learning content in inaccessible formats. None of the VLEs supported text-only navigation. In all the VLEs, the reader view was not available, which just like the screen reader removes all the extra items on the page and centers the text and images in the article for better readability. This might have been done so that users do not miss advertisements on the platforms. In addition, none of the 6 VLEs had organized, structured, and made content clear for screen reader users, and none provided learning content in accessible formats. There were four main sources of problems in the parts of the VLEs we investigated–perceptual difﬁculties, lack of sufﬁcient alternative text for users of screen readers, challenges navigating June 2021 \| Volume 3 \| Article 638275 10 Making Virtual Learning Environments Accessible Baguma and Wolters the VLEs and inaccessible learning content. Perceptual difﬁculties were mostly due to low color contrast, and small font sizes, which make it hard or impossible for people with low vision to read the text. Lack of sufﬁcient alternative information for screen readers was pervasive and ranged from uninformative link text, to missing alternative image/ﬁgure descriptions. Challenges navigating the VLEs ranged from lack of support for keyboard and text only navigation, redundant links, duplicate alternative text, missed ﬁrst level headings, skipped heading levels, and poor content structuring. eLearning Accessibility in Context When heading levels are skipped, keyboard users including users of screen readers get confused or ﬁnd difﬁculty navigating the site because headings provide the semantic structure of the document. Unless authored with accessibility in mind, PDF, Word and PowerPoint documents often have accessibility issues. Also, such documents are typically viewed using a separate application, and can thus cause confusion and navigation difﬁculties. Additionally, videos without synchronized captions are inaccessible to people with hearing impairments. Beyene, Mekonnen and Giannoumu (2020) explored institutional problems to the accessibility of educational resources to learners with visual impairments in the Ethiopian context. They noted that lack of educational resources in alternative formats is a common problem in Higher Education Institutions (HEIs) in developing countries, and recommended sensitising teachers and university staff on the needs of students with disabilities. Frontiers in Computer Science \| www.frontiersin.org Manual Evaluation Also, the learning content provided in the VLEs was in inaccessible formats such as PDFs and PowerPoint presentations that were not optimised for accessibility, and videos without synchronised captions. We believe that a number of accessibility gaps are due to non-consideration of accessibility during conﬁguration, use and administration of the VLEs given the vacuum in the regulatory environment in the country. particularly at conﬁguration level also exist. Therefore, national policies and guidelines on inclusive education and accessible ICTs and ICT based services should emphasize awareness, training as well as monitoring and evaluation of eLearning at platform conﬁguration and content authoring levels. The effort for accessible design of off-the-shelf eLearning platforms is driven by global human rights mandates such as the UNCRPD and the 2030 Agenda as well as global Web Accessibility standards such as WCAG 1.0 and 2.0. Hence, efforts at national level, need to focus on conﬁguration that supports accessibility for all users including people with disabilities and accessible content authoring. Since many of the issues we uncovered will also make content more difﬁcult to process for people without disabilities, a stronger emphasis on accessibility will ultimately make University VLEs more usable for everyone. For example, Moodle options that easily support screen readers, such as reader view, are not enabled. Cover all Disabilities in the Communications Act In our detailed analysis of accessibility problems, we highlighted several issues that can be addressed with appropriate in-house training and best practice guidelines. A case in point are low color contrast and small font size, which can be addressed through setting an in-house style guide that mandates minimum font sizes and high color contrast options. Rogers recommends pairing all non-text content with a text alternative. This means using ‘Alt tags’ for images/ﬁgures to clearly describe what the image/ﬁgure depicts; videos with captions describing the audio track; audio with a text transcript; and labeling Form inputs. Provision of alternative text extends to lecture notes. For example, a student reported that some teachers include illustrations in lecture presentations and fail to thoroughly explain the illustrations, yet it is not possible to record illustrations (Beyene et al., 2020). There is need to ensure that all disabilities affected by the accessibility of communication services are covered by the Communications Act. Relevant communication services include mass media, telecommunication, and the Internet, especially since the borders between those three types of communication channels have been signiﬁcantly reduced. The convergence of media technologies and the digital forms of access and delivery offer more ways for the audiences to engage with the media (Shah, 2020), and these forms of access are extensively used in ODeL (video, podcasts, interactive presentations). Link Existing Policy Frameworks and Create a Policy on eLearning Accessibility to People with Disabilities Currently special needs and inclusive education on one hand and ICT and disability present as two separate policy ecosystems. Given the enormous potential of ICT based learning for all learners, and given that accessible education technology is empowering and emancipating for those with disabilities, there is a clear need to link these policy ecosystems. However, when it comes to alternative text for graphics, providing this text can be difﬁcult. It is not enough to copy ﬁgure captions instead of providing meaningful descriptions of the ﬁgures, just to meet the technical accessibility requirements. Graphics play an important role in teaching, particularly in the sciences and engineering, by providing important supplementary or complementary information to the main text. Hersh (2008) recommends development of guidelines on what constitutes good alternative text descriptions and examples, and investigation of different types of graphics and associated learning aims to enable suggestions to be made of the different types of text description that are appropriate in each case. Cover all Disabilities in the Communications Act In addition, educational content authors should examine the role of graphics in their teaching material to ensure that learners have full access to this learning content, whether through the provision of alternative text descriptions or in some other way. A relevant policy on eLearning accessibility can either be a standalone policy, or part of a related policy such as the Special Needs and Inclusive Education Policy, which is before the Ugandan Cabinet for approval. Such a policy should provide focused direction and guidance to different stakeholders on making eLearning accessible. At the minimum, the policy should address requirements by global and national disability laws, and the Web Content Accessibility Guidelines by the World Wide Web Consortium (W3C). Limitations As this is the ﬁrst study of accessibility for people with disabilities who study at Ugandan universities, it has several limitations of scope. First of all, we only performed a high level sample of each VLE. It is possible that individual programmes and courses at one or more of the six institutions studied would perform better or worse. Our evaluation also focused mostly on accessibility to people with visual, and to some extent hearing impairments, and those with motor impairments who ﬁnd it difﬁcult to use a mouse. This was due to the still limited use of digital video and audio materials The study ﬁndings show that the accessibility challenges of the studied University VLEs in Uganda are mostly content related, but a substantial number of platform accessibility barriers June 2021 \| Volume 3 \| Article 638275 Frontiers in Computer Science \| www.frontiersin.org 11 Making Virtual Learning Environments Accessible Baguma and Wolters in teaching and learning in Uganda, and the maturity of automated and manual assessment of visual accessibility. progress being made. Based on the gaps in an institution’s current level of eLearning accessibility, institutional action plans will be needed covering milestones, tasks, and target dates to bridge the gap. Pearson et al. (2019) suggest that Accessibility Coordinators can play an important role in the delivery of such action plans. Implementation Level Teams and Action Plans Since eLearning design and delivery is a team effort, making eLearning accessible requires different stakeholders working together (Seale, 2006). This can be achieved by identifying representatives from key stakeholder groups namely: educators, administrators, developers, IT support team members and people with disabilities. These ambassadors can be the face of the accessibility initiative and bring attention to the In-House Training and Best Practice Guides In-House Training and Best Practice Guides To ensure accessibility of eLearning content, University Administrators, web developers, IT administrators, educators, student leaders and other stakeholders who play an active role in decision making in universities, developing and maintaining an accessible eLearning platform and developing eLearning content should be sensitized and trained based on their current skills and attitudes, and what actions need to be taken. This can be achieved through a combination of seminars, workshops, manuals, and eLearning courses. There is need to give more visibility to people with disabilities in ICT policies in order to cover key issues that affects them regarding use of ICTs and ICT enabled services like eLearning. People with disabilities should be made a standalone category of stakeholders in education and ICT policies instead of treating them as one of several special interest groups. In order to achieve this, people with a variety of disabilities should be closely involved in policy making. REFERENCES Haegele, J. A., and Hodge, S. (2016). Disability Discourse: Overview and Critiques of the Medical and Social Models. Quest. 68 (2), 193–206. doi:10.1080/ 00336297.2016.1143849 Abimanyi-Ochom, J., and Mannan, H. (2014). “Uganda’s Disability Journey: Progress and Challenges”. Afr. J. Disabil. 3 (1), 108. doi:10.4102/ajod. v3i1.108 Hanson, V. L., Cavender, A., Cavender, A., and Trewin, S. (2015). Writing about Accessibility. interactions. 22 (6), 62–65. doi:10.1145/2828432 Hersh, M. (2008). “Accessibility and Usability of Virtual Learning Environments,” in the 8th IEEE International Conference on Advanced Learning Technologies, ICALT 2008, July 1st- July 5th, 2008. (Santander, Cantabria: Spain). doi:10. 1109/icalt.2008.82 Access to Information for the Visually Impaired has been Made Possible (2020). Available at: https://afri-can.org/access-to-information-for-the-visually- impaired-has-been-made-possible/ (Accessed November 10, 2020). ICT for Disability Policy 2nd draft 2017 (2017). Available at: https://www.ict.go.ug/ wp-content/uploads/2018/06/ICTs-for-Disability-Policy-Draft.pdf (Accessed November 2, 2020). Acosta-Vargas, P., Luján-Mora, S., and Salvador-Ullauri, L. (2016). “Evaluation of the Web Accessibility of Higher-Education Websites”. In 2016 15th International Conference on Information Technology Based Higher Education and Training (ITHET), 1–6. doi:10.1109/ithet.2016.7760703 Mayoka, G., and Kyeyune, R. (2012). An Analysis of E-Learning Information System Adoption in Ugandan Universities. Case of Makerere University Business School. Inform. Technol. Res. J. 2 (1), 1–7. Anderson, G. (2020). Accessibility Suffers during Pandemic. Available at: https:// www.insidehighered.com/news/2020/04/06/remote-learning-shift-leaves-students- disabilities-behind (Accessed October 10, 2020). doi:10.1007/s42413-020- 00070-x National Population and Housing Census (2014). Available at: https://www.ubos. org/wp-content/uploads/publications/03_20182014_National_Census_Main_ Report.pdf. Baguma, R. (2019). Using WhatsApp in Teaching to Develop Higher Order Thinking Skills-A Literature Review Using the Activity Theory Lens. IJEDICT 15 (2), 98–116. Pearson, V., Lister, K., and Coughlan, T. (2019). “Accessibility Coordinators: A Model for Embedded, Sustainable Change towards Inclusive Higher Education”. in Proceedings of the 12th Annual International Conference of Education, Research and Innovation (ICERI 2019), (Seville, Spain: IATED), 3127–3136. http://oro.open.ac.uk/68402/ Baguma, R. (2017). An Audit of Inclusive ICTs for Education in Uganda in Proceedings of ICEGOV 2017. New Delhi, India. doi:10.1145/3047273.3047339 Bagarukayo, E., Ssentamu, N. P., Mayisela, T., and Brown, C. (2016). Activity theory as a lens to understand how Facebook develops knowledge application skills. IJEDICT 2. Pope, J. (2020). Broken ARIA Reference – Example. https://blog.pope.tech/2020/03/ 20/broken-aria-reference-example/ Beyene, W. M., Mekonnen, A. T., and Giannoumis, G. A. (2020). Inclusion, Access, and Accessibility of Educational Resources in Higher Education Institutions: Exploring the Ethiopian Context. Int. J. Inclusive Education. 0 (0), 1–17. doi:10. 1080/13603116.2020.1817580 Ro’fahanjarwati, A., and Suprihatiningrum, J. (2020). “Is Online Learning Accessible during COVID-19 Pandemic? Voices and Experiences of UIN Sunan Kalijaga Students with Disabilities”. DATA AVAILABILITY STATEMENT impairment. This is consistent with international results, even though our review of the legal and policy landscape in Uganda identiﬁed numerous issues that may be addressed in other countries. There are two areas for future work that we wish to highlight. The ﬁrst area relates to coverage of impairment. The deﬁnition of disability cited earlier highlights physical and mental impairments as well as sensory disabilities, and yet our analysis focuses mostly on sensory impairments. There is a need to extend the accessibility analysis to cover the needs of people with mobility impairment (e.g., those who have lost the full use of arms, hands, or legs due to trafﬁc accidents or illness), and people who are neurodiverse (e.g., those with autism or ADHD). The second area relates to learning design in general. Anderson (2020) noted that course design in an online environment requires pedagogical proﬁciency in utilizing the VLE features that promote active learning and higher order thinking skills, administrative and technical skills. As has been found repeatedly in the inclusive design literature, we would expect that good instructional design also serves to make ODeL more accessible. The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. CONCLUSION To the best of our knowledge, this is the ﬁrst paper to study the accessibility of VLEs used in Ugandan Universities for people with disabilities. We found numerous accessibility issues, even though we mostly focused on accessibility to people with visual June 2021 \| Volume 3 \| Article 638275 Frontiers in Computer Science \| www.frontiersin.org 12 Baguma and Wolters Making Virtual Learning Environments Accessible FUNDING MW acknowledges funding from the Alan Turing Institute (EPSRC) and Lift-Health-UG. EPSRC EP/N510129/1 and Lift-Health UG GCRF/SFC Theme Development Fund TDF_20 GCRF 05. AUTHOR CONTRIBUTIONS RB conceived the original idea for the paper, conducted the policy survey and accessibility evaluation, and wrote the ﬁrst full draft of the paper. MW supported RB in reshaping the original idea, conducted additional background research, and produced the ﬁnal draft together with RB. Uganda DPOs Present Critical Rights Issues to CRPD Committee (2016). Available at: https://disabilityrightsfund.org/uganda-dpos-present-critical-rights-issues- to-crpd-committee/ Wozniak, S., Moses, O., and Bernard, S. (2020). “Uganda’s Disability Data Landscape and the Economic Inclusion of Persons with Disabilities”. Development Initiatives. Available at: https://devinit.org/resources/uganda-disability-data-landscape- economic-inclusion-persons-with-disabilities/. doi:10.5194/acp-2020-1070-rc1 Zongozzi, J. N. (2020). Accessible Quality Higher Education for Students with Disabilities in a South African Open Distance and E-Learning Institution: Challenges. Int. J. Disabil. Development Education 0 (0), 1–13. doi:10.1080/1034912x.2020.1822518 Frontiers in Computer Science \| www.frontiersin.org June 2021 \| Volume 3 \| Article 638275 Uganda DPOs Present Critical Rights Issues to CRPD Committee (2016). Available at: https://disabilityrightsfund.org/uganda-dpos-present-critical-rights-issues- to-crpd-committee/ Wozniak, S., Moses, O., and Bernard, S. (2020). “Uganda’s Disability Data Landscape and the Economic Inclusion of Persons with Disabilities”. Development Initiatives. Available at: https://devinit.org/resources/uganda-disability-data-landscape- economic-inclusion-persons-with-disabilities/. doi:10.5194/acp-2020-1070-rc1 Zongozzi, J. N. (2020). Accessible Quality Higher Education for Students with Disabilities in a South African Open Distance and E-Learning Institution: Challenges. Int. J. Disabil. Development Education 0 (0), 1–13. doi:10.1080/1034912x.2020.1822518 REFERENCES Nadwa. 14 (1), 1–38. doi:10. 21580/nw.2020.14.1.5672 Seale, J. (2006). “A Contextualised Model of Accessible E-Learning Practice in Higher Education Institutions”. Australas. J. Educ. Technology. 22 (2), 268–288. doi:10.14742/ajet.1302 Convention on the Rights of Persons with Disabilities (CRPD) (2020). Available at: https://www.un.org/development/desa/disabilities/convention-on-the-rights-of- persons-with-disabilities.html (Accessed November 20, 2020). Shah, S. (2020). ICT: Convergence of Information and Communication Technology. Available at: https://www.sociologydiscussion.com/science/ict-convergence-of- information-and-communication-technology/723 (Accessed October 13, 2020). doi:10.1109/rteict49044.2020.9315618 Disability Rights Fund (2016). Available at: https://disabilityrightsfund.org/ uganda-dpos-present-critical-rights-issues-to-crpd-committee/ Education and Sports Sector Strategic Plan (2017/18 - 2019). Available at: http:// npa.go.ug/wp-content/uploads/2018/11/EDUCATION-AND-SPORTS-SECTOR- STRATEGIC-PLAN.pdf (Accessed November 5, 2020). Singal, N., Ware, H., and Bhutani, S. K. (2017). Inclusive Quality Education for Children with Disabilities. United Kingdom: University of Cambridge. Guglielman, E. (2010). “E-learning and Disability: Accessibility as a Contribute to Inclusion,” in Proceedings of the 5th Doctoral Consortium at the European Conference on Technology Enhanced Learning, September 29, 201 (Barcelona, Spain). The Independent (2019). Gov’t Urged to Hasten Inclusive Education Policy. Available at: https://www.independent.co.ug/govt-urged-to-hasten-inclusive- education-policy/ (Accessed October 2, 2020). June 2021 \| Volume 3 \| Article 638275 Frontiers in Computer Science \| www.frontiersin.org 13 Baguma and Wolters Making Virtual Learning Environments Accessible 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. Copyright © 2021 Baguma and Wolters. 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. June 2021 \| Volume 3 \| Article 638275 Frontiers in Computer Science \| www.frontiersin.org 14
https://openalex.org/W4236006384	https://www.researchsquare.com/article/rs-3907/v1.pdf	English	null	The ECM and CAFs interact to differentially regulate PDAC parenchymal (CPC) and CSC phenotypes to determine pancreatic ductal adenocarcinoma (PDAC) progression	Research Square (Research Square)	2,019	cc-by	5,978	The ECM and CAFs interact to differentially regulate PDAC parenchymal (CPC) and CSC phenotypes to determine pancreatic ductal adenocarcinoma (PDAC) progression Stefania Cannone University of Bari Background Pancreatic Ductal Adenocarcinoma (PDAC) is one of the most lethal cancers having a five-year survival rate of less than 8% [1], [2] and will become the second cause of cancer deaths in the coming years [3], [4]. One of the aggressive characteristics of PDAC is their highly reactive and tumor-supportive stromal microenvironment named desmoplasia [5]. Desmoplasia is a dense extracellular matrix (ECM), which makes up to 90% of PDAC tissue [6, 7] [8, 9], [10] and is characterized by increasing collagen type I levels as the tumor progresses and which contains the cancer cells and their accessory cells, including cancer associated fibroblasts (CAFs) [11], [12]. Collagen I makes up to 80% of the tumor space in PDAC and is associated with a worsened outcome [13, 14] and stimulates malignant cell properties to promote tumor growth, early metastasis and chemo-radiation resistance [15] [14] [9] [16]. Cancer stem cells (CSCs), which drive tumor heterogeneity and influence tumorigenesis, metastasis and drug resistance through their self-renewal and multi-lineage differentiation capabilities (stemness) (for review see [17]). Recent studies have demonstrated that the stromal ECM composition “per se” produces important cues that guide the expression of different PDAC phenotypes in both parenchymal cancer cells (CPCs) [18],[5] and CSCs [5]. In particular, ECM composition differently regulates growth, morphology, invasive, angiogenic capacities and secretome profiles in PDAC CPCs and their derived CSCs [5]. Importantly, in that study only the CSCs secreted factors known to activate and maintain CAFs19], [20]. This suggests that the described dual “symbiotic”, mutual support interaction between tumor cells and CAFs is maintained primarily through the CSC population through the existence of a tighter relationship between CSCs and CAFs than between the CPCs and CAFs. However, this has yet to be demonstrated. Once activated, CAFs enhance the development, progression and invasion of PDAC through their extensive crosstalk with the tumor, resulting in reciprocal stimulation and therapy resistance [21], [22]. Recent data in PDAC has shown that CAF cells, via their secretome, can increase parenchymal tumor cell (CPC) invasion [23], [24], [25], [26], reduce their growth [23], [27] and modify their epigenetic and metabolic phenotypes [24, 27]. Only one study [27] measured the effect of the combination of CAF secretome and high levels of ECM collagen I on parenchymal PDAC cell lines (CPCs) but did not determine the individual roles of collagen I and the CAF (Conditioned Medium). Research article Page 1/17 Keywords: Desmoplastic Reaction, Cancer stem cells, Cancer Associated Fibroblasts, Pancreatic Adenocarcinoma, Vasculogenic Mimicry, 3D organotypic cultures Posted Date: August 20th, 2019 DOI: https://doi.org/10.21203/rs.2.13193/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Keywords: Desmoplastic Reaction, Cancer stem cells, Cancer Associated Fibroblasts, Pancreatic Adenocarcinoma, Vasculogenic Mimicry, 3D organotypic cultures Posted Date: August 20th, 2019 DOI: https://doi.org/10.21203/rs.2.13193/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. Read Full License Page 1/17 Abstract Summary CAFs and acellular stromal ECM components interact to drive metastatic progression by stimulating the hallmark behaviors of each tumor cell type that contribute to metastasis: invasion in the CPCs and growth and angiogenesis in the CSCs. Cell lines The human pancreatic adenocarcinoma parenchymal (CPC) cell line, Panc1, and Panc1 CSCs, generated as previously described [28], were grown and maintained in standard conditions as previously described [5]. Panc1 cells express the mutated PDAC driver genes KRAS, CDKN2A, MAP2K4, and TP53 [29], [30]. Cancer Associated Fibroblasts (CAFs) The experimental procedure relating to the use of patient-derived pancreatic tumor pieces was performed after approval from the South Mediterranean Personal Protection Committee, under the reference 2011- A01439–32. CAFs isolation and culture were processed as previously described [31]. Briefly, pancreatic tissues obtained during pancreatic surgery from patients with resectable pancreatic adenocarcinoma were cut into small pieces of 1 mm3 using a razor blade. The dissociation of these tissue pieces and the culture to isolate CAFs were performed as in [31] and primary CAF were verified by positive α-SMA staining and negative KRT19 immunofluorescence staining. To collect conditioned media (CM), 1.5x105 CAF cells/well were seeded in 24-well cell culture plates and medium changed every 3 days. When the monolayer reached approximately 80% confluence, they were incubated with 1 ml of medium with 1% FBS lacking growth factors and antibiotics for 30 hours. The CM were collected, centrifuged, protein concentration assayed with the Bradford protein assay reagent (Pierce, Milan, Italy) using bovine serum albumin as a standard and stored in liquid nitrogen. Background Importantly, those in vitro experiments were not performed on CSCs and, therefore, the effect of CAFs on CSC behavior in PDAC is completely unknown. Nor is the contribution of the ECM in the modulation of the CAF-driven determination of CPC and CSC phenotypic plasticity and behaviour known. Page 2/17 Given the complex interactions between cancer cells and CAFs, more work is needed to investigate the contributions of CAFs in enabling or maintaining hallmark behaviors in CSCs. Here, we analyzed the role of ECM collagen I in modulating the effect of CAF-derived signals with conditioned medium from primary cultured CAFs derived from patients with PDAC on the parenchymal (CPC) and CSC populations in a previously described three-dimensional (3D) organotypic model of PDAC [5] [18] mimicking the ECM of early (low collagen I levels) and late (high collagen I levels) stage PDAC tumors. Given the complex interactions between cancer cells and CAFs, more work is needed to investigate the contributions of CAFs in enabling or maintaining hallmark behaviors in CSCs. Here, we analyzed the role of ECM collagen I in modulating the effect of CAF-derived signals with conditioned medium from primary cultured CAFs derived from patients with PDAC on the parenchymal (CPC) and CSC populations in a previously described three-dimensional (3D) organotypic model of PDAC [5] [18] mimicking the ECM of early (low collagen I levels) and late (high collagen I levels) stage PDAC tumors. These data reveal that the cellular (CAFs) and acellular (ECM) stromal components interact to differently modulate the hallmark tumor behaviors in the CSCs and CPCs. In particular, the CAFs reduce the growth of CPCs while favoring the growth of CSCs, which would trigger a positive feedback mechanism to stimulate CSC growth and make for a more malignant, persistent and immortal tumor. Indirect co-culture To condition cells, both CPCs and CSCs were grown for 1 day on 90%M:10%C or 20%M:80%C in their corresponding complete culture media and for the subsequent 5 days in either 100% CM collected from the primary patient CAFs or in the CM diluted at 50% with the same complete culture media used to grow the cells. A change of medium was conducted midweek. To analyze if the CAF CMs could change the growth and/or the growth phenotype (morphology) of the cells, growth was assessed by the Resazurin cell viability assay and morphology was examined microscopically. Growth measurements Curves of CPCs and CSCs were calculated from Resazurin (Immunological Sciences, Rome, Italy) reduction assays for cell growth as previously described [32]. Standard curves obtained by fluorescence readings of Resazurin on serial dilutions of both CPCs and CSCs were used to calculate cell number. Invadopodia proteolytic activity Experiments to measure invadopodia focal ECM proteolysis were conducted in cells seeded onto a ECM layer in which quenched BODIPY linked to BSA (DQ-Green-BSA, Thermo Fisher Scientific) was mixed at a ﬁnal concentration of 30 µg/ml as previously described [5]. were then incubated at 37° C with 5% CO2 for 1 hour to allow the mixture to create a thin gel on the bottom of the wells. 1.5x104 cells/well were seeded on the top of the matrix and cultured as described above. were then incubated at 37° C with 5% CO2 for 1 hour to allow the mixture to create a thin gel on the bottom of the wells. 1.5x104 cells/well were seeded on the top of the matrix and cultured as described above. Vascular network analysis CSCs and CPCs were grown on the 90%Matrigel:10%Collagen I (90%M:10%C) ECM mixture. After 5 days in these growth conditions, vascular channel networks were photographed using the TE200 microscope (Nikon USA, Garden City, NY, USA) and analysed as previously described [5]. To study paracrine CAF regulation of vascular parameters, CSCs or parenchymal cells were cultured as above and after 24 hrs, to permit their adherence, the cultures were incubated with CAF CM as described above for indirect co- culture. 3D culture models The 90% Matrigel–10% Collagen I and 20% Matrigel - 80% Collagen I ECM mixtures were prepared as previously described [5]. In all cases,100 µl of the mixture were plated in 96-well cell culture plates, which Page 3/17 were then incubated at 37° C with 5% CO2 for 1 hour to allow the mixture to create a thin gel on the bottom of the wells. 1.5x104 cells/well were seeded on the top of the matrix and cultured as described above. Statistical analysis GraphPad Prism 5 (GraphPad Software) to perform a two-tailed Student’s t test assuming unequal variances to compare the effects of CAF CM on Panc1 CPCs and CSCs and to determine whether the Page 4/17 Page 4/17 differences between two groups were statistically significant. P-values < 0.05, 0.01, or 0.001 are indicated as , * or **, respectively when compared to each control for each matrix and †, †† or ††† compared to the same CM treatment on 90% Matrigel:10% Collagen I. differences between two groups were statistically significant. P-values < 0.05, 0.01, or 0.001 are indicated as , or , respectively when compared to each control for each matrix and †, †† or ††† compared to the same CM treatment on 90% Matrigel:10% Collagen I. Results Given the above described crosstalk between CAFs and cancer cells and the reported role of the ECM composition in regulating the phenotypes of both PDAC CPCs [5] [18] and CSCs [5], we evaluated the role of the ECM composition on the effect of two concentrations of Conditioned Medium (CM) derived from CAFs isolated from a PDAC patient on the growth, invasive capacity and vascular morphology of each cell type. The experiments were performed with the cells cultured on either 90% Matrigel:10% Collagen I or 20%Matrigel:80% Collagen I since these combinations very well mimic the ECM of early and late stages of PDAC progression, respectively (see Materials and Methods and protocol scheme in Supplemental Figure 1). CAF CM reduces CPC growth and increases CSC growth on all of the substrates. When CPCs or CSCs were incubated with two concentrations of CAF CM on the two substrates, we observed that CAF CM reduces growth of the CPCs and increases growth of the CSC in a progressive, dose-dependent manner that was independent of ECM composition with a similar behavior on both early (90%M:10%C) and late (20%M:80%C) ECM compositions (Figure 1). This would favor the expansion of the CSCs over the CPCs, in line with the report that only the CSC population secreted factors known to activate CAFs [5] and supports the hypothesis of a mutual “symbiotic” support between these two cell types as in other tumor types [33] [34]. Further, this would support the reports that CAFs drive an increase in general ‘stemness’ of the tumor in various other tumor types and would suggest that, at least in PDAC, this occurs by a net favoring of CSC growth over CPC growth. Effect of primary CAF CM on CPC and CSC vasculogenic mimicry We have previously reported that vascular-like structures correspondent with in vivo Vasculogenic Mimicry (VM) were formed by the CSCs on 100% Matrigel and were reduced as Collagen I levels increased and completely disrupted as Collagen I levels surpassed 30% of the ECM composition [5]. We asked whether the CAF CM-driven stimulation of CSC growth observed in Figure 1 would also result in an increase of VM organization in the CSCs and, perhaps, in a slight increase in VM in the CPC population. Indeed, when CSCs or CPCs were incubated with the two concentrations of CAF CM on the 90%M:10%C substrate, we observed that CAF CM increases both the mean number of lacunae per well and number of capillary connections to nodes per field of the CSCs in a progressive, dose-dependent manner that resulted in the formation of very tight, organized vascular structures. Interestingly, CAF-CM somewhat stimulated a capillary-like phenotype also in the CPCs but only at the higher concentration of CAF CM, suggesting that it may induce the epithelial-mesenchymal transition of CPCs. Effect of primary CAF CM on CPC and CSC invadopodia i i We have previously observed that the PDAC CPCs have higher levels of invadopodia-driven invasion than do the CSCs and that this phenomenon is greater on Matrigel rich ECMs than on collagen I ECMs [5]. We, therefore, next analyzed the role of the ECM composition in modulating the CAF CM-dependent regulation of the invasive phenotype of the CPCs and CSCs, by measuring their invadopodia-mediated ECM proteolytic activity defined by their Digestion Index [35] (Figure 2B). As previously reported [5], we found that (i) the control CPCs had a much higher ability to form functional invadopodia and digest the two types of ECM compared to control CSCs and (ii) both cell types had a higher Digestion Index when cultured on 90%M:10%C with respect to 20%M:80%C. Page 5/17 Page 5/17 The effects of the CAF CM on the formation and activity of invadopodia were very complex. On 90%M:10%C, treatment with 50% CM produced a small but not significant increase in the CPC invadopodia activity while 100% CM stimulated CPC invadopodial activity 2.5-fold. CAF CM had no significant effect on CSC invadopodia activity at either concentration. Interestingly, when cultured on 20%M:80%C neither cell line responded significantly to CAF CM at either concentration. Discussion Utilizing a 3D organotypic culture model representing the ECM changes observed during in vivo PDAC progression from a laminin-rich to a collagen I-rich environment [36], we have previously found that the ECM composition drove the very early development of metastatic behaviors of both the parenchymal tumor cell (CPC) and CSC populations which is another of the important PDAC characteristics [14], [37], [38]. Indeed, when growing in an early tumor ECM modeled by the Matrigel rich ECM (containing laminin, collagen IV and entactin), the CSCs secreted high levels of pro-angiogenic/growth factors, which activate their transdifferentiation into an endothelial-like VM network via a VEGF/VEGFR–2 mediated cascade. At the same time, when on Matrigel the more differentiated CPC population has a high invadopodia-driven invasive capacity that is stimulated by EGF [39], which is highly secreted by the CSCs. This concerted and reciprocal activation of these two malignant phenotypes, i.e. (1) the high rate of local invasion executed by CPCs into the (2) aberrant vascular network created by the CSC-derived vascular signals suggested a symbiotic relationship between the CPCs and the CSCs underlies the initiation and maintenance of early PDAC infiltration and metastasis. In this way, parenchymal CPC cells and CSCs interact to contribute to the vascular [40] and invasive phenotype of the early stage tumor. However, tumor growth and phenotypes are influenced not only by their own gene expression-related and ECM-related factors, but also via the interaction of the tumor cells with the accessory cells found in the Page 6/17 tumor stroma. In PDAC, the major stroma cell driving progression is considered to be the Cancer Associated Fibroblasts (CAFs) and due to the sparse distribution of tumor cells the interaction between the different cell types occurs principally via the soluble factors secreted by both tumor cells and CAFs [23] [24] [25] [26]. While in other tumor types, the interaction of CSCs with CAFs has been documented, in PDAC the role of CAFs in determining and/or modulating the balance between the parenchymal and CSC tumor populations is still unknown. Here, utilizing our organotypic cultures mimicking the changes in ECM composition with PDAC progression, we characterized the role of CAFs on both CPC and CSC phenotypes when cultured on ECM compositions known to drive specific growth and phenotype patterns in the two tumor cell types. We find that when growing on an early tumor ECM (modeled by 90%M:10%C), the CAFs increased both CSC growth (Fig. Discussion Altogether, these factors support their high growth rate necessary to form the vascular network. This would both mobilize CSCs (together with endothelial cells, pericytes etc) into a vascular niche and further stimulate the CSCs endothelial-like differentiation program, in-order- to participate in vasculogenic mimicry, and their angiogenic secretome to stimulate endothelial angiogenesis [41]. This enhanced ability of the CAFs to stimulate the previously described CSC growth/pro-angiogenic program [5], especially in response to an early stage tumor microenvironment (eg. growth on Matrigel), is in line with in vivo experiments in which (a) subcutaneous tumors originating from CSCs gave rise to amore abundant vascular network composed of larger vessels than the tumors originating from CPCs, (b) the tumors of the CSC-injected mice had increased Ki–67 staining for mitotic index [5] and (c) grow faster than those in mice injected with CPCs [28]. Discussion 1A) and their assembly into a VM network (Fig. 3). Together, this increases their dedicated programing towards the preparation of a vascular niche and eventual transdifferentiation into an endothelial-like network [5]. At the same time, when on Matrigel the CAFs further reduced the growth rate of the more differentiated CPC cell population (Fig. 1B) but greatly increased their already high invadopodia-driven invasive capacity (Fig. 2). This concerted over-activation of these two malignant phenotypes by the CAFs, i.e. (1) the high rate of CPC local invasion into the (2) CSC-derived vasculogenic network suggest that the CAFs specifically activate the previously described symbiotic relationship between the parenchymal CPC cells and the CSCs that underlies early PDAC infiltration and metastasis [5]. It has been previous reported [5] that CSCs develop their vascular phenotype on Matrigel via the coordinated interaction of two factors: (i) the intrinsic over-expression of genes for endothelial factors and vascular receptors and (ii) the high secretion of VEGF from both the CSCs and CPCs. Based on the findings reported here, this program is further stimulated by a third factor, the secretion by CAFs of various pro-angiogenic/growth factors. Altogether, these factors support their high growth rate necessary to form the vascular network. This would both mobilize CSCs (together with endothelial cells, pericytes etc) into a vascular niche and further stimulate the CSCs endothelial-like differentiation program, in-order- to participate in vasculogenic mimicry, and their angiogenic secretome to stimulate endothelial angiogenesis [41]. This enhanced ability of the CAFs to stimulate the previously described CSC growth/pro-angiogenic program [5], especially in response to an early stage tumor microenvironment (eg. growth on Matrigel), is in line with in vivo experiments in which (a) subcutaneous tumors originating from CSCs gave rise to amore abundant vascular network composed of larger vessels than the tumors originating from CPCs, (b) the tumors of the CSC-injected mice had increased Ki–67 staining for mitotic index [5] and (c) grow faster than those in mice injected with CPCs [28]. It has been previous reported [5] that CSCs develop their vascular phenotype on Matrigel via the coordinated interaction of two factors: (i) the intrinsic over-expression of genes for endothelial factors and vascular receptors and (ii) the high secretion of VEGF from both the CSCs and CPCs. Based on the findings reported here, this program is further stimulated by a third factor, the secretion by CAFs of various pro-angiogenic/growth factors. Conclusions In conclusion, our study sheds light on the role of the CAFs in driving the very early metastatic development, one of the most representative PDAC hallmarks [38]. We find that the CAFs and acellular stromal components interact to modulate the hallmark tumor behaviors of the CPC and CSC cell types Page 7/17 and drive metastatic progression by stimulating the hallmarks of each tumor cell type that contribute to metastasis: invasion in the CPCs and growth & angiogenesis in the CSCs (Figure 4). Altogether, these findings propose a scenario in which the ECM composition and the cellular secretomes of the two tumor cell types and the CAFs cooperate to jointly regulate both growth and morphology of the CPC and CSC cell lines and, by modulating the highly dynamic interactions between them, establishes a continuum between tumor initiation and progression in primary PDAC tumors. This suggests that also in PDAC, such as in other tumor types, CAFs drive progression by favoring the growth and vascular plasticity of the CSC population. Consent for publication Not applicable Ethics approval and consent to participate The experimental procedure relating to the use of patient-derived pancreatic tumor pieces was performed after approval from the South Mediterranean Personal Protection Committee, under the reference 2011- A01439–32. Availability of data and material: The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Abbreviations PDAC: Pancreatic Ductual AdenoCarcinoma; CAF: Cancer Associated Fibroblasts; ECM: ExtraCellular Matrix; CPC: Cancer Parenchymal Cells; CSC: Cancer Stem Cells; 3D: three dimensional culture; VM: Vasculogenic Mimicry; M: Matrigel; C: Collagen I; CM: Conditioned Medium Funding Page 8/17 Page 8/17 Page 8/17 This work was supported by Fondation de France, (2013–00038330; 2015–00059283), Fondation ARC (PJA 20161204740), Ligue contre le Cancer (GB/MA/IQ–10607) (OS, HG). KZ was a fellow of the Marie Curie Initial Training Network IonTraC (FP7-PEOPLE–2011-ITN Grant Agreement No. 289648) to SJR. The SJR laboratory is part of the Italian network “Istituto Nazionale Biostrutture e Biosistemi” (INBB), and the project BioBoP of the Region Puglia. The funding bodies had no involvement in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the article for publication. Acknowledgements Not applicable Authors’ contributions SJR, MRG, RAC, and KZ organized the project and designed the experiments. SJR, RAC and VC wrote the manuscript. MRG, SC, conducted the experiments, performed the image analyses and statistical evaluation. RT provided the CAF population from patient tissue and OS and HG provided the conditioned medium from the CAF cell cultures. All authors have read and approved the manuscript References [1] Hong SM, Park JY, Hruban RH, Goggins M. Molecular signatures of pancreatic cancer. Arch Pathol Lab Med. 2011;135:716–27. [2] Di Marco M, Grassi E, Durante S, Vecchiarelli S, Palloni A, Macchini M, et al. State of the art biological therapies in pancreatic cancer. World J Gastrointest Oncol. 2016;8:55–66. [3] Siegel RL, Miller KD, Jemal A. 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Stromal cues regulate the pancreatic cancer epigenome and metabolome. Proc Natl Acad Sci U S A. 2017;114:1129–34. References Cancer stem cells and angiogenesis. Int J Dev Biol. 2011;55:477–82. References [28] Dalla Pozza E, Dando I, Biondani G, Brandi J, Costanzo C, Zoratti E, et al. Pancreatic ductal adenocarcinoma cell lines display a plastic ability to bidirectionally convert into cancer stem cells. Int J Oncol. 2014;46:1099–108. [29] Deer EL, Gonzalez-Hernandez J, Coursen JD, Shea JE, Ngatia J, Scaife CL, et al. Phenotype and genotype of pancreatic cancer cell lines. Pancreas. 2010;39:425–35. [30] Kim Y, Han D, Min H, Jin J, Yi EC. Comparative proteomic profiling of pancreatic ductal adenocarcinoma cell lines. Mol Cells. 2014;37:888–98. [31] Leca J, Martinez S, Lac S, Nigri J, Secq V, Rubis M, et al. Cancer-associated fibroblast-derived annexin A6+ extracellular vesicles support pancreatic cancer aggressiveness. J Clin Invest. 2016;126:4140–56. 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Figures Page 12/17 Page 12/17 Page 12/17 Figure 1 Figure 1. CAF CM inhibits CPC growth and stimulates CSC growth on both ECM compositions. A) Representative microscopic images of growth morphology of Panc1 CPCs and their derived CSCs cultured on organotypic cultures composed of 90% Matrigel:10% Collagen I (left panel) and 20% Matrigel:80% Collagen I (right panel). Scale bar represents 500µm for all images. (B) CPC and CSC growth rates in organotypic cultures of 90% Matrigel:10% Collagen I (left panel) and 20% Matrigel:80% Collagen I (right panel). Growth rates of CPCs and CSCs cultured on the different ECMs were calculated from Resazurin reduction assays according to the standard curves obtained by fluorescence readings of Resazurin and are normalized to the control as 100 as described in Materials and Methods. Data are mean ± SEM, n=5, p<0.05, p<0.01, p<0.001 to the control of each cell type on each matrix. Fi 1 Figure 1 Figure 1. CAF CM inhibits CPC growth and stimulates CSC growth on both ECM compositions. A) Representative microscopic images of growth morphology of Panc1 CPCs and their derived CSCs cultured on organotypic cultures composed of 90% Matrigel:10% Collagen I (left panel) and 20% Matrigel:80% Collagen I (right panel). Scale bar represents 500µm for all images. (B) CPC and CSC growth rates in organotypic cultures of 90% Matrigel:10% Collagen I (left panel) and 20% Matrigel:80% Collagen I (right panel). Growth rates of CPCs and CSCs cultured on the different ECMs were calculated from Resazurin reduction assays according to the standard curves obtained by fluorescence readings of Resazurin and are normalized to the control as 100 as described in Materials and Methods. Data are mean ± SEM, n=5, p<0.05, p<0.01, p<0.001 to the control of each cell type on each matrix. Page 13/17 Page 14/17 Figure 2 Figure 1. CAF CM inhibits CPC growth and stimulates CSC growth on both ECM compositions. A) Representative microscopic images of growth morphology of Panc1 CPCs and their derived CSCs cultured on organotypic cultures composed of 90% Matrigel:10% Collagen I (left panel) and 20% Matrigel:80% Collagen I (right panel). Scale bar represents 500µm for all images. (B) CPC and CSC growth rates in organotypic cultures of 90% Matrigel:10% Collagen I (left panel) and 20% Matrigel:80% Figure 2 Figure 1. CAF CM inhibits CPC growth and stimulates CSC growth on both ECM compositions. A) Representative microscopic images of growth morphology of Panc1 CPCs and their derived CSCs cultured on organotypic cultures composed of 90% Matrigel:10% Collagen I (left panel) and 20% Matrigel:80% Collagen I (right panel). Scale bar represents 500µm for all images. (B) CPC and CSC growth rates in organotypic cultures of 90% Matrigel:10% Collagen I (left panel) and 20% Matrigel:80% Page 14/17 Collagen I (right panel). Growth rates of CPCs and CSCs cultured on the different ECMs were calculated from Resazurin reduction assays according to the standard curves obtained by fluorescence readings of Resazurin and are normalized to the control as 100 as described in Materials and Methods. Data are mean ± SEM, n=5, p<0.05, p<0.01, p<0.001 to the control of each cell type on each matrix. y g y g Resazurin and are normalized to the control as 100 as described in Materials and Methods. Data are mean ± SEM, n=5, p<0.05, p<0.01, p<0.001 to the control of each cell type on each matrix. Figure 3 Figure 3 Figure 3 Page 15/17 Page 15/17 Figure 3. CAF secretome stimulates the vascular-like morphology (VM) of both CSCs and CPCs grown on 90% Matrigel:10%Collagen I. ECM composition modifies the effect of CAF CM on vasculogenic mimicry in CPCs and CSCs. Cells were grown on 90% Matrigel:10% Collagen I and after 24 hrs to permit their adherence the cultures were incubated with CAF CM as described in Materials and Methods. A) Representative microscopic images of growth morphology of CPCs and their derived CSCs cultured on organotypic cultures composed of 90% Matrigel:10% Collagen I for 5 days with their growth medium or Figure 3. CAF secretome stimulates the vascular-like morphology (VM) of both CSCs and CPCs grown on 90% Matrigel:10%Collagen I. ECM composition modifies the effect of CAF CM on vasculogenic mimicry in CPCs and CSCs. Cells were grown on 90% Matrigel:10% Collagen I and after 24 hrs to permit their adherence the cultures were incubated with CAF CM as described in Materials and Methods. A) Representative microscopic images of growth morphology of CPCs and their derived CSCs cultured on organotypic cultures composed of 90% Matrigel:10% Collagen I for 5 days with their growth medium or with either 50% or 100% of the CAF conditioned medium. Scale bar represents 500µm for all images. Figure 2 (B) After 7 days in these growth conditions, vascular channel networks were analysed as described in Material and Methods for mean number of lacunae per well (left panel) and mean number of capillary connections per field (right panel). Mean ± SEM from three independent experiments, ns, non significant, p<0.01, **p<0.001 compared to the CPC control; †††p<0.001 CSCs compared to their control. Page 16/17 Figure 4 Figure 4. Model of Influence of the ECM composition on the CAF-dependent regulation of CPC and CSC plasticity. Matrigel induces the formation of autocrine loops in CSCs. Indeed, on Matrigel (1) CSCs secrete a high amount of potent proangiogenic and growth factors (ie. PDGF, MMP9, IL8, EGF, HGF, bFGF, ET-1) [5], which, via paracrine mechanisms, stimulate CAF growth, which in turn secrete factors that increase CSC growth/self-renewal and vasculogenic mimicry while decreasing CPC growth but increasing CPC Figure 4 Page 16/17 Figure 4. Model of Influence of the ECM composition on the CAF-dependent regulation of CPC and CSC plasticity. Matrigel induces the formation of autocrine loops in CSCs. Indeed, on Matrigel (1) CSCs secrete a high amount of potent proangiogenic and growth factors (ie. PDGF, MMP9, IL8, EGF, HGF, bFGF, ET-1) [5], which, via paracrine mechanisms, stimulate CAF growth, which in turn secrete factors that increase CSC growth/self-renewal and vasculogenic mimicry while decreasing CPC growth but increasing CPC invasion. This creates a vicious positive-feedback growth cycle between the CAFs and CSCs to increase the stemness of the tumor while exacerbating the aggressive angiogenesis phenotype of the CSCs and invasive phenotype of the CPCs. invasion. This creates a vicious positive-feedback growth cycle between the CAFs and CSCs to increase the stemness of the tumor while exacerbating the aggressive angiogenesis phenotype of the CSCs and invasive phenotype of the CPCs. supplement1.tif Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Page 17/17
https://openalex.org/W4210450407	http://dogadergi.ksu.edu.tr/tr/download/article-file/1877716	English	null	Scutellaria orientalis subsp. virens ve Scutellaria salviifolia üzerinde Anatomik, Mikromorfolojik, Karyolojik ve Biyokimyasal bir çalışma	Kahramanmaraş Sütçü İmam Üniversitesi tarım ve doğa dergisi	2,022	cc-by	8,509	KSÜ Tarım ve Doğa Derg 25 (Ek Sayı 1): 125-136, 2022 KSU J. Agric Nat 25 (Suppl 1): 125-136, 2022 https://doi.org/10.18016/ksutarimdoga.vi.970571 KSÜ Tarım ve Doğa Derg 25 (Ek Sayı 1): 125-136, 2022 KSU J. Agric Nat 25 (Suppl 1): 125-136, 2022 https://doi.org/10.18016/ksutarimdoga.vi.970571 KSÜ Tarım ve Doğa Derg 25 (Ek Sayı 1): 125-136, 2022 KSU J. Agric Nat 25 (Suppl 1): 125-136, 2022 https://doi.org/10.18016/ksutarimdoga.vi.970571 KSÜ Tarım ve Doğa Derg 25 (Ek Sayı 1): 125-136, 2022 KSU J. Agric Nat 25 (Suppl 1): 125-136, 2022 https://doi.org/10.18016/ksutarimdoga.vi.970571 Anatomical, Micromorphological, Karyological and Biochemical study of Scutellaria orientalis subsp. virens and Scutellaria salviifolia Anatomical, Micromorphological, Karyological and Biochemical study of Scutellaria orientalis subsp. virens and Scutellaria salviifolia Mikail AÇAR¹, Neslihan TAŞAR2, Gülçin BEKER AKBULUT3 Mikail AÇAR¹, Neslihan TAŞAR2, Gülçin BEKER AKBULUT3 1,2Department of Plant and Animal Production, Tunceli Vocational School, Munzur University, Tunceli, Türkiye, 2Malatya Turgut Özal University, Battalgazi Vocational School, Department of Park and Horticulture, Malatya, Türkiye 1https://orcid.org/ 0000-0003-3848-5798, 2https://orcid.org/ 0000-0002-0417-4660, 3https://orcid.org/0000-0002-4964-6780 : ntasar@munzur.edu.tr 1,2Department of Plant and Animal Production, Tunceli Vocational School, Munzur University, Tunceli, Türkiye, 2Malatya Turgut Özal University, Battalgazi Vocational School, Department of Park and Horticulture, Malatya, Türkiye 1https://orcid.org/ 0000-0003-3848-5798, 2https://orcid.org/ 0000-0002-0417-4660, 3https://orcid.org/0000-0002-4964-6780 : ntasar@munzur edu tr Botanic Research Article Article History Received : 14.07.2021 Accepted : 31.12.2021 Keywords Trichome Lipid peroxidation Total carbohydrate Micromorphology Scutellaria - ABSTRACT In this study, the anatomical and micromorphological structure, karyological characteristics and biochemical content of Scutellaria orientalis subsp. virens and endemic Scutellaria salviifolia, whose distributions areas overlap, were compared. Some anatomical and micromorphological differences were observed on the taxa; scleranchymatic pericycle layer on the stem, stomata density, distribution of trichomes, as well as the main vascular bundle and general shape of the petiole. The chromosome numbers of both taxa were determined as 2n = 22. However, there was a difference between chromosome length range and total chromosome length. The chromosome numbers and chromosome morphologies of these species have been defined for the first time in this paper. Differences in biochemical content were observed between species. Chlorophyll a (Chl a), total chlorophyll (Total Chl), total carbohydrate and malondialdehyde (MDA) contents were determined higher in leaf and stem samples of S. orientalis subsp. virens than S. salviifolia. There was no significant difference between the two taxa in terms of chlorophyll b (Chl b) content. Carotenoid (Car) content was detected higher in leaves samples of S. orientalis subsp. virens, but no significant difference was found between stems samples. Also, the effect of taxa on biochemical contents in relation to the habitat they live in is given in this study. Scutellaria orientalis subsp. virens ve Scutellaria salviifolia üzerinde Anatomik, Mikromorfolojik, Karyolojik ve Biyokimyasal bir çalışma Botanik Araştırma Makalesi Makale Tarihçesi Received : 14.07.2021 Accepted : 31.12.2021 Anahtar Kelimeler Trikom Lipid peroksidasyon Toplam karbonhidrat Mikromorfoloji Scutellaria Botanik Araştırma Makalesi Makale Tarihçesi Received : 14.07.2021 Accepted : 31.12.2021 Anahtar Kelimeler Trikom Lipid peroksidasyon Toplam karbonhidrat Mikromorfoloji Scutellaria Scutellaria orientalis subsp. virens ve Scutellaria salviifolia üzerinde Anatomik, Mikromorfolojik, Karyolojik ve Biyokimyasal bir çalışma INTRODUCTION Carbohydrates are direct photosynthetic activity products and are structural building blocks as well as energy sources and metabolites. They act as a source of energy and provide carbon necessary to produce new tissue (Trouvelot et al., 2014; Homayoonzadeh et al., 2020). Lipid peroxidation is the reaction that occurs in unsaturated fatty acids of cell membrane phospholipids. Products such as malondialdehyde resulting from lipid peroxidation event determine the severity of peroxidation (Abdelrahim et al., 2020; Álvarez-Robles et al., 2020). Lamiaceae is the third largest family in terms of number of taxa in Turkey. Lamiaceae family is represented by 48 genera and 782 taxa (603 species, 178 subspecies and varieties) in Türkiye. 44% of these taxa are endemic. In addition, the family has 23 hybrid species and 19 of them are endemic (%83). Studies of Turkey's Lamiaceae result has proven to be one of the centers of diversity in the Old World. In addition, in Turkey, there are about 10% of all species of Lamiaceae (Celep and Dirmenci, 2017). Being from the Lamiaceae family, the Scutellaria L. genus is one of the semi-cosmopolitan genera of the Lamiaceae family with its 471 species identified (Yilmaz, et al., 2020). Due to the suspicious species status of some taxa defined from the Soviet Union and China, in reality this number is close to 360 (Paton, 1990). The Scutellaria species usually grow in stony and rocky slopes in Turkey. The genus Scutellaria has 39 taxa (17 species, 23 subspecies, 2 varieties and 1 hybrid) were represented in Turkey (Güner et al., 2012, Yilmaz et al., 2020). 17 taxa are endemic (44%) in Turkey (Davis, 1988; Duman, 2000; Güner et al., 2012; Celep and Dirmenci, 2017). The genus Scutellaria is taxonomically complex because it has many species and especially some taxa closely in morphologically. Therefore, anatomical findings, karyo-morphological analysis of taxa are important for the systematics. Also, the effect of taxa on biochemical contents in relation to the habitat they live in is given in this study. This study aims to reveal the anatomical structure and karyomorphological features of S. orientalis L. subsp. virens (Boiss. & Kotschy) J.R. Edm. and S. salviifolia Benth. taxa whose overlapping areas of distribution, and to improve the knowledge on their biochemical content. Thus, the results obtained from this study will provide data for future studies. INTRODUCTION Chlorophylls are the main pigments that drive photosynthesis by absorbing light and converting it into chemical energy (Agathokleous et al., 2020). Carotenoids absorb certain wavelengths of light energy in the photosynthetic system and then transfer it to the chlorophyll molecule and thus contribute to the photosynthesis process. In addition, according to some researchers, carotenoids prevent the breakdown of chlorophyll (photo oxidation) in an environment with excessive light and oxygen and protect the plant against physiological tissue injuries caused by excessive light (Leiva-Ampuero et al., 2020; ABSTRACT Bu çalışmada, yayılış alanları örtüşen Scutellaria orientalis subsp. virens ve endemik Scutellaria salviifolia'nın anatomik ve mikromorfolojik yapısı, karyolojik özellikleri ve biyokimyasal içeriği karşılaştırılmıştır. Taksonlarda bazı anatomik ve mikromorfolojik farklılıklar gözlenmiştir. Bunlar; gövdede sklerankimatik periskl tabakası, stoma yoğunluğu, trikomların dağılımı, petiyolün genel şekli ve ayrıca ana iletim demetinde birtakım farklılıklar şeklindedir. Her iki taksonun kromozom sayıları 2n=22 olarak belirlenmesine rağmen kromozom uzunluk aralığı ile toplam kromozom uzunluğu arasında fark görülmüştür. Bu türlerin kromozom sayıları ve kromozom morfolojileri ilk kez bu çalışmada tanımlanmıştır. Türler arasında biyokimyasal içerik farklılıkları da gözlenmiştir. S. orientalis subsp. virens'in yaprak ve gövde örneklerinde klorofil a (Chl a), toplam klorofil (Toplam Chl), toplam karbonhidrat ve malondialdehit (MDA) içeriğinin S. salviifolia'ya göre daha yüksek olduğu belirlenmiştir. Klorofil b (Chl b) içeriği açısından iki takson arasında önemli bir fark tespit edilmemiştir. S. orientalis subsp. virens'in yaprak örneklerinde karotenoid (Car) içeriği daha yüksek saptanmış, ancak gövde örnekleri arasında önemli bir fark bulunmamıştır. Çalışmada taksonların yaşadıkları habitata göre biyokimyasal içerikleri üzerindeki etkisi de verilmiştir. KSÜ Tarım ve Doğa Derg 25 (Ek Sayı 1): 125-136, 2022 KSU J. Agric Nat 25 (Suppl 1): 125-136, 2022 Araştırma Makalesi Research Article To Cite: Açar M, Taşar N, Beker-Akbulut G 2022. Anatomical, Micromorphological, Karyological and Biochemical study of Scutellaria orientalis subsp. virens and Scutellaria salviifolia. KSU J. Agric Nat 25 (Suppl 1): 125- 136. https://doi.org/10.18016/ksutarimdoga.vi.970571. p g g Atıf Şekli: Açar M, Taşar N, Beker-Akbulut G 2022. Scutellaria orientalis subsp. virens ve Scutellaria salviifolia üzerinde Anatomik, Mikromorfolojik, Karyolojik ve Biyokimyasal bir çalışma. KSÜ Tarım ve Doğa Derg 25 (Ek Sayı 1): 125-136. https://doi.org/10.18016/ksutarimdoga.vi.970571. Zhang et al., 2020). MATERIAL and METHOD Scutellaria taxa, the study material, were collected from their natural habitats in the field. The localities of taxa are given in Table 1 and Figure 1. The collected samples have been turned into herbarium material and are kept in the herbarium of Munzur University. Table 1. The localities and collector number of taxa Çizelge 1. Taksonların lokaliteleri ve numaraları Taxon Locality S. orientalis subsp. virens B7 Tunceli: Between Tunceli center and Ovacık, roadsides, 1000 m, May 2020, MA 2000 S. salviifolia B7 Tunceli: Between Tunceli center and Ovacık, Aktuluk neighborhood, the roadsides May 2020, 950 m, MA 2001 Table 1. The localities and collector number of taxa Table 1. The localities and collector number of taxa Çizelge 1. Taksonların lokaliteleri ve numaraları it was made into a permanent preparation with entellan (Tardif and Conciatori, 2015). Anatomical examinations were made under an Olympus BX53 microscope. Nutlet was examined in SEM (JCM-5000) and microphotographs were taken. Anatomical studies was made on samples kept in 70% ethyl alcohol. And performed on at least two individuals to represent the population for each taxon. Anatomical sections were carried out manually. In order to better distinguish the tissues and cells were stained with safranin-fastgreen. After, Nutlets of the studied taxa were collected from their 126 KSÜ Tarım ve Doğa Derg 25 (Ek Sayı 1): 125-136, 2022 KSU J. Agric Nat 25 (Suppl 1): 125-136, 2022 Araştırma Makalesi Research Article natural habitats for karyological analysis. In order to obtain somatic chromosomes, the germination environment was provided by sowing in an oven at 22 ° C. After the seed germinated, the cut root tips were kept in colchicine solution for 2 hours at room temperature (Elçi, 1982; Gedik et al., 2014). Root tips kept in colchicine solution for 2 hours were taken from this solution and placed in Farmer's solution (3: 1). Root tips were fixed in acetic alcohol at +4 ° C in a refrigerator for 24 hours. At the end of 24 hours, the root tips were hydrolyzed in 1N HCl for 8-10 minutes in an oven set at 60 ° C. After the hydrolysis process was completed, the root tips were dyed with feulgen dye for 1 hour at 22 ºC in a dark environment. The meristems taken for chromosome examinations were broken up in a drop of aceto orcein dye with a sharp razor blade and the coverslip was closed (Levan et al., 1964). Lipid Peroxidation Analysis The method was done according to Heath and Packer (1968). 0.5 gram of leaf tissue was homogenized in 5 mL of 0.1% TCA. The homogenate was centrifuged at 10,000 g for 10 minutes. 2 mL of 0.5% TBA (prepared in 20% TCA) was added to 2 mL of this solution and kept in a water bath at 95 ° C for 30 min. than samples were centrifuged at 10,000 gram for 15 min again. MDA content was calculated at 532 nm and 600 nm. Statistical Analysis Statistical evaluation of the obtained data was made using the SPSS 21.0 program. In this program, variance analysis was performed and Duncan test (Duncan, 1955) was applied within the scope of significance test. Biochemical analysis Plant parts were stored at -80° C until analysis. Pigment analysis, total carbohydrate and lipid peroxidation analysis were determined. Photosynthetic Pigment Analysis Method suggested by De-Kok and Graham (1980) was used for pigment analysis. 0.5 gr of each leaf and stem samples ground in the blender were taken for extraction in 3 replicates for each sample and placed in a glass mortar and homogenized in 25 ml of acetone. They were homogenized in a shaking oven for 30 minutes. These samples were then stored for 24 hours at 4 ° C. Then samples were filtered and 1/5 water was added. Samples were centrifuged at 3000 rpm for 10 minutes. The absorbance values of samples were read at 470 nm, 645nm and 662 nm according to Lichtenthaler and Welburn (1983) and Chl-a, Chl-b, Car and Total Chl contents were determined. MATERIAL and METHOD Levan's naming system was used in determining the location of the centromere (Levan et al., 1964). Intra-chromosomal asymmetric index (A1) and inter-chromosomal asymmetric index (A2) were calculated according to Zarco (1986). Figure 1. General view of taxa. A,B- Scutellaria orientalis subsp. virens C,D- S. salviifolia Şekil 1. Taksonların genel görünümü. A,B- Scutellaria orientalis subsp. virens C,D- S. salviifolia Figure 1. General view of taxa. A,B- Scutellaria orientalis subsp. virens C,D- S. salviifolia Şekil 1. Taksonların genel görünümü. A,B- Scutellaria orientalis subsp. virens C,D- S. salviifolia Biochemical analysis have been calculated corresponding to the standard values entered on the computer in the Slide program. Leaf anatomy and micromorphology ep: epidermis, co: collenchyma, pa: parenchyma, sc: sclerenchyma, ph: phloem, xy: xylem, pi: pith Leaf anatomy and micromorphology Leaf anatomical structure of taxa differs more than stem anatomical structure. Generally, in both taxa midrib has developed and swollen in abaxial direction Figure 3. The anatomical structure of the stem S. salviifolia e: epidermis, co: collenchyma, pa: parenchyma, ph: phloem, xy: xylem Şekil 3. S. salviifolia’ nın kök anatomisi. co: kollenkima, pa: parankima, sc: sklerankima, ph: floem, xy: ksilem, pi: öz Figure 3. The anatomical structure of the stem S. salviifolia e: epidermis, co: collenchyma, pa: parenchyma ph: phloem xy: xylem Figure 3. The anatomical structure of the stem S. salviifolia e: epidermis, co: collenchyma, pa: parenchyma, ph: phloem, xy: xylem Şekil 3. S. salviifolia’ nın kök anatomisi. co: kollenkima, pa: parankima, sc: sklerankima, ph: floem, xy: ksilem, pi: öz (Figure 4-5). Midrib area; it is arranged as, below the upper epidermis, collenchyma, below it parenchyma, xylem, phloem, parenchyma, again collenchyma and lower epidermis. Collenchyma layer is less developed in S. salviifolia. In both taxa, the mesophyll consists of only the palisade parenchyma, while in S. orientalis subsp. virens it consists of 4-5 rows and the parenchyma cells in the abaxial direction can be a little more oval. In S. salviifolia, there are 4 rows of palisade parenchyma. While the epidermis is mostly rectangular shape in S. orientalis subsp. virens, it is more oval shape in S. salviifolia. In addition, stomata are located on both the upper and lower surfaces in both taxa, while it is dense on the upper surface in S. orientalis subsp. virens, while it is dense on the lower surface in S. salviifolia While in S. orientalis subsp. virens the leaf bottom surface is densely covered with non-glandular trichome, it is not so in S. salviifolia. Both non-glandular trichome and glandular trichome were observed in both taxa, and the non-glandular trichome is denser in S. orientalis subsp. virens and the glandular trichome is denser in S. salviifolia. In addition, B2 type has been observed only in S. salviifolia and P type is quite intense.In addition, while the non-glandular trichomes are curved in S. orientalis subsp. virens, they are relatively not curved in S. salviifolia. Figure 2. The anatomical structure of the stem S. orientalis subsp. virens. ep: epidermis, co: collenchyma, pa: parenchyma, sc: sclerenchyma, ph: phloem, xy: xylem, pi: pith Ş kil 2 S i li b Vi ’ i kök i i Figure 2. The anatomical structure of the stem S. orientalis subsp. virens. Stem anatomy and micromorphology The total carbohydrate content was assayed according to Rosenberg (1980). Anthrone method was used for colorimetric method of determining the concentration of the total carbohydrate. Absorbance was measured at 620 nm (Shimadzu UV-1201V). Glucose values The stem anatomical structure of taxa shows the general characteristics of the family. The stem has 4 corners and vascular bundles at the corners are developed (Figure 2-3). In addition, developed collenchyma layer was seen in the corners. S. 127 KSÜ Tarım ve Doğa Derg 25 (Ek Sayı 1): 125-136, 2022 KSU J. Agric Nat 25 (Suppl 1): 125-136, 2022 Araştırma Makalesi Research Article orientalis subsp. virens, scleranchymatic pericycle surround partly the stem, whereas in S. salviifolia there is no scleranchymatic pericycle layer. Endodermis cannot be distinguished from parenchyma tissue completely. Between the corners, parenchyma tissue in S. salviifolia occupies relatively more area. Although the collenchymatic hypodermis layer is seen between the corners in both taxa, it was seen more in S. orientalis subsp. virens. In addition, although vascular cambium was observed in S. salviifolia, it is not obvious in S. orientalis subsp. virens. Non-glandular trichomes and glandular trichomes have seen in both taxa; Non-glandular trichomes have been observed as a multicellular with micropapillae and developed cell wall. Non-glandular trichomes cell wall thickness more in S. orientalis subsp. virens. The glandular trichomes are; both taxa were observed as Labiatae type (peltate) and capitate type glandular trichome. Capitate glandular trichomes are of three subtypes. Rounded head with a short stalk cell and a broad round head shape (A type) and with a neck or without neck structure and with 2 stalk cells and multicellular round head shape (B1 type) and long 2-3 cell stalk and with neck or without neck structure and rounded head shape (B2 type). Although these two types of glandular trichomes are observed in taxa, relatively long capitate (B1 type) is more dense in S. salviifolia. Short large headed capitate (A type) was more intense in S. orientalis subsp. virens. Besides, in the stem structure, B2 type only observed in S. orientalis subsp. virens. Only in S. salviifolia, a cup-shaped head structure was commonly seen after releasing secretion B1 type capitate trichomes. However, this head cell shape was not seen in S. orientalis subsp. virens. Leaf anatomy and micromorphology Petiole anatomy and micromorphology The petiole is flattened in the adaxial direction in S. orientalis subsp. virens. In S. salviifolia, it is hollowed in the adaxial direction and swollen and ribbed from Şekil 2. S. orientalis subsp. Virens’ in kök anatomisi. co: kollenkima, pa: parankima, sc: sklerankima, ph: floem, xy: ksilem, pi: öz 128 KSÜ Tarım ve Doğa Derg 25 (Ek Sayı 1): 125-136, 2022 KSU J. Agric Nat 25 (Suppl 1): 125-136, 2022 Araştırma Makalesi Research Article the edges. There are 3 vascular bundles in both taxa. One is in the middle and the other two are on the edges. The middle vein is developed. Both taxa are in arc shape and consist of almost two lobes in S. salviifolia. Chlorenchymatic cells were seen in the corners (Figure 6-7). glandular trichomes were observed in both taxa, while A, P, B1 and B2 types were observed in S. orientalis subsp. virens; A, B1 and B2 types were observed in S. salviifolia and P type was not observed (Figure 8). Figure 4. S. orientalis subsp. virens, anatomical structure of the leaf ade: adaxial epidermis, abe: abaxial epidermis, co: collenchyma, vb: vascular bundle, pa: parenchyma, pp: palisade parenchyma, tr: trichome Şekil 4. S. orientalis subsp. Virens yaprağının anatomik yapısı. ade: adaksiyel epidermis, abe: abaksiyel epidermis co: kollenkima Figure 6. S. orientalis subsp. virens, anatomical structure of petiole, A and C- General view, B-, D- Middle vein, e: epidermis, co: collenchyma, vb: vascular bundle, pa: parenchyma, pp: palisade parenchyma, tr: trichome Figure 6. S. orientalis subsp. virens, anatomical structure of petiole, A and C- General view, B-, D- Middle vein, e: epidermis, co: collenchyma, vb: vascular bundle, pa: parenchyma, pp: palisade parenchyma, tr: trichome Figure 4. S. orientalis subsp. virens, anatomical structure of the leaf ade: adaxial epidermis, abe: abaxial epidermis, co: collenchyma, vb: vascular bundle, pa: parenchyma, pp: palisade parenchyma, tr: trichome Figure 4. S. orientalis subsp. virens, anatomical structure of the leaf ade: adaxial epidermis, abe: abaxial epidermis, co: collenchyma, vb: vascular bundle, pa: parenchyma, pp: palisade parenchyma, tr: trichome Şekil 6. S. orientalis subsp. Virens. Yaprark sapının anatomik yapısı. Ave C genel görünüş, B-, D Şekil 4. S. orientalis subsp. Virens yaprağının anatomik yapısı. ade: adaksiyel epidermis, abe: abaksiyel epidermis, co: kollenkima, vb: vasküler demet, pa: parankima, pp: palisat parankiması, tr: trikom öşe gö ü ü ü e: epidermis, co: kollenkima, vb: vasküler damar, pa: parankima, pp: palizet parankiması, tr: trikıom 1 Figure 5. Petiole anatomy and micromorphology S.salviifolia , anatomical structure of the leaf ade: adaxial epidermis, abe: abaxial epidermis, co: collenchyma, vb: vascular bundle, pa: parenchyma, pp: palisade parenchyma, tr: trichome Figure 7. S. salviifolia, anatomical structure of petiole, A and C- General view, B- Corner view, D- Middle vein, e: epidermis, co: collenchyma, vb: vascular bundle, pa: parenchyma, pp: palisade parenchyma, tr: trichome Figure 7. S. salviifolia, anatomical structure of petiole, A and C- General view, B- Corner view, D- Middle vein, e: epidermis, co: collenchyma, vb: vascular bundle, pa: parenchyma, pp: palisade parenchyma, tr: trichome Şekil 5. S.salviifolia yaprağının anatomik yapısı. ade: adaksiyel epidermis, abe: abaksiyel epidermis, co: kollenkima, vb: vasküler demet, pa: parankima, pp: palisat parankiması, tr: trikom Şekil 7. S. salviifolia. Yaprark sapının anatomik yapısı. Ave C genel görünüş, B-, D köşe görünümü e: epidermis, co: kollenkima, vb: vasküler damar, pa: parankima, pp: palizet parankiması, tr: trikom e: epidermis, co: kollenkima, vb: vasküler damar, pa: parankima, pp: palizet parankiması, tr: trikom Vascular bundles in the corners are surrounded by a parenchymatic sheath. Both non-glandular and Vascular bundles in the corners are surrounded by a parenchymatic sheath. Both non-glandular and Nutlet Structure Nutlet Structure 129 KSÜ Tarım ve Doğa Derg 25 (Ek Sayı 1): 125-136, 2022 KSU J. Agric Nat 25 (Suppl 1): 125-136, 2022 Araştırma Makalesi Research Article Karyomorphological finding are given in Figure 9-10 and Table 2-3. The nutlet surface structure of taxa could not be observed since it is covered with dense trichomes. However, the non-glandular trichomes differ from each other. In S. orientalis subsp. virens, surface completely covered with ashy hairlets. In S. orientalis subsp virens, there are curved non-glandular trichomes; It is flat in S. salviifolia. Nutlet shape is obovate in both taxa. Figure 9. A and C- General view of S. orientalis subsp. virens and S. salviifolia, B and D- Nutlet surface of S. orientalis subsp. virens and S. salviifolia Şekil 9. A ve C- S. orientalis subsp. virens ve S. salviifolia’ nın genel görünüşü. B ve D S. orientalis subsp. virens ve S. salviifolia’ nın nutlet yüzeyi Figure 8. Glandular trichome structure of taxa (top; S. orientalis subsp. virens, under; S. salviifolia Şekil 8. Taksonların glandüler trikom yapısı (üstte; S. orientalis subsp. virens, altta; S. salviifolia) Figure 9. A and C- General view of S. orientalis subsp. virens and S. salviifolia, B and D- Nutlet surface of S. orientalis subsp. virens and S. salviifolia Figure 8. As analysis of pigment was evaluated in S. salviifolia, Petiole anatomy and micromorphology Glandular trichome structure of taxa (top; S. orientalis subsp. virens, under; S. salviifolia Şekil 9. A ve C- S. orientalis subsp. virens ve S. salviifolia’ nın genel görünüşü. B ve D S. orientalis subsp. virens ve S. salviifolia’ nın nutlet yüzeyi Şekil 9. A ve C- S. orientalis subsp. virens ve S. salviifolia’ nın genel görünüşü. B ve D S. orientalis subsp. virens ve S. salviifolia’ nın nutlet yüzeyi Şekil 8. Taksonların glandüler trikom yapısı (üstte; S. orientalis subsp. virens, altta; S. salviifolia) Karyomorphological Findings Figure 11. Metaphase chromosomes belonging to the taxa studied; 1. S. orientalis subsp. virens 2. S. salviifolia (scale bar 10 µm) Şekil 11. Incelenen taksonlara ait metafaz kromozomları; 1. S. orientalis subsp. virens 2. S. salviifolia (scale bar 10 µm) Karyomorphological Findings Karyomorphological Findings Figure 11. Metaphase chromosomes belonging to the taxa studied; 1. S. orientalis subsp. virens 2. S. salviifolia (scale bar 10 µm) Şekil 11. Incelenen taksonlara ait metafaz kromozomları; 1. S. orientalis subsp. virens 2. S. salviifolia (scale bar 10 µm) Figure 11. Metaphase chromosomes belonging to the taxa studied; 1. S. orientalis subsp. virens 2. S. salviifolia (scale bar 10 µm) Şekil 11. Incelenen taksonlara ait metafaz kromozomları; 1. S. orientalis subsp. virens 2. S. salviifolia (scale bar 10 µm) Figure 11. Metaphase chromosomes belonging to the taxa studied; 1. S. orientalis subsp. virens 2. S. salviifolia (scale bar 10 µm) Şekil 11. Incelenen taksonlara ait metafaz kromozomları; 1. S. orientalis subsp. virens 2. S. salviifolia (scale bar 10 µm) Table 2. Somatic chromosome number, polyploid level, karyotype formula, chromosome length range, total chromosome length (TKL) and asymmetric index (A1, A2) of the examined taxa. Table 2. Somatic chromosome number, polyploid level, karyotype formula, chromosome length range, total chromosome length (TKL) and asymmetric index (A1, A2) of the examined taxa. chromosome length (TKL) and asymmetric index (A1, A2) of the examined taxa. Çizelge 2. İncelenen taksonların somatik kromozom sayısı, poliploid düzeyi, karyotip formülü, kromozom uzunluk aralığı, toplam kromozom uzunluğu ve asimetrik indeks (A1, A2) Taxon 2n Polyploid level Karyotype formula Chromosome length range (µm) TKL (µm) A1 A2 S. orientalis subsp. virens 22 2x 1M+10m 2.18-3.72 30.50 3.6 2.77 S. salviifolia 22 2x 2M+ 2sm+8m 2.38-3.65 34.24 15.8 3.11 Pigmentation Results As analysis of pigment was evaluated in S salviifolia Chl a content in leaves and stems samples were determined respectively 6.45 µg g-1 and 2.32 µg g-1. Petiole anatomy and micromorphology g y , n taksonların somatik kromozom sayısı, poliploid düzeyi, karyotip formülü, kromozo alığı, toplam kromozom uzunluğu ve asimetrik indeks (A1, A2) Chl a content in leaves and stems samples were determined respectively 6.45 µg g-1 and 2.32 µg g-1. As analysis of pigment was evaluated in S. salviifolia, 130 KSÜ Tarım ve Doğa Derg 25 (Ek Sayı 1): 125-136, 2022 KSU J. Agric Nat 25 (Suppl 1): 125-136, 2022 Araştırma Makalesi Research Article detected on the leaves as 0.82 μg g-1 and on the stems as 0.48 μg g-1. When we compared the Chl a content in S. salviifolia and S. orientalis subsp. virens, the Chl a and Total Chl contents were determined higher in the leaves and stems samples of S. orientalis subsp. virens. There was no significant difference between the two plants in terms of Chl b content. When the Car content was evaluated, it was determined higher in leaves samples of S. orientalis subsp. virens, but no significant difference was found between stems patterns (p <0.05) (Figure 12). Chl b content was found 2.60 µg g-1 in the leaves and 0.45 µg g-1 in the stems. Total Chl content in leaves and stems samples were measured as 9.05 µg g-1 and 2.76 µg g-1 respectively. Car content was found as 0.71 µg g-1 in leaves and 0.44 µg g-1 in stems samples. When the Chl a content in leaves and stem samples in S. orientalis subsp. virens were determined respectively 7.37 µg g-1 and 2.54 µg g-1. The concentration of Chl b was contained in leaves at 2.58 μg g-1 and stems at 0.48 μg g-1. Total Chl content was calculated in leaves and stems samples as 9.95 µg g- and 3.01 µg g-1 respectively. Car concentrations were Table 3. Karyomorphological parameters of the examined taxa: (NB: Relative length, L / S: arm ratio, CI: centromere index, SD: Centromere status,M:median point, m: median, sm: submedian) Çizelge 3. Incelenen taksonların karyomorfolojik parametreleri: (NB: nispi boy, L / S: kol oranı, CI: sentromer indeksi, , SD: sentromer durumu, M:noktalı medyan, m: medyan, sm: submedyan) Table 3. Karyomorphological parameters of the examined taxa: (NB: Relative length, L / S: arm ratio, CI: centromere index, SD: Centromere status,M:median point, m: median, sm: submedian) Çizelge 3. Petiole anatomy and micromorphology Incelenen taksonların karyomorfolojik parametreleri: (NB: nispi boy, L / S: kol oranı, CI: sentromer indeksi, , SD: sentromer durumu, M:noktalı medyan, m: medyan, sm: submedyan) Table 3. Karyomorphological parameters of the examined taxa: (NB: Relative length, L / S: arm ratio, CI: centromere index, SD: Centromere status,M:median point, m: median, sm: submedian) Çizelge 3. Incelenen taksonların karyomorfolojik parametreleri: (NB: nispi boy, L / S: kol oranı, CI: sentromer indeksi, , SD: sentromer durumu, M:noktalı medyan, m: medyan, sm: submedyan) Table 3. Karyomorphological parameters of the examined taxa: (NB: Relative length, L / S: arm ratio, CI: centromere index, SD: Centromere status,M:median point, m: median, sm: submedian) Çizelge 3. Incelenen taksonların karyomorfolojik parametreleri: (NB: nispi boy, L / S: kol oranı, CI: sentromer indeksi, , SD: sentromer durumu, M:noktalı medyan, m: medyan, sm: submedyan) S. orintalis subsp. virens Haploid Total Lenght Long Arm L Small Arm S Arm Ratio (AR) Centromere İndex İ=100(S/C) Relative Length N.P. Centromere Status S.D 1 3.72 1.90 1.82 1.04 48.92 12.20 m 2 3.38 1.78 1.60 1.11 47.34 11.08 m 3 3.12 1.60 1.52 1.05 48.72 10.23 m 4 3.05 1.58 1.47 1.07 48.20 10.00 m 5 2.91 1.50 1.41 1.06 48.45 9.54 m 6 2.85 1.47 1.38 1.07 48.42 9.34 m 7 2.75 1.40 1.35 1.04 49.09 9.02 m 8 2.47 1.25 1.22 1.02 49.39 8.10 m 9 2.18 1.05 1.13 0.93 51.83 7.15 m 10 2.04 1.02 1.02 1.00 50.00 6.69 M 11 2.03 1.03 1.00 1.03 49.26 6.66 m S. salviifolia Haploid Total Lenght Long Arm L Small Arm S Arm Ratio (AR) Centromere İndex İ=100(S/C) Relative Length N.P. Centromere Status S.D 1 3.65 2.40 1.25 1.92 34.25 10.66 sm 2 3.50 2.28 1.22 1.87 34.86 10.22 sm 3 3.95 2.15 1.80 1.19 45.57 11.54 m 4 3.25 1.92 1.33 1.44 40.92 9.49 m 5 3.72 1.86 1.86 1.00 50.00 10.86 M 6 3.18 1.63 1.55 1.05 48.74 9.29 m 7 2.91 1.45 1.46 0.99 50.17 8.50 m 8 2.74 1.37 1.37 1.00 50.00 8.00 M 9 2.53 1.32 1.21 1.09 47.83 7.39 m 10 2.43 1.28 1.15 1.11 47.33 7.10 m 11 2.38 1.25 1.13 1.11 47.48 6.95 m S. orintalis subsp. virens 131 KSÜ Tarım ve Doğa Derg 25 (Ek Sayı 1): 125-136, 2022 KSU J. Agric Nat 25 (Suppl 1): 125-136, 2022 Araştırma Makalesi Research Article Figure 12. Haploid idiograms belonging to the taxa studied; 1. S. orientalis subsp. Total Carbohydrate Results Farklı harflerle gösterilen değerler istatiksel olarak anlamlı (p <0.05),aynı harflerle gösterilen değerler anlamsız bulundu (Duncan, 1955) MDA/ g fresh weight in stems samples. When we evaluated the MDA content in S. salviifolia and S. orientalis subsp. virens, MDAcontent in S. orientalis subsp. virens was found higher in leaves and stems samples. Statistically, these changes were found to be significant (p <0.05) (Figure 14). MDA/ g fresh weight in stems samples. When we evaluated the MDA content in S. salviifolia and S. orientalis subsp. virens, MDAcontent in S. orientalis subsp. virens was found higher in leaves and stems samples. Statistically, these changes were found to be significant (p <0.05) (Figure 14). Petiole anatomy and micromorphology virens 2. S. salviifolia Şekil 12. Incelenen taksonlara ait haploid idiyogramlar; 1. S. orientalis subsp. virens 2. S. salviifolia Figure 12. Haploid idiograms belonging to the taxa studied; 1. S. orientalis subsp. virens 2. S. salviifolia Şekil 12. Incelenen taksonlara ait haploid idiyogramlar; 1. S. orientalis subsp. virens 2. S. salviifolia Total Carbohydrate Results Total Carbohydrate Results 0.69 µg g-1 respectively. When we compared the total carbohydrate content in S. salviifolia and S. orientalis subsp. virens. Total carbohydrate content in S. orientalis subsp virens was found higher in leaves and stems samples. Statistically, these changes were determined significant (p <0.05) (Figure 13). 0.69 µg g-1 respectively. When we compared the total carbohydrate content in S. salviifolia and S. orientalis subsp. virens. Total carbohydrate content in S. orientalis subsp virens was found higher in leaves and stems samples. Statistically, these changes were determined significant (p <0.05) (Figure 13). The total carbohydrate content of S. salviifolia plant was determined as 1.27 µg g-1 and 0.55 µg g-1, respectively, in leaves and stems samples. The total carbohydrate content of S. orientalis subsp. virens leaf and stem samples was found to be 1.46 µg g-1 and Figure 13. Changes in pigment contents in S. salviifolia and S. orientalis subsp. virens. The values shown with different letters were found to be statistically significant (p <0.05), the values shown with the same letters were found to be insignificant (Duncan, 1955) b c a d b c a d b c a c b c a c 0,00 2,00 4,00 6,00 8,00 10,00 Chl a Chl b T Chl Car µg g-1 Pigmentation S. salviifolia leaves S. salviifolia stems S. orientalis subsp. virens leaves S. orientalis subsp. virens stems b c a d b c a d b c a c b c a c 0,00 2,00 4,00 6,00 8,00 10,00 Chl a Chl b T Chl Car µg g-1 Pigmentation S. salviifolia leaves S. salviifolia stems S. orientalis subsp. virens leaves S. orientalis subsp. virens stems Figure 13. Changes in pigment contents in S. salviifolia and S. orientalis subsp. virens. The values shown with different letters were found to be statistically significant (p <0.05), the values shown with the same letters were found to be insignificant (Duncan, 1955) g ( , ) Şekil 13. S. salviifolia ve S. orientalis subsp. virens’ in pigment içeriğindeki değişiklikler. Farklı harflerle gösterilen değerler istatiksel olarak anlamlı (p <0.05),aynı harflerle gösterilen değerler anlamsız bulundu (Duncan, 1955) g , Şekil 13. S. salviifolia ve S. orientalis subsp. virens’ in pigment içeriğindeki değişiklikler. Farklı harflerle gösterilen değerler istatiksel olarak anlamlı (p <0.05),aynı harflerle gösterilen değerler anlamsız bulundu (Duncan 1955) Şekil 13. S. salviifolia ve S. orientalis subsp. virens’ in pigment içeriğindeki değişiklikler. MDA Results addition, the fact that the petiole main vascular bundle consists of one piece reveals its difference from in this study. In addition, type II C glandular hair seen only in calyx in his study was not observed in this leaf stem and petiole. Özdemir and Altan (2005) stated in their study that the vascular bundles in Scutellaria orientalis subsp. santolinoides and S. orientalis subsp. bicolor petioles were surrounded by scleranchymatic cells, but in this study, these scleranchymatic cells were not observed in the other subspecies of this species, S. orientalis subsp virens. In the stem, it has been stated that the vascular bundles are surrounded by scleranchymatic cells, and in the case it is seen that it is in partly form. It is also stated that the leaf mesophyll is bifacial, in the case it is unifacial. In S. orientalis subsp. virens, the leaf consists only of the palisade parenchyma. However, in some observations, it was also observed that the palisades on the lower surface were slightly more oval. Anatomical and micromorphological studies conducted on Lamiaceae family recently can provide useful characters in revealing their similarities and differences in distinguishing taxa (Açar and Satıl, 2019; Ecevit-Genç et al., 2018; Polat et al., 2017; Kaya et al., 2013; Selvi et al., 2013; Satıl and Kaya, 2007; Satıl et al., 2011). The vascular structure of the petiole carries a taxonomic character. In the cross sections, the middle vein curves in the form of a half-moon or it creates an annular structure by further curling; the arrangement of small bundles of vascular vessel or circular structure of small bundles; The number and sequence of small vascular bundles at the ends of the petiole (wing) are systematically important characters in identifying genera and species (Metcalfe and Chalk, 1950). Karyological studies have been carried out on some species belonging to the genus Scutellaria. The chromosome number of S. tomentosa Bertol., S. theobromina Rech.f., S. araxensis Grossh., S. platystegia Juz., S. nepetifolia Benth., S., S. persica Bornm. and S. pinnatifida has been reported as 2n = 2x = 22 (Ranjbar and Mahmoudi, 2013) In another study, the chromosome number of endemic S. orientalis subsp. bicolor species was determined as 2n = 22 (Gedik et al., 2016). However, there is no information about the chromosome number and structure of S. salviifolia species, which is an endemic taxon. According to the study by Akçın et al. MDA Results The MDA content of S. salviifolia plant was found as 2.55 µmol MDA/ g fresh weight (FW) and 1.07 µmol MDA/ g FW, respectively, in leaves and stem samples. The MDA content of S. orientalis subsp virens was determined as µmol MDA/ g FW in leaves and µmol 132 KSÜ Tarım ve Doğa Derg 25 (Ek Sayı 1): 125-136, 2022 KSU J. Agric Nat 25 (Suppl 1): 125-136, 2022 https://doi.org/10.18016/ksutarimdoga.vi.970571 Figure 14. Changes in Total carbohydrate contents in S. salviifolia and S. orientalis subsp. virens. The values shown with different letters were found to be statistically significant (p <0.05), the values shown with the same letters were found to be insignificant (Duncan, 1955) Şekil 14. S. salviifolia ve S. orientalis subsp. virens’ deki toplam karbonhidrat içeriğindeki değişiklikler. Farklı harflerle gösterilen değerler istatiksel olarak anlamlı (p <0.05),aynı harflerle gösterilen değerler b d a c 0,00 0,20 0,40 0,60 0,80 1,00 1,20 1,40 1,60 1,80 S. salviifolia leaves S. salviifolia stems S. orientalis subsp. virens leaves S. orientalis subsp. virens stems µg g-1 Total carbohydrate KSÜ Tarım ve Doğa Derg 25 (Ek Sayı 1): 125-136, 2022 KSU J. Agric Nat 25 (Suppl 1): 125-136, 2022 https://doi.org/10.18016/ksutarimdoga.vi.970571 b d a c 0,00 0,20 0,40 0,60 0,80 1,00 1,20 1,40 1,60 1,80 S. salviifolia leaves S. salviifolia stems S. orientalis subsp. virens leaves S. orientalis subsp. virens stems µg g-1 Total carbohydrate b d a c 0,00 0,20 0,40 0,60 0,80 1,00 1,20 1,40 1,60 1,80 S. salviifolia leaves S. salviifolia stems S. orientalis subsp. virens leaves S. orientalis subsp. virens stems µg g-1 Total carbohydrate Total carbohydrate Figure 14. Changes in Total carbohydrate contents in S. salviifolia and S. orientalis subsp. virens. The values shown with different letters were found to be statistically significant (p <0.05), the values shown with the same letters were found to be insignificant (Duncan, 1955) Şekil 14. S. salviifolia ve S. orientalis subsp. virens’ deki toplam karbonhidrat içeriğindeki değişiklikler. Farklı harflerle gösterilen değerler istatiksel olarak anlamlı (p <0.05),aynı harflerle gösterilen değerler anlamsız bulundu (Duncan, 1955) In this study; the anatomical and karyological characteristics and biochemical content of the samples taken from S. orientalis subsp. virens and S. salviifolia belonging to the genus of Scutellaria, Lamiaceae family, which are frequently used in traditional medicine, pharmacology, cosmetics and food industry and are of great economic importance, were investigated. MDA Results (2011), the S. salviifolia petiole gave similar results to the research we conducted, but the vascular bundle in the middle part in this study differs in that it consists of almost two parts and the absence of peltate-type hair. Çalı (2017) stated in his study that there were sclerenchymatic cells in the stem of S. salviifolia, but sclerenchyma was not found in this study. The fact that the mesophyll is made entirely of palisade is similar to the stoma being on both surfaces. In The chromosome number of S. salviifolia species, which is an endemic species was determined as 2n = 22. It is seen that the total chromosome lengths of KSÜ Tarım ve Doğa Derg 25 (Ek Sayı 1): 125-136, 2022 KSU J. Agric Nat 25 (Suppl 1): 125-136, 2022 Araştırma Makalesi Research Article this species vary between 2.38-3.65 μm and arm ratios between 1.11-1.92 μm. The karyotype formula is 7m + 2sm + 2M. The total chromosome length is 34.24 um. It has been determined that this species has a satellite on its second chromosome. The karyology of the S. salviifolia species was first determined in this study (Figure 11-12). content was dound higher in the leaf samples of S. orientalis subsp. virens, but no significant difference was found between the patterns of stems. Statistically, it was observed that these changing were substantial (p <0.05) (Figure 13). The most important product of lipid peroxidation is MDA. It leads to binding of ion permeability and enzyme it causes negative consequences such as change of activity. Due to this feature of MDA, it can be used to protect DNA with nitrogen bases. React and hence mutagenic, cell genotoxic and carcinogenic for cultures (Hafez et al., 2020; Khoubnasabjafari and Jouyban, 2020; Nilsson and Liu, 2020). MDA content in S. orientalis subsp. virens was found higher in leaves and stems samples than S. salviifolia. Statistically, it was observed that these changing were substantial (p <0.05) (Figure 14). The chromosome number of S. orientalis subsp. virens was determined as 2n = 22. It is seen that the total chromosome lengths vary between 2.08-3.72 μm and arm ratios between 1.03-1.04 μm. The karyotype formula is 10m + 1M. Total chromosome length is 30.50 μm. The S. orintalis subsp. virens taxon was first discussed in terms of chromosome number and chromosome morphology in this study (Table 2). MDA Results Chlorophylls absorb light energy of certain wavelengths and either convert this energy into another wavelength used in photosynthesis or transfer it directly to the compounds required for photosynthesis. Also they act like a catholyzer in the stages of photosynthesis. The spectral distribution of light sources in crop production plays an important role in finding photomorphogenic reactions. It has been noted that the plant grows in direct proportion as the pigment system absorbs the sunlight (Amoozgar et al., 2017; Izzo et al., 2019). Carotenoids are not only one of the plant pigments but also important antioxidants that play a role in oxidative stress tolerance. There are studies showing that the carotenoid oxidation products as stress signals for plants (Berru et al., 2021; Xia et al., 2021). In this study, the contents of Chl a and Total Chl were higher in the leaves and stem samples of S. orientalis subsp. virens, There was no significant difference in the content of Chl b between the two plants. Car Carbohydrates are organic compounds containing carbon, oxygen and hydrogen atoms in their structures. It is also stated that carbohydrates act as signal molecules similar to hormones (Shah et al., 2019; Smeekens et al., 2000). Total carbohydrate content in S. orientalis subsp. virens was found higher in leaves and stems samples than S. salviifolia. Statistically, these changes have been determined to be significant (p <0.05) (Figure15). Pigment, total carbohydrate and MDA contents are used as important markers in plants under stress conditions. The biochemical characteristics of the plant are important in the response and adaptation to stress in ecological conditions. In this study, it was determined that the biochemical composition of S. orientalis subsp. virens was found higher than that of S. salviifolia. Figure 15. Changes in MDA contents in S. salviifolia and S. orientalis subsp. virens. The values shown wi different letters were found to be statistically significant (p <0.05), the values shown with the sam letters were found to be insignificant (Duncan, 1955) b d a c 0,00 0,50 1,00 1,50 2,00 2,50 3,00 3,50 S. salviifolia leaves S. salviifolia stems S. orientalis subsp. virens leaves S. orientalis subsp. virens stems µmol MDA /g FW MDA content b d a c 0,00 0,50 1,00 1,50 2,00 2,50 3,00 3,50 S. salviifolia leaves S. salviifolia stems S. orientalis subsp. virens leaves S. orientalis subsp. REFERENCES Abdelrahim DN, Takruri HR, Al-Ismail KM 2020. Effect of introducing the Jordanian common rue (Ruta chalepensis L.) on blood lipid profile in adult male Sprague Dawley rats toxified with Paracetamol. J Saudi Soc Food Nutr, 13(1): 17-23. Elçi Ş 1982. 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https://openalex.org/W3139033944	https://bmcresnotes.biomedcentral.com/track/pdf/10.1186/s13104-021-05610-y	English	null	Genome data of four Pythium insidiosum strains from the phylogenetically-distinct clades I, II, and III	BMC research notes	2,021	cc-by	3,025	© The Author(s) 2021. 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://crea- tivecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdo- main/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Abstract Objectives: We employed the Illumina NGS platform to sequence genomes of 4 different strains of the pathogenic oomycete Pythium insidiosum, the causative agent of pythiosis. These strains were isolated from humans in Thailand (n = 3) and the United States (n = 1), and phylogenetically classified into clade-I, -II, and -III. Our study augmented the completeness of the P. insidiosum genome database for exploration of the biology, evolution, and pathogenesis of the pathogen. Data description: One paired-end library (180-bp insert) was prepared from a gDNA sample of P. insidiosum strains ATCC200269 (clade-I), Pi19 (clade-II), MCC18 (clade-II), and SIMI4763 (clade-III) for whole-genome sequencing by Illumina HiSeq2000/HiSeq2500 NGS platform. A range of 28.4–59.4 million raw reads, accounted for 3.0–7.3 Gb, were obtained and assembled into the genome sizes of 47.1 Mb (15,153 contigs; 85% completeness; 19,329 open reading frames [ORFs]) for strain ATCC200269, 35.4 Mb (14,576 contigs; 83% completeness; 13,895 ORFs) for strain Pi19, 34.5 Mb (11,084 contigs; 84% completeness; 13,249 ORFs) for strain MCC18, and 47.1 Mb (15,162 contigs; 85% completeness; 19,340 ORFs) for strain SIMI4763. The genome data can be downloaded from the NCBI/DDBJ data- bases under the accessions BCFN00000000.1 (ATCC200269), BCFS00000000.1 (Pi19), BCFT00000000.1 (MCC18), and BCFU00000000.1 (SIMI4763). Keywords: Pythium insidiosum, Pythiosis, Genome sequence, Next-generation sequencing of a pathogen of interest. Besides, such data could serve as a comprehensive genetic resource for the identifica- tion of diagnostic and therapeutic microbial markers. Here, we employed the Illumina HiSeq2000/HiSeq2500 NGS platform to sequence the genomes of 4 different strains (i.e., ATCC200269, Pi19, MCC18, and SIMI4763) of Pythium insidiosum, a prominent pathogenic oomy- cete microorganism that infects humans and animals worldwide and causes an infectious condition with high mortality and morbidity, called pythiosis [2–4]. These strains were isolated from human patients with pythio- sis from Thailand (n = 3) and the United States (n = 1), and have been phylogenetically classified into clade-I (n = 1), clade-II (n = 2), and clade-III (n = 1), based on Genome data of four Pythium insidiosum strains from the phylogenetically‑distinct clades I, II, and III Theerapong Krajaejun1* , Weerayuth Kittichotirat2, Preecha Patumcharoenpol3, Thidarat Rujirawat4, Tassanee Lohnoo4 and Wanta Yingyong4 Krajaejun et al. BMC Res Notes (2021) 14:197 https://doi.org/10.1186/s13104-021-05610-y Krajaejun et al. BMC Res Notes (2021) 14:197 https://doi.org/10.1186/s13104-021-05610-y BMC Research Notes BMC Research Notes Objective Next-generation sequencing (NGS) is a sophisticated technology that facilitates multiple genome sequenc- ing of different strains of the same microbial species, in a short duration, and at a low cost [1]. Obtained data promise extensive comparative genomic analyses to bet- ter understand the biology, evolution, and pathogenesis Correspondence: mr_en@hotmail.com; weerayuth.kit@kmutt.ac.th 1 Department of Pathology, Faculty of Medicine, Ramathibodi Hospita Mahidol University, Bangkok, Thailand 2 Systems Biology and Bioinformatics Research Group, Pilot Plant Development and Training Institute, King Mongkut’s University of Technology Thonburi, Bangkhuntien, Bangkok, Thailand Full list of author information is available at the end of the article insidiosum, which could provide knowledge that can be adapted for the development of preventive measures, reliable diagnostic assay, and effective thera- peutic modality for pythiosis. Krajaejun et al. BMC Res Notes (2021) 14:197 Krajaejun et al. BMC Res Notes (2021) 14:197 Page 2 of 4 CLC Genomics Workbench software trimmed raw reads to ensure a read length of at least 35 bases. Cuta- dapt 1.8.1 [14] removed the adaptor sequences from all reads. A total of 59,442,302 raw reads (average length: 122.2 bases) from the strain ATCC200269; 30,517,195 raw reads (average length: 92.5 bases) from the strain Pi19; 28,443,839 raw reads (average length: 94.7 bases) from the strain MCC18; and 28,531,434 raw reads (average length: 122.3 bases) from the strain SIMI4763 were obtained. Velvet 1.2.10 [15] assembled the raw reads of the strain ATCC200269 into 15,153 contigs [average length: 3111.1 (range: 300–182,581); N50: 11,266; total bases: 47,142,494; %N: 0.7%; genome cov- erage: 154×]; the strain Pi19 into 14,576 contigs [aver- age length: 2426.8 (range: 300–111,336); N50: 6208; total bases: 35,372,432; %N: 2.4%; genome coverage: 91×]; the strain MCC18 into 11,084 contigs [average length: 3116.3 (range: 300–150,908); N50: 8946; total bases: 34,541,218; %N: 2.3%; genome coverage: 87×]; and the strain SIMI4763 into 15,162 contigs [average length: 3109.2 (range: 300–182,337); N50: 11,187; total bases: 47,141,692; %N: 0.7%; genome coverage: 74×]. BLAST search analyses of the assembled sequences of the strains ATCC200269, Pi19, MCC18 and SIMI4763, using the “Core Eukaryotic Genes Mapping Approach (CEGMA)” panel (containing 248 highly-conserved eukaryotic genes) [16] demonstrated 85%, 83%, 84%, and 85% genome completeness, respectively. MAKER2 pipeline [17] assigned 19,329; 13,895; 13,249 and 19,340 open reading frames (ORFs) in the genomes of the strains ATCC200269, Pi19, MCC18 and SIMI4763, respectively. All contig sequences have been depos- ited in the National Center for Biotechnology Infor- mation (NCBI) and DNA Data Bank of Japan (DDBJ) databases under the accessions BCFN00000000.1 (for strain ATCC200269), BCFS00000000.1 (Pi19), BCFT00000000.1 (MCC18), and BCFU00000000.1 (SIMI4763) (Table 1). the ribosomal deoxyribonucleic acid (rDNA) sequence analysis [5]. So far, the draft genome sequences from 7 strains of P. insidiosum (including the synonym spe- cies Pythium destruens), isolated from humans, horses, and the environment in various countries, are available in the public databases [6–12]. This study contributed additional genomic data to augment the completeness of the public P. insidiosum genome database. Research- ers around the world can use this genome data as a basis to explore the biology, evolution, and pathogen- esis of P. Consent for publication Not applicable. Consent for publication Not applicable. Limitations We used the Illumina HiSeq2000/HiSeq2500 short- read NGS platform to sequence 4 genomes of P. insidiosum (strains ATCC200269, Pi19, MCC18, and SIMI4763). Users of the genome data should be aware that the sequencing-by-synthesis technique in the Illumina platforms constructs a library base on DNA amplification, which could result in sequence coverage biases and substitution errors. As seen in the genome data of these P. insidiosum strains, the total bases ranged from 3.0 to 7.3 Gb, and the genome sequence coverages ranged from 74× to 154×. Another limita- tion of the study is the number and type of the DNA library. The genome sequences of each P. insidiosum strain were obtained from only one paired-end library. As expected, all strains showed a less complete genome (83–85% CEGMA-based genome completeness), a higher number of contigs (11,084–15,162 contigs), and a smaller genome size (34.5–47.1 Mb), when compared with the P. insidiosum’s reference genome (92% com- pleteness; 1192 contigs; 53.2-Mb size) generated from one paired-end and three mate-pair libraries [8]. Availability of data and materials Please see Table 1 and references [18–21] for details and links to the data. The draft genome sequence of the P. insidiosum strain ATCC200269 comprising 15,153 contigs (accession numbers BCFN01000001- BCFN01015153), is available in GenBank here: https://identifiers.org/ncbi/ insdc:BCFN00000000.1 [18]. The draft genome sequence of the P. insidiosum strain Pi19 comprising 14,576 contigs (accession numbers BCFS01000001-BCFS01014576), is available in GenBank here: https://identifiers.org/ncbi/insdc:BCFS000000 00.1 [19]. The draft genome sequence of the P. insidiosum strain MCC18 compris- ing 11,084 contigs (accession numbers BCFT01000001-BCFT01011084), is available in GenBank here: https://identifiers.org/ncbi/insdc:BCFT000000 00.1 [20]. The draft genome sequence of the P. insidiosum strain SIMI4763 compris- ing 15,162 contigs (accession numbers BCFU01000001-BCFU01015162), is available in GenBank here: https://identifiers.org/ncbi/insdc:BCFU000000 00.1 [21]. Acknowledgements Not applicable. 3. Krajaejun T, Sathapatayavongs B, Pracharktam R, Nitiyanant P, Leela- chaikul P, Wanachiwanawin W, et al. Clinical and epidemiological anal- yses of human pythiosis in Thailand. Clin Infect Dis. 2006;43:569–76. Data descriptionh The P. insidiosum strain ATCC200269 (phyloge- netic clade-I) was isolated from a human patient in the United States, while the strains Pi19 (clade-II), MCC18 (clade-II), and SIMI4763 (clade-III) were iso- lated from human patients in Thailand. The identity (i.e., species) and genotype (i.e., clade) of each strain were confirmed by the rDNA sequence analysis [acces- sion numbers: AB898108 (for strain ATCC200269), AB898113 (Pi19), AB971183 (MCC18), and AB971189 (SIMI4763)] [5]. These organisms were cultured in Sab- ouraud dextrose broth with shaking (50–150 rounds per min) for one week at 37 °C. The resulting hyphal material of each strain was harvested and subjected to genomic deoxyribonucleic acid (gDNA) extraction, using an established method [13]. The identity of each strain was re-assessed by the rDNA sequence analysis, using the obtained gDNA [5]. One paired-end library with a 180-bp gap was prepared for each gDNA sample before proceeding to whole-genome sequencing by the Illumina HiSeq2000 (for strains Pi19 and MCC18) and HiSeq2500 (for strains ATCC200269 and SIMI4763) NGS platforms (Yourgene Bioscience, Taiwan), as pre- viously described [6, 7, 10, 12]. In brief, the Qiagen Table 1 Overview of data files/data sets Label Name of data file/data set File types (file extension) Data repository and identifier (DOI or accession number) Data file 1 Pythium insidiosum strain ATCC200269, whole genome shotgun sequencing project FASTA GenBank (https://identifiers.org/ncbi/insdc:BCFN00000000.1) [18] Data file 2 Pythium insidiosum strain Pi19, whole genome shotgun sequencing project FASTA GenBank (https://identifiers.org/ncbi/insdc:BCFS00000000.1) [19] Data file 3 Pythium insidiosum strain MCC18, whole genome shotgun sequencing project FASTA GenBank (https://identifiers.org/ncbi/insdc:BCFT00000000.1) [20] Data file 4 Pythium insidiosum strain SIMI4763, whole genome shotgun sequencing project FASTA GenBank (https://identifiers.org/ncbi/insdc:BCFU00000000.1) [21] Krajaejun et al. BMC Res Notes (2021) 14:197 Krajaejun et al. BMC Res Notes (2021) 14:197 Krajaejun et al. BMC Res Notes (2021) 14:197 Page 3 of 4 In summary, the draft genomes of P. insidiosum strains ATCC200269 (genome size: 47.1 Mb), Pi19 (35.4 Mb), MCC18 (34.5 Mb), and SIMI4763 (47.1 Mb) isolated from human patients with pythiosis living in Thailand and the United States, have been gener- ated and publicly available. The obtained genome data could be a useful dataset to enhance the exploration of the biology, evolution, and pathogenesis of P. insidio- sum, which can lead to clinical applications for better management of patients with pythiosis. Author details 1 Department of Pathology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand. 2 Systems Biology and Bioinformat- ics Research Group, Pilot Plant Development and Training Institute, King Mongkut’s University of Technology Thonburi, Bangkhuntien, Bangkok, Thailand. 3 Interdisciplinary Graduate Program in Bioscience, Faculty of Sci- ence, Kasetsart University, Bangkok, Thailand. 4 Research Center, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand. Received: 8 March 2021 Accepted: 11 May 2021 Received: 8 March 2021 Accepted: 11 May 2021 Authors’ contributions 4. Chitasombat MN, Jongkhajornpong P, Lekhanont K, Krajaejun T. Recent update in diagnosis and treatment of human pythiosis. PeerJ. 2020;8:e8555. W.K. and T.K. conceived the project. W.K., P.P., T.R., T.L., and W.Y. performed the experiments. W.K., P.P., T.R., and T.K. analyzed the data. W.K. and T.K. wrote the manuscript. All authors reviewed the manuscript. W.K. and T.K. acquired the research funds. 5. Rujirawat T, Sridapan T, Lohnoo T, Yingyong W, Kumsang Y, Sae-Chew P, et al. Single nucleotide polymorphism-based multiplex PCR for identification and genotyping of the oomycete Pythium insidiosum from humans, animals and the environment. Infect Genet Evol. 2017;54:429–36. References 1 Ki i h CEGMA: Core Eukaryotic Genes Mapping Approach; DDBJ: DNA Data Bank of Japan; gDNA: Genomic deoxyribonucleic acid; NCBI: National Center for Biotechnology Information; NGS: Next-generation sequencing; ORF: Open reading frame; rDNA: Ribosomal deoxyribonucleic acid. 1. Kittichotirat W, Krajaejun T. Application of genome sequenc- ing to study infectious diseases. J Infect Dis Antimicrob Agents. 2019;36:47–58. 2. Gaastra W, Lipman LJ, De Cock AW, Exel TK, Pegge RB, Scheurwater J, et al. Pythium insidiosum: an overview. Vet Microbiol. 2010;146:1–16. Ethics approval and consent to participate This study was approved by the Human Research Ethics Committee, Faculty of Medicine, Ramathibodi Hospital, Mahidol University (approval numbers: MURA2020/966). Consent for publication Not applicable. Funding This study obtained financial supports from the Faculty of Medicine, Ramathibodi Hospital, Mahidol University [Grant number CF_63008], the Thailand Research Fund [Grant number RSA6280092], and the King Mongkut’s University of Technology Thonburi through the "KMUTT 55th Anniversary Commemorative Fund". The funders had no role in the design of the study and collection, analysis, and interpretation of data and in writ- ing the manuscript. 6. Kittichotirat W, Patumcharoenpol P, Rujirawat T, Lohnoo T, Yingyong W, Krajaejun T. Draft genome and sequence variant data of the oomycete Pythium insidiosum strain Pi45 from the phylogenetically-distinct Clade-III. Data Brief. 2017;15:896–900. Page 4 of 4 Krajaejun et al. BMC Res Notes (2021) 14:197 Krajaejun et al. BMC Res Notes (2021) 14:197 7. Patumcharoenpol P, Rujirawat T, Lohnoo T, Yingyong W, Vanittanakom N, Kittichotirat W, et al. Draft genome sequences of the oomycete Pythium insidiosum strain CBS 573.85 from a horse with pythiosis and strain CR02 from the environment. Data Brief. 2018;16:47–50. 7. Patumcharoenpol P, Rujirawat T, Lohnoo T, Yingyong W, Vanittanakom N, Kittichotirat W, et al. Draft genome sequences of the oomycete Pythium insidiosum strain CBS 573.85 from a horse with pythiosis and strain CR02 from the environment. Data Brief. 2018;16:47–50. 14. Martin M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet J. 2011;17:10. 15. Zerbino DR, Birney E. Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res. 2008;18:821–9. 15. Zerbino DR, Birney E. Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res. 2008;18:821–9. 8. Rujirawat T, Patumcharoenpol P, Lohnoo T, Yingyong W, Lerksuthirat T, Tangphatsornruang S, et al. Draft genome sequence of the patho- genic oomycete Pythium insidiosum Strain Pi-S, isolated from a patient with pythiosis. Genome Announc. 2015;3:e00574-e615. 16. Parra G, Bradnam K, Korf I. CEGMA: a pipeline to accurately annotate core genes in eukaryotic genomes. Bioinformatics. 2007;23:1061–7. 16. Parra G, Bradnam K, Korf I. CEGMA: a pipeline to accurately annotate core genes in eukaryotic genomes. Bioinformatics. 2007;23:1061–7. 17. Holt C, Yandell M. MAKER2: an annotation pipeline and genome-data- base management tool for second-generation genome projects. BMC Bioinform. 2011;12:491. 17. Holt C, Yandell M. MAKER2: an annotation pipeline and genome-data- base management tool for second-generation genome projects. BMC Bioinform. 2011;12:491. 9. Ascunce MS, Huguet-Tapia JC, Braun EL, Ortiz-Urquiza A, Keyhani NO, Goss EM. Funding Whole genome sequence of the emerging oomycete patho- gen Pythium insidiosum strain CDC-B5653 isolated from an infected human in the USA. Genomics Data. 2016;7:60–1. 18. Rujirawat T, Patumcharoenpol P, Kittichotirat W, Krajaejun T. Pythium insid- iosum strain ATCC200269, whole genome shotgun sequencing project. GenBank. 2019. https://www.ncbi.nlm.nih.gov/nuccore/BCFN00000000.1. 18. Rujirawat T, Patumcharoenpol P, Kittichotirat W, Krajaejun T. Pythium insid- iosum strain ATCC200269, whole genome shotgun sequencing project. GenBank. 2019. https://www.ncbi.nlm.nih.gov/nuccore/BCFN00000000.1. g 19. Rujirawat T, Patumcharoenpol P, Kittichotirat W, Krajaejun T. Pythium insid- iosum strain Pi19, whole genome shotgun sequencing project. GenBank. 2019. https://www.ncbi.nlm.nih.gov/nuccore/BCFS00000000.1. 19. Rujirawat T, Patumcharoenpol P, Kittichotirat W, Krajaejun T. Pythium insid- iosum strain Pi19, whole genome shotgun sequencing project. GenBank. 2019. https://www.ncbi.nlm.nih.gov/nuccore/BCFS00000000.1. 10. Krajaejun T, Kittichotirat W, Patumcharoenpol P, Rujirawat T, Lohnoo T, Yingyong W. Data on whole genome sequencing of the oomycete Pythium insidiosum strain CBS 101555 from a horse with pythiosis in Brazil. BMC Res Notes. 2018;11:880. 20. Rujirawat T, Patumcharoenpol P, Kittichotirat W, Krajaejun T. Pythium insidiosum strain MCC18, whole genome shotgun sequencing project. GenBank. 2019. https://www.ncbi.nlm.nih.gov/nuccore/BCFT00000000.1. 20. Rujirawat T, Patumcharoenpol P, Kittichotirat W, Krajaejun T. Pythium insidiosum strain MCC18, whole genome shotgun sequencing project. GenBank. 2019. https://www.ncbi.nlm.nih.gov/nuccore/BCFT00000000.1. 11. Rujirawat T, Patumcharoenpol P, Lohnoo T, Yingyong W, Kumsang Y, Payattikul P, et al. Probing the phylogenomics and putative pathogenic- ity genes of Pythium insidiosum by oomycete genome analyses. Sci Rep. 2018;8:4135. p g 21. Rujirawat T, Patumcharoenpol P, Kittichotirat W, Krajaejun T. Pythium insidiosum strain SIMI4763, whole genome shotgun sequencing project. GenBank. 2019. https://www.ncbi.nlm.nih.gov/nuccore/BCFU00000000.1. p g 21. Rujirawat T, Patumcharoenpol P, Kittichotirat W, Krajaejun T. Pythium insidiosum strain SIMI4763, whole genome shotgun sequencing project. GenBank. 2019. https://www.ncbi.nlm.nih.gov/nuccore/BCFU00000000.1. 12. Krajaejun T, Kittichotirat W, Patumcharoenpol P, Rujirawat T, Lohnoo T, Yingyong W. Draft genome sequence of the oomycete Pythium destruens strain ATCC 64221 from a horse with pythiosis in Australia. BMC Res Notes. 2020;13:329. 12. Krajaejun T, Kittichotirat W, Patumcharoenpol P, Rujirawat T, Lohnoo T, Yingyong W. Draft genome sequence of the oomycete Pythium destruens strain ATCC 64221 from a horse with pythiosis in Australia. BMC Res Notes. 2020;13:329. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. 13. Lohnoo T, Jongruja N, Rujirawat T, Yingyon W, Lerksuthirat T, Nampoon U, et al. Efficiency comparison of three methods for extracting genomic DNA of the pathogenic oomycete Pythium insidiosum. 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https://openalex.org/W2172339454	https://escholarship.umassmed.edu/cgi/viewcontent.cgi?article=1013&context=thompson	English	null	Modulation of calcium-induced cell death in human neural stem cells by the novel peptidylarginine deiminase–AIF pathway	Biochimica et biophysica acta. Molecular cell research	2,014	cc-by	10,622	a r t i c l e i n f o Article history: Received 13 December 2013 Received in revised form 19 February 2014 Accepted 24 February 2014 Available online 5 March 2014 Keywords: Apoptosis inducing factor (AIF) Cell death Citrullination–deimination Human neural stem cell Peptidylarginine deiminase (PAD, PADI) Vimentin PADs (peptidylarginine deiminases) are calcium-dependent enzymes that change protein-bound arginine to cit- rulline (citrullination/deimination) affecting protein conformation and function. PAD up-regulation following chick spinal cord injury has been linked to extensive tissue damage and loss of regenerative capability. Having found that human neural stem cells (hNSCs) expressed PAD2 and PAD3, we studied PAD function in these cells and investigated PAD3 as a potential target for neuroprotection by mimicking calcium-induced secondary injury responses. We show that PAD3, rather than PAD2 is a modulator of cell growth/death and that PAD activity is not associated with caspase-3-dependent cell death, but is required for AIF (apoptosis inducing factor)-mediated ap- optosis. PAD inhibition prevents association of PAD3 with AIF and AIF cleavage required for its translocation to the nucleus. Finally, PAD inhibition also hinders calcium-induced cytoskeleton disassembly and association of PAD3 with vimentin, that we show to be associated also with AIF; together this suggests that PAD-dependent cytoskel- eton disassembly may play a role in AIF translocation to the nucleus. This is the ﬁrst study highlighting a role of PAD activity in balancing hNSC survival/death, identifying PAD3 as an important upstream regulator of calcium- induced apoptosis, which could be targeted to reduce neural loss, and shedding light on the mechanisms involved. © 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/). Inhibition of citrullination can reduce disease onset or severity in mouse models of multiple sclerosis, rheumatoid arthritis and ulcerative colitis [23–25]. Signiﬁcantly, the PAD inhibitor Cl-amidine reduces neu- ral damage in a chick spinal cord injury model and in a neonatal mouse hypoxia model [6,7,26]. In the chick, PAD3 appears to be the main PAD involved in secondary injury response, that includes increased intracel- lular Ca2+, leading to apoptosis and consequent neural tissue loss. Dis- ruption of a known PAD target, vimentin, reduces viability of HEL cells, an effect inhibited by Ca2+ chelators [27,28]. Ca2+-dependent cell death is not executed by caspase 3, and translocation to the nucleus of the mitochondrial protein, apoptosis inducing factor (AIF), mediates apoptosis in injured brains [29,30]. a r t i c l e i n f o AIF down-regulation appears to be neuroprotective; hence effective targeting of this pathway could be valuable in pathologies involving Ca2+ homeostasis dysregulation ei- ther during embryonic development or post-natally, such as traumatic injuries, hypoxia–ischemia and damage to neural precursors caused by radiotherapy in young brains [7,30–35]. Modulation of calcium-induced cell death in human neural stem cells by the novel peptidylarginine deiminase–AIF pathway Kin Pong U a, Venkataraman Subramanian b, Antony P. Nicholas c, Paul R. Thompson b, Patrizia Ferretti a,⁎ a Developmental Biology Unit, UCL Institute of Child Health, London WC1N 1EH, UK b Department of Chemistry, TSRI, Scripps Florida, FL 33458, USA a Developmental Biology Unit, UCL Institute of Child Health, London WC1N 1EH, UK b Department of Chemistry, TSRI, Scripps Florida, FL 33458, USA a Developmental Biology Unit, UCL Institute of Child Health, London WC1N 1EH, UK b Department of Chemistry, TSRI, Scripps Florida, FL 33458, USA c Department of Neurology, University of Alabama at Birmingham and Birmingham VA Medical Center, Birmingham, AL 35294, USA Contents lists available at ScienceDirect Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/bbamcr journal homepage: www.elsevier.com/locate/bbamcr ⁎ Corresponding author at: Developmental Biology Unit, UCL Institute of Child Health, 30 Guilford Street, London WC1N 1EH, UK. Tel.: +44 020 7905 2372; fax: +44 020 7905 2953. http://dx.doi.org/10.1016/j.bbamcr.2014.02.018 0167-4889/© 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/). E-mail address: p.ferretti@ucl.ac.uk (P. Ferretti). Modulation of calcium-induced cell death in human neural stem cells by the novel peptidylarginine deiminase-AIF pathway. Item Type Journal Article Authors U, Kin Pong; Subramanian, Venkataraman; Nicholas, Antony P.; Thompson, Paul R; Ferretti, Patrizia Citation Biochim Biophys Acta. 2014 Jun;1843(6):1162-71. doi: 10.1016/ j.bbamcr.2014.02.018. Epub 2014 Mar 5. <a href="http:// dx.doi.org/10.1016/j.bbamcr.2014.02.018">Link to article on publisher's site</a> DOI 10.1016/j.bbamcr.2014.02.018 Rights © 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http:// creativecommons.org/licenses/by/3.0/). Download date 24/10/2024 03:57:42 Item License http://creativecommons.org/licenses/by/3.0/ Link to Item http://hdl.handle.net/20.500.14038/50002 Biochimica et Biophysica Acta 1843 (2014) 1162–1171 ⁎ Corresponding author at: Developmental Biology Unit, UCL Institute of Child Health, 30 Guilford Street, London WC1N 1EH, UK. Tel.: +44 020 7905 2372; fax: +44 020 7905 2953. E-mail address: p.ferretti@ucl.ac.uk (P. Ferretti). 2.6. PAD activity assay The BAEE (Nα-benzoyl-L-arginine ethyl ester; Sigma) colorimetric assay was used to assess PAD activity in 5 μg (1 μg protein/μl) of protein extract per sample with minor modiﬁcations from previously described protocols [43]. The optical density of the colorimetric reaction detecting citrulline was measured at 490 nm using a microplate reader (Revela- tion v4.21 Dynex Technologies, Inc). The background (reading at time point 0) was subtracted from each sample measurement. In situ hybridization was carried out using digoxigenin-labeled ribo- probes essentially as previously described [38]. In brief, de-waxed sec- tions digested with proteinase K (20 mg/ml) were re-ﬁxed in 4% PFA solution, treated with 0.1 M triethanolamine containing 0.25% acetic an- hydride and hybridized overnight at 65°°C. After high stringency washes, the riboprobes were localized using an alkaline phosphatase- conjugated sheep anti-digoxigenin Fab fragment (Roche) and detected by incubation in nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP, Roche). 2.7. Reverse-transcription-polymerase chain reaction (RT-PCR) and quantitative real-time polymerase chain reaction (qPCR) Total RNA was extracted from tissues (for developmental studies at least three human embryos at each stage of gestation were used) and cells using TRIzol reagent (Invitrogen), according to manufacturer's in- struction; MLTV-Reverse Transcriptase (Promega) was used to prepare cDNA [44]. In reverse transcription-polymerase chain reaction experi- ments, all cDNAs were ampliﬁed for 35 cycles except for the house- keeping gene GAPDH (glyceraldehyde-3-phosphate dehydrogenase; 30 cycles) using the following conditions: 5 min 95 °C, 30 s 95 °C, 30 s 52 °C–68 °C (depending on primers), and 5 min 72 °C and 4 °C. PCR products were resolved on 1.5% agarose gel. Primer sequences for RT- PCR and qPCR and ampliﬁcation conditions used are shown in Supple- mentary Table 1. 2.1. Cell lines and treatments Human tissues were supplied by Human Developmental Biology Re- sources under ethical approval. Human neural stem cell lines (hNSCs) from either the brain or the spinal cord of embryos between Carnegie stage (CS) 18 (gestation age 37–42 days) and CS22 (gestational age 54–56 days) were established and grown as previously described [36]. HEK293T (Human Embryonic Kidney 293) cells were grown DMEM– High Glucose–GlutaMAX (Gibco) supplemented with 10% fetal calf serum. The PAD inhibitor Cl-amidine [37] was dissolved in phosphate buffer saline (PBS) and used at different concentrations (0.1–500 μM ﬁnal concentration). Thapsigargin (Sigma) dissolved in ethanol was used at different concentrations (1–25 μM ﬁnal concentration). Cells were treated with Cl-amidine 15 min before the addition of thapsigargin. In some experiments the inhibitory effect of Cl-amidine treatment 15 min after the addition of thapsigargin was also assessed. Immunoprecipitation was carried out using the Millipore 17-500 Catch and Release Immunoprecipitation kit according to the manufacturer's instructions (Upstate). Three hundred to seven hun- dred microgram of hNSC proteins were immunoprecipitated with the anti-PAD3 antibody (5 μg), the anti-AIF antibody (2 μg), or the F95 antibody (10 μg) and the immunoprecipitated fractions ana- lyzed by Western blotting. 2.5. Assessment of cell growth cell growth and death The methylene blue (MB) assay was used to carry out cell growth analysis in 96 well plates as previously described [42]. Propidium iodide (PI, 2 μg/ml ﬁnal concentration; Sigma) staining was used to assess cell death either in live cultures or in PFA-ﬁxed cells. Nuclei were counter- stained with Hoechst 33258. Staining was visualized either using a ﬂuo- rescent inverted microscope (Olympus 1X71) with a monochrome ORCA R2 digital camera (Hamamatsu) or as for immunocytochemistry. 2.4. Western blot and immunoprecipitation Proteins were extracted from either cell pellets or tissues as previ- ously described [41]. For Western-blot analysis, 20–40 μg of proteins per lane were separated by 10% or 12% SDS-PAGE and electrotransferred to nitrocellulose membranes (GE Healthcare) using a TransBlot-SD (BioRad). The primary antibodies used were the same as for immunocy- tochemistry. The secondary antibodies were: horseradish peroxidase- conjugated anti-rabbit immunoglobulin IgG (Dako, Denmark; 1:4000) and anti-mouse immunoglobulin IgM (Serotec, 1: 4000). Bound anti- bodies were visualized using the ECL Western Blotting reagents (Amersham Biosciences). 2.2. In-situ hybridization Spinal cords from human embryos at 46 days of gestation were ﬁxed in 4% PFA (par-formaldehyde), parafﬁn embedded and sectioned (7 μm thickness). PAD2 and PAD3 probes were produced by PCR ampliﬁcation of the regions corresponding to exon 1 and exon 7 of PAD2 and PAD3 cDNAs, and ligation into the pGEMT-Easy vector (Promega). The pGEMT-Easy vectors containing either the PAD2 or PAD3 insert were digested with SpeI or NcoI restriction endonuclease and transcribed using T7 and Sp6 RNA polymerase to produce DIG labeled riboprobes. 2.3. Immunocytochemistry hNSCs plated on either coverslip or chamber-slides (PAA) were ﬁxed with either 4% PFA or 100% ice cold methanol depending on the antigen to be detected, for 15 and 5 min, respectively. Immunocytochemistry was carried out at room temperature essentially as previously described [39]. The primary antibodies used were rabbit anti-PAD3 (Covalab), rab- bit anti-PAD2 (Covalab), rabbit anti-Cleaved Caspase 3 (Cell Signaling), goat anti-AIF (Santa Cruz), mouse anti-ß3-tubulin (Promega), mouse anti-vimentin (Dako), and Alexa 568-conjugated anti-Annexin V (Life Technologies). Filamentous actin was detected with Alexa Fluor 488- conjugated phalloidin (0.02 unit/μl; Invitrogen). The secondary anti- bodies were donkey anti-mouse Alexa 488, donkey or goat anti-rabbit Alexa 488 or 568, and donkey anti-goat Alexa 594 (Life Technology). Nuclei were counterstained with Hoechst 33258 (2.5 μg/ml ﬁnal 2.8. PAD3 overexpression and siRNA knockdown 1. Introduction The importance of citrullination/deimination, the hydrolysis of protein-bound arginine to citrulline, in several neural pathologies is be- coming increasingly apparent [1–7]. These include traumatic injury, hypoxia and neurodegenerative diseases, such as Alzheimer's disease and multiple sclerosis, where an increase in Ca2+ levels is considered to play a relevant role in neural tissue loss [8–11]. Citrullination is carried out by a family of calcium-dependent en- zymes, peptidylarginine deiminases (PADs) that have different tissue distributions, often overlapping, and are believed to have distinct sub- strate speciﬁcity [12–17]. PAD activity has been reported in the cyto- plasm, including mitochondrial and microsomal fractions, as well as in the nucleus [18]. Among the known PAD substrates are cytoskeletal proteins and histones [19,20]. PAD2, the ancestral and more widely expressed PAD, is the main PAD in the central nervous system (CNS), though expression of other PADs has been reported in various neural cell types [15,21,22]. Having ﬁrst established that PAD3 is expressed in the developing human nervous system and in human neural stem cells, as in the chick [6], we investigated whether this pathway may be a novel key reg- ulator of human neural cell death/survival focusing on its potential in- volvement in calcium-induced cell damage. We show that whereas PAD inhibition signiﬁcantly increases hNSC growth, raising intracellular K.P. U et al. / Biochimica et Biophysica Acta 1843 (2014) 1162–1171 1163 Ca2+ with thapsigargin increases PAD3 activity and cell death that is greatly reduced by a PAD inhibitor or PAD3 siRNA. We also show that thapsigargin-induced death in hNSCs is dependent on AIF, not caspase 3, and that cleavage of AIF, required for its translocation to the nucleus, is PAD3-dependent. Finally, we show that vimentin becomes associated with PAD3 upon cytoplasmic Ca2+ increase, that disrupts cytoskeleton integrity, and that vimentin is also associated with AIF, suggesting a pos- sible role in AIF stabilization in the mitochondria. Altogether our ﬁnd- ings support a role for human PADs in the regulation of cell death/ survival, identify PAD3 as an upstream regulator of Ca2+-dependent cell death, and shed light on the pathway(s) involved. concentration). Stained cells were visualized and digitally scanned using an Axiophot 2 (Zeiss) with Hamamatsu ORCA-ER digital camera or by confocal laser scanning microscopy using an LSM 710 (Zeiss). Image collection and analysis were performed using Openlab (Perkin Elmer-Improvision) or ImageJ software [40]. 2.8. PAD3 overexpression and siRNA knockdown The pET16b-hPAD3 plasmid [45] was used as a template for PCR am- pliﬁcation of human PAD3 (PADI3) using forward and reverse primers K.P. U et al. / Biochimica et Biophysica Acta 1843 (2014) 1162–1171 1164 expected molecular size in Western blots (Supplementary Fig. S1B) were used to assess PAD2 and PAD3 protein expression. Both PAD2 and PAD3 proteins could be detected by Western blot in the developing human brains and spinal cords at the stages tested, but their levels of ex- pression were more difﬁcult to quantify given the limited amount of human material available (Fig. 1C). that contained BglII and EcoRI restriction sites, respectively, and ligated into EGFP-N2 plasmid using these restriction sites. The PAD3 construct lacking the enzyme active site, ΔPAD3-EGFP, was made by digesting the PAD3-EGFP plasmid sequentially with Eco53kI restriction endonu- clease at 37 °C and SmaI restriction endonuclease at 24 °C. Constructs were sequenced to ensure they contained the correct sequences. HEK293T and hNSC at 60% conﬂuency were transfected with the PAD3-EGFP, ΔPADI3-EGFP or EGFP only plasmid using Lipofectamine LTX (Invitrogen). For siRNA inhibition studies, hNSC cells were transfected with validated human PADI3 siRNA (s28546), or PADI2 siRNA (s223214), or negative control siRNA 2 (Ambion) at a ﬁnal con- centration of 250 nM with Lipofectamine LTX. Knockdown efﬁciency was assessed by RT-qPCR analysis. PAD mRNA expression was then assessed in 4 hNSCs lines we gener- ated from embryonic human brain and spinal cord (Fig. 2A). All hNSC lines expressed PAD2 and PAD3, but were PAD1 and PAD4-negative (Supplementary Fig. S1A). Immunocytochemistry shows the presence of PAD2 and PAD3 proteins in both the nucleus and cytoplasm of hNSCs (Fig. 2B). Although PAD2 is known to be present in the nucleus [46], this is the ﬁrst time that human PAD3 has been shown to localize also to the nucleus. Nuclear localization of PAD2 and PAD3 in hNSCs, to- gether with evidence of Cit-H3 in these cells, and previously reported detection of the chick PAD3 in nuclear fractions by Western blot, strengthen mounting evidence that translocation to the nucleus of these PADs does not require a classical nuclear localization signal, that is found only in PAD4 [18,47,48]. 2.9. Statistical analysis Each experiment was performed at least 3 times; each experimental group was n = 3–6. Statistical signiﬁcance was evaluated by ANOVA and Student's t-test; p b 0.05 was taken to be signiﬁcant. 3.2. PAD inhibition in normal hNSC increases cell growth 3.1. PAD2 and PAD3 isozymes are expressed in the developing human nervous system and in human neural stem cells (hNSCs) We investigated the effect of reducing PAD basal activity on hNSC behavior by monitoring cell growth at different times following treat- ment with the PAD inhibitor, Cl-amidine. A signiﬁcant increase in cell growth was observed in Cl-amidine treated hNSCs at 48 and 96 h, with 100 μM being the most effective concentration, suggesting a role for PAD in the modulation of cell growth (Fig. 2C, Supplementary Fig. S2). Increased hNSC growth could also be induced by PAD3 siRNA, but neither by scrambled nor PAD2 siRNA (Fig. 2D). These results sug- gest a role for PAD3 in the modulation of cell growth. PAD mRNA expression was assessed in the human central nervous system (CNS), brain and spinal cord, at different developmental stages (Fig. 1). Only PAD2 and PAD3 were detected in the developing brain and spinal cord at all developmental stages studied (46, 63 and 70 days of gestation), whereas the developing human liver, which was used as a control, also expressed PAD1 and PAD4 (Supplementary Fig. S1A). An increase in the PAD2 and a decrease in the PAD3 transcript are observed with development, but both are detected in the brains and spinal cords at all stages of gestation studied. Analysis of PAD2 and PAD3 expression by in situ hybridization at 46 days of gestation is consistent with this ﬁnding and shows that PAD3 expression in the developing human spinal cord is particularly high in the germinal zone (Fig. 2B). Antibodies to PAD2 and PAD3, that reacted with proteins of the 3.3. Cell death and PAD3 expression in hNSCs are induced by increasing intracellular Ca2+ 3.3. Cell death and PAD3 expression in hNSCs are induced by increasing intracellular Ca2+ D) Analysis of cell mined by the methylene blue assay after transfection with siRNA against PAD2 (siPAD2) and PAD3 (siPAD3) or scrambled siRNA. A signiﬁcant increase in cell growth as com- ols is observed at 48 h only in cells transfected with siPAD3 (p b 0.05; two-way ANOVA). Error bars indicate SDM; n ≥3. 1165 K.P. U et al. / Biochimica et Biophysica Acta 1843 (2014) 1162–1171 K.P. U et al. / Biochimica et Biophysica Acta 1843 (2014) 1162–1171 Fig. 2. PADs are expressed in human neural stem cells (hNSCs) and PAD3 inhibition increases hNSC proliferation. A) PAD2 and PAD3 transcript detected by RT-qPCR in hNSC derived from embryonic brain and spinal cord (SC). B) Detection of PAD2 and PAD3 by immunocytochemistry (red) in hNSCs: both proteins are detected in cytoplasm and nucleus (counterstained with Hoechst dye). Scale bars: 25 μm. All pictures are at the same magniﬁcation. C) Analysis of cell growth determined by the methylene blue assay after treatment with 100 μM Cl-amidine for 24, 48 or 96 h. Cl-amidine signiﬁcantly increases hNSC proliferation as compared to controls at 48 and 96 h. * = p b 0.05, = p b 0.01 by ANOVA and Student's t-test. D) Analysis of cell growth determined by the methylene blue assay after transfection with siRNA against PAD2 (siPAD2) and PAD3 (siPAD3) or scrambled siRNA. A signiﬁcant increase in cell growth as com- pared to controls is observed at 48 h only in cells transfected with siPAD3 (p b 0.05; two-way ANOVA). Error bars indicate SDM; n ≥3. Fig. 3. Cl-amidine treatment reduces dose-dependent cell death induced by thapsigargin in hNSCs. A) Quantiﬁcation of cell death induced by 5 μM thapsigargin (Thaps) detected by propidium iodide (PI) staining. The number of PI-positive cells increases upon thapsigargin treatment and is reduced both by pre-treatment or post-treatment with 100 μM Cl-amidine (Cl-am); n = 8; error bars = s.d.; = p b 0.01 by ANOVA and Student's t-test. B) Cell survival determined by methylene blue assay after 24 hour treatment with different concentrations of thapsigargin either alone or following 100 μM Cl-amidine treatment (n ≥3; error bars = SDM). Cl-amidine treatment signiﬁ- cantly increases cell survival (p b 0.05; ANOVA). the increase in PAD expression and activity observed in response to in- jury in the chick spinal cord. 3.3. Cell death and PAD3 expression in hNSCs are induced by increasing intracellular Ca2+ We then wished to establish whether increasing cytoplasmic Ca2+ in hNSC with thapsigargin induced cell death in hNSCs, and mimicked Fig. 1. PAD expression in the developing human central nervous system (brain and spinal cord) and in hNSCs. A) Real time RT-PCR analysis of PAD3 and PAD2 in fetal brains (left panel) and spinal cords (right panel) from human embryos at 42, 63 and 70 days of gestation. PAD2 expression increases while PAD3 expression decreases with development. Human liver was used as a positive control for all PADs. Asterisk indicates statistically signiﬁcant differences (p b 0.05). B) PAD2 and PAD3 transcript detected by in situ hybridization in human spinal cord at 46 days of gestation (dg). Scale bars are 200 μm. C) PAD2 and PAD3 protein detected by Western blot in developing human brain (Br) and spinal cord (SC); no dramatic change in PAD protein expression is observed. Fig. 1. PAD expression in the developing human central nervous system (brain and spinal cord) and in hNSCs. A) Real time RT-PCR analysis of PAD3 and PAD2 in fetal brains (left panel) and spinal cords (right panel) from human embryos at 42, 63 and 70 days of gestation. PAD2 expression increases while PAD3 expression decreases with development. Human liver was used as a positive control for all PADs. Asterisk indicates statistically signiﬁcant differences (p b 0.05). B) PAD2 and PAD3 transcript detected by in situ hybridization in human spinal cord at 46 days of gestation (dg). Scale bars are 200 μm. C) PAD2 and PAD3 protein detected by Western blot in developing human brain (Br) and spinal cord (SC); no dramatic change in PAD protein expression is observed. 1165 e expressed in human neural stem cells (hNSCs) and PAD3 inhibition increases hNSC proliferation. A) PAD2 and PAD3 transcript detected by RT-qPCR in hNSC derived from in and spinal cord (SC). B) Detection of PAD2 and PAD3 by immunocytochemistry (red) in hNSCs: both proteins are detected in cytoplasm and nucleus (counterstained with Scale bars: 25 μm. All pictures are at the same magniﬁcation. C) Analysis of cell growth determined by the methylene blue assay after treatment with 100 μM Cl-amidine for Cl-amidine signiﬁcantly increases hNSC proliferation as compared to controls at 48 and 96 h. * = p b 0.05, ** = p b 0.01 by ANOVA and Student's t-test. 3.5. PAD3 is involved in AIF translocation and cytoskeleton disassembly The effect of PAD over-expression in hNSCs was then assessed in hNSC. As for HEK293T cells, PAD3-EGFP reduced survival of thapsigargin-treated cells as compared to cells transfected ΔPAD3- EGFP, EGFP alone, or untransfected (Fig. 6A). Consistent with this ob- servation, a signiﬁcantly higher percentage of PAD3-EGFP-positve cells, versus cells carrying ΔPAD3-EGFP or an empty EGFP vector control, were annexin-V-positive following thapsigargin treatment (Fig. 6B). To further conﬁrm that PAD3 rather than PAD2 is the Fig. 4. Effect of thapsigargin on PAD expression and citrullination in hNSCs. A) RT-qPCR analysis of PAD2 and PAD3 transcripts after thapsigargin treatment: note up-regulation of PAD3, but not PAD2 transcript, in treated cells; * = p b 0.05 by ANOVA and Student's t-test (n ≥3; error bars indicate SDM). B) Western blot analysis of citrullinated proteins detected by F95 monoclonal antibody and of citrullinated histone H3 (Cit-H3) following treatment with either Cl-amidine (100 μM) or thapsigargin (5 μM) alone, or both com- pounds for 24 h. Cl-amidine was added to the culture medium 15 min before thapsigargin treatment. Actin was used as a loading control. Note that PAD activation by thapsigargin results in protein citrullination and this is reduced by the PAD inhibitor, Cl-amidine. Fig. 5. PAD activity is increased in HEK293T cells expressing PAD3-EGFP A) Live images of cells transfected either with EGFP alone or PAD3-EGFP 40 h after transfection with corre- sponding nuclear staining and detection of endogenous PAD3 (ePAD3) and PAD3-EGFP (P3-G) by Western blot in untransfected cells (1), cell transfected with the EGFP plasmid (2) and cells transfected with PAD3-EGFP. All panels are at the same magniﬁcation. Scale bar is 50 μm. B) PAD activity in HEK293T whole cell lysate assessed by the BAEE assay 24 h after transfection. A signiﬁcant increase (p b 0.05; two-way ANOVA) in PAD activity is ob- served in HEK293T cell expressing PAD3-EGFP, but not in HEK293T cells expressing the mutated PAD3-EGFP lacking enzymatic activity (ΔPAD3-EGFP) where activity is as in untransfected (WT) cells. C) Cl-amidine (10 μM) signiﬁcantly (p b 0.05; two-way ANOVA) reduces PAD activity in PAD3-EGFP HEK293T cell lysate in the BAEE assay. D) Cell death determined by propidium iodide (PI) staining 40 h after transfection with PAD3-EGFP (P3); * = p b 0.05, ** = p b 0.01 by Anova and Student's t-test. E) Cell survival determined by methylene blue assay after 24 hour treatment with thapsigargin. 3.5. PAD3 is involved in AIF translocation and cytoskeleton disassembly 3.5. PAD3 is involved in AIF translocation and cytoskeleton disassembly In order to identify possible mechanisms of PAD3-induced cell death, we stained thapsigargin-treated cells for cleaved caspase 3 and AIF. This treatment did not induce caspase 3 activation, whereas staurosporine treatment did. In contrast, both treatments induced AIF translocation from the cytoplasm to the nucleus, that was detectable by 2h, with AIF being largely nuclear at 8h (Fig. 7A, Supplementary Fig. 7). Therefore we tested the hypothesis that PAD3-induced cell Fig. 5. PAD activity is increased in HEK293T cells expressing PAD3-EGFP A) Live images of cells transfected either with EGFP alone or PAD3-EGFP 40 h after transfection with corre- sponding nuclear staining and detection of endogenous PAD3 (ePAD3) and PAD3-EGFP (P3-G) by Western blot in untransfected cells (1), cell transfected with the EGFP plasmid (2) and cells transfected with PAD3-EGFP. All panels are at the same magniﬁcation. Scale bar is 50 μm. B) PAD activity in HEK293T whole cell lysate assessed by the BAEE assay 24 h after transfection. A signiﬁcant increase (p b 0.05; two-way ANOVA) in PAD activity is ob- served in HEK293T cell expressing PAD3-EGFP, but not in HEK293T cells expressing the mutated PAD3-EGFP lacking enzymatic activity (ΔPAD3-EGFP) where activity is as in untransfected (WT) cells. C) Cl-amidine (10 μM) signiﬁcantly (p b 0.05; two-way ANOVA) reduces PAD activity in PAD3-EGFP HEK293T cell lysate in the BAEE assay. D) Cell death determined by propidium iodide (PI) staining 40 h after transfection with PAD3-EGFP (P3); * = p b 0.05, = p b 0.01 by Anova and Student's t-test. E) Cell survival determined by methylene blue assay after 24 hour treatment with thapsigargin. Signiﬁ- cantly (p b 0.05; two-way ANOVA) higher cell death is observed in PAD3-EGFP cells than in HEK293T cells transfected with the EGFP, ΔPAD3-EGFP or WT (labels are as in (B)). Error bars indicate SDM; n ≥3. We then assessed the effect of PAD3 over-expression in control and thapsigargin-treated cells. Cell death, as detected by propidium iodide (PI), was higher in HEK293T cells transfected with PAD3-EGFP than in untransfected controls. Upon thapsigargin treatment a signiﬁcantly higher number of PI-positive cells was observed in PAD3-EGFP as com- pared to thapsigargin-treated untransfected controls (Fig. 5D). At all the concentrations of thapsigargin tested, cell survival was signiﬁcantly lower in PAD3-EGFP cells than in cells carrying ΔPAD3-EGFP or EGFP alone (Fig. 5E). 3.3. Cell death and PAD3 expression in hNSCs are induced by increasing intracellular Ca2+ / Biochimica et Biophysica Acta 1843 (2014) 1162–1171 1166 protein citrullination was reduced by treatment with Cl-amidine (Fig. 4B). Altogether these data suggest that increased PAD activity in thapsigargin-treated hNSCs is due to PAD3 rather than PAD2. protein citrullination was reduced by treatment with Cl-amidine (Fig. 4B). Altogether these data suggest that increased PAD activity in thapsigargin-treated hNSCs is due to PAD3 rather than PAD2. main mediator of thapsigargin-induced death, we assessed the effect of PAD3 and PAD2 siRNAs (250 nM) on thapsigargin-induced cell death. Both siRNAs speciﬁcally reduced the levels of their respective transcripts by at least 50% as compared to scrambled siRNA or con- trols (Supplementary Fig. S6); PAD2 siRNA did not show any effect on PAD3 transcript levels, but some effect of PAD3 siRNA on PAD2 levels was observed, though much smaller than that on PAD3. Treat- ment with PAD3 siRNA rescued thapsigargin-induced cell death, but no such effect was observed with the highly selective PAD2 siRNA, (Fig. 6C). Together these results indicate that PAD3 is the main PAD modulating this response. 3.4. Cell death is increased by overexpressing PAD3 and reduced by PAD3 inhibition To further investigate the relative contribution of the PAD isozymes to thapsigargin-induced cell death, we constructed a PAD3-EGFP plas- mid and a truncated PAD3-EGFP (ΔPAD3-EGFP) lacking the C-terminal active site of PAD3 (Fig. 5 and Supplementary Fig. S5). We initially tested these construct in HEK293T cells. These cells express PAD3 though at lower levels than hNSCs (Supplementary Fig. S1C). Expression of EGFP control vector and PAD3-EGFP in HEK293T cells was detected as early as 6 h after transfection. It increased over 48 h and PAD3-EGFP was clearly detected by the PAD3 antibody by Western blot, consistent with EGFP detection in live cells (Fig. 5A). Afterward the number of PAD3-EGFP-positive cells, unlike ΔPAD3-EGFP-positive cells, decreased quite rapidly. To ensure that the protein produced by the PAD3-EGFP plasmid was functional, we assessed PAD activity by the BAEE activity assay in protein extracts. PAD activity was much higher in PAD3-EGFP than in ΔPAD3-EGFP and EGFP transfected HEK293T cells, consistent with an active PAD3, and this activity was reduced by Cl-amidine (Fig. 5B, C). 3.3. Cell death and PAD3 expression in hNSCs are induced by increasing intracellular Ca2+ The ability of different concentrations of thapsigargin to induce hNSC death at 24 h was assessed by propidium iodide (Fig. 3A and Sup- plementary Fig. S3) and methylene blue assay (Fig. 3B). Reduced hNSC survival was observed with 5 μM (LD50) thapsigargin and survival was further decreased by increasing concentrations of the compound (Fig. 3B). Thapsigargin-induced cell death was signiﬁcantly reduced by the PAD inhibitor, Cl-amidine. PAD inhibition also greatly reduced cyto- skeleton disassembly induced by thapsigargin, as assessed by staining for actin and vimentin (Supplementary Fig. S4). Altogether this suggests that PAD activation plays an important role in cell homeostasis. Fig. 3. Cl-amidine treatment reduces dose-dependent cell death induced by thapsigargin in hNSCs. A) Quantiﬁcation of cell death induced by 5 μM thapsigargin (Thaps) detected by propidium iodide (PI) staining. The number of PI-positive cells increases upon thapsigargin treatment and is reduced both by pre-treatment or post-treatment with 100 μM Cl-amidine (Cl-am); n = 8; error bars = s.d.; = p b 0.01 by ANOVA and Student's t-test. B) Cell survival determined by methylene blue assay after 24 hour treatment with different concentrations of thapsigargin either alone or following 100 μM Cl-amidine treatment (n ≥3; error bars = SDM). Cl-amidine treatment signiﬁ- cantly increases cell survival (p b 0.05; ANOVA). Fig. 3. Cl-amidine treatment reduces dose-dependent cell death induced by thapsigargin in hNSCs. A) Quantiﬁcation of cell death induced by 5 μM thapsigargin (Thaps) detected by propidium iodide (PI) staining. The number of PI-positive cells increases upon thapsigargin treatment and is reduced both by pre-treatment or post-treatment with 100 μM Cl-amidine (Cl-am); n = 8; error bars = s.d.; ** = p b 0.01 by ANOVA and Student's t-test. B) Cell survival determined by methylene blue assay after 24 hour treatment with different concentrations of thapsigargin either alone or following 100 μM Cl-amidine treatment (n ≥3; error bars = SDM). Cl-amidine treatment signiﬁ- cantly increases cell survival (p b 0.05; ANOVA). Analysis of PAD expression in thapsigargin-treated hNSC showed a signiﬁcant increase in PAD3 transcript levels, whereas no change in PAD2 expression was detected (Fig. 4A). Up-regulation of the PAD3 transcript was paralleled by increased protein citrullination, as shown by Western blot using an antibody to protein-bound citrul- line. Consistent with PAD nuclear localization we also observed an increase in citrullinated histone 3 (Cit-H3). Thapsigargin-induced K.P. U et al. 4.1. PAD2 and PAD3 are developmentally regulated in the human CNS death is mediated through the AIF pathway. As shown in Fig. 7B, trans- location of AIF from the mitochondria to the nucleus was inhibited in thapsigargin-treated hNSCs by the PAD inhibitor, Cl-amidine. We showed that at embryonic stages and early fetal stages of human CNS development only PAD2 and PAD3 transcripts are detected. This is consistent with PAD4 localization to the myelin sheath in the human CNS, a structure that has not yet formed at the developmental stages that we were able to examine [12]. The overall levels of PAD2 and PAD3 transcript change in opposite fashion with development, with the former increasing and the latter decreasing. This parallel previous observation in the chick embryo where PAD3 in the germinal zone de- creases with development and is found in a subset of neurones at later developmental stages. We then wished to gain further insight into the mechanisms by which PAD may regulate cell death. We therefore assessed whether AIF was associated with PAD3. Immunoprecipitation with the PAD3 an- tibody showed that AIF co-precipitated with PAD3 only in hNSCs treat- ed with thapsigargin, though PAD3 was immunoprecipitated under all conditions tested (Fig. 7C, D). In addition, association of PAD3 with AIF was largely abolished by Cl-amidine pre-treatment, and so was a lower size AIF band observed in thapsigargin-treated lysates. We further assessed the effect of thapsigargin on AIF cleavage by Western blotting (Fig. 7E). The cleaved form of AIF was indeed present in thapsigargin treated-cells, but was not detected when PAD activity was inhibited by Cl-amidine, indicating that PAD activation is required for the release of AIF from the mitochondria. In order to conﬁrm that as- sociation of PAD3 with AIF resulted in citrullination of this protein, we investigated whether AIF co-immunoprecipated with the F95 antibody to citrullinated proteins, and this was found to be the case (Fig. 7F). Vimentin, a well-known target of PADs, was also immunoprecipated by F95 (Fig. 7F). Expression of PAD3 at early stages of development in the germinal region may seem counterintuitive given the evidence of PAD3 involve- ment in the apoptotic pathway. As the human embryo is not a very trac- table model for functional studies, at this stage we can only speculate on the possible functions of PADs in the developing human CNS. 3.5. PAD3 is involved in AIF translocation and cytoskeleton disassembly Signiﬁ- cantly (p b 0.05; two-way ANOVA) higher cell death is observed in PAD3-EGFP cells than in HEK293T cells transfected with the EGFP, ΔPAD3-EGFP or WT (labels are as in (B)). Error bars indicate SDM; n ≥3. Fig. 4. Effect of thapsigargin on PAD expression and citrullination in hNSCs. A) RT-qPCR analysis of PAD2 and PAD3 transcripts after thapsigargin treatment: note up-regulation of PAD3, but not PAD2 transcript, in treated cells; * = p b 0.05 by ANOVA and Student's t-test (n ≥3; error bars indicate SDM). B) Western blot analysis of citrullinated proteins detected by F95 monoclonal antibody and of citrullinated histone H3 (Cit-H3) following treatment with either Cl-amidine (100 μM) or thapsigargin (5 μM) alone, or both com- pounds for 24 h. Cl-amidine was added to the culture medium 15 min before thapsigargin treatment. Actin was used as a loading control. Note that PAD activation by thapsigargin results in protein citrullination and this is reduced by the PAD inhibitor, Cl-amidine. K.P. U et al. / Biochimica et Biophysica Acta 1843 (2014) 1162–1171 1167 Fig. 6. PAD3 is the main PAD involved in hNSC cell death. Death/survival of hNSCs treated for 24 h with thapsigargin was determined by the methylene blue assay and staining for the apoptosis marker annexin V. A) Cell survival is signiﬁcantly (p b 0.05; two-way ANOVA) reduced in hNSCs carrying PAD3-EGFP as compared to cells transfected with PAD3 lacking the active site (ΔPAD3-EGFP), EGFP alone, or no transfection (WT). B) Example of cells expressing PAD3-EGFP (PAD, green) and Annexin-V (AnV, red) counted for quantiﬁcation (arrows); note the signiﬁcantly higher percentage of PAD3-EGFP/Annexin-V-positive cells; * = p b 0.05 by ANOVA and Student's t-test. C) Cell survival is signiﬁcantly (p b 0.05; two-way ANOVA) increased in hNSC transfected with PAD3 but not PAD2 siRNA. Scale bars are 50 μmin B. Error bars indicate SDM; n ≥3. Fig. 6. PAD3 is the main PAD involved in hNSC cell death. Death/survival of hNSCs treated for 24 h with thapsigargin was determined by the methylene blue assay and staining for the apoptosis marker annexin V. A) Cell survival is signiﬁcantly (p b 0.05; two-way ANOVA) reduced in hNSCs carrying PAD3-EGFP as compared to cells transfected with PAD3 lacking the active site (ΔPAD3-EGFP), EGFP alone, or no transfection (WT). 4.2. PAD3 is the main PAD modulating cell death in hNSCs 4.2. PAD3 is the main PAD modulating cell death in hNSCs Both PAD2 and PAD3 are expressed in hNSCs. However, they are differently regulated at the transcriptional level: whereas the PAD3 transcript is up-regulated by increased intracellular calcium, PAD2 is not affected. This difference in human PAD2 and PAD3 regulation by Ca2+ at the transcriptional level in the human cells parallels that observed in the chick following spinal cord injury, where only the PAD3 isozyme was found to be up-regulated in a microarray screen aimed at identifying gene expression changes in response to injury [6]. Ca2+-induced increase in PAD3 transcript levels in neural cells is also consistent with regulation of epidermal PAD3 by Ca2+ in keratinocytes, where extensive analysis of PAD3 transcriptional reg- ulation has been carried out [50–53]. 4.1. PAD2 and PAD3 are developmentally regulated in the human CNS During de- velopment, dynamic spontaneous Ca2+ transients in the rat ventricular region have been proposed to play a role in neural progenitor prolifera- tion, apical nuclear migration and early differentiation [32,49]. Brief PAD activation in response to Ca2+ oscillations may therefore be required to modify target proteins involved in some of these processes, but not suf- ﬁcient for extensive citrullination leading to cell death. This is consistent with the presence of low levels of citrullinated proteins in untreated chick embryos and with evidence that an increase in intracellular Ca2+ lasting at least 10 min is required for AIF translocation leading to cell death [29]. As we have found that PAD inhibition increases hNSC growth, one could hypothesize that cyclical PAD activation in response to physiological Ca2+ transients may contribute to the regulation of neural progenitor proliferation/differentiation program(s). As cytoskeletal protein integrity was affected by thapsigargin (Supplementary Fig. S4), we also assessed whether vimentin and actin, like AIF, were immunoprecipitated by PAD3 only in thapsigargin- treated cells. This was indeed the case, and this association was inhibited by Cl-amidine pre-treatment, that partly restored cytoskeletal organiza- tion as well as AIF cytoplasmic localization in the thapsigargin-treated cells (Supplementary Fig. S4, Fig. 7A, C). In these experiments, double immunostaining for AIF and either vimentin or actin in hNSCs suggested a possible co-localization of AIF and vimentin particularly in control cells (Fig 8A–B). We therefore assessed whether there was any direct interac- tion of AIF with the cytoskeleton by AIF immunoprecipitation. Vimentin, but not actin, was found to be associated with AIF (Fig 8C); this might re- ﬂect a role of this intermediate ﬁlament in stabilizing AIF in the mitochondria. 3.5. PAD3 is involved in AIF translocation and cytoskeleton disassembly B) Example of cells expressing PAD3-EGFP (PAD, green) and Annexin-V (AnV, red) counted for quantiﬁcation (arrows); note the signiﬁcantly higher percentage of PAD3-EGFP/Annexin-V-positive cells; * = p b 0.05 by ANOVA and Student's t-test. C) Cell survival is signiﬁcantly (p b 0.05; two-way ANOVA) increased in hNSC transfected with PAD3 but not PAD2 siRNA. Scale bars are 50 μmin B. Error bars indicate SDM; n ≥3. 4.1. PAD2 and PAD3 are developmentally regulated in the human CNS 4. Discussion Together, the ﬁndings reported here suggest that ﬁne tuning of PAD activity is important for hNSC homeostasis, and that modulation of PAD3 levels/activity plays a role in balancing their growth and death. This is consistent with PAD3 association with AIF in the presence of cal- cium. It has been proposed that identiﬁcation and targeting of upstream regulators of AIF release would provide a valuable therapeutic approach. Our study suggests that PAD3 is such a regulator. K.P. U et al. / Biochimica et Biophysica Acta 1843 (2014) 1162–1171 1168 Inhibition of PAD3 activity reduces thapsigargin-induced cell death in human cells and this is consistent with the in vivo injury response re- ported in the chick [6]. We have several lines of evidence supporting the hypothesis that PAD3 and not PAD2, which is the other PAD present in hNSCs, mediates Ca2+-induced cell death. Increased cell death is ob- served in cells over-expressing PAD3, and PAD3 siRNA can signiﬁcantly reduce thapsigargin-induced cell death. In contrast, in these experi- ments PAD2 knockdown does not increase cell survival in treated cells, clearly suggesting different functions for these two PADs in hNSCs. 4.3. PAD3 is a key upstream regulator of AIF-dependent/caspase 3-independent cell death Thapsigargin is a Ca2+-ATPase inhibitor that increases cytoplasmic Ca2+ by reducing its up-take into the endoplasmic reticulum stores. As shown here, thapsigargin induces cell death in hNSCs in a caspase 3-independent manner. In contrast, thapsigargin treatment promotes translocation of AIF (apoptosis inducing factor) to the nucleus, a path- way known to be activated by increased intracellular Ca2+. AIF is a mi- tochondrial intermembrane ﬂavoprotein that plays a vital role in mitochondrial function in addition to its role in cell death execution, where, upon translocation from the mitochondria to the nucleus and binding to DNA, induces chromatin condensation and DNA degradation [54,55]. AIF-mediated cell death depends on the cell type and nature of the apoptotic insult, which may determine the mechanisms of AIF re- lease from the mitochondria [56–59]. Signiﬁcantly, AIF has been report- ed to play a role in neural tissue damage following hypoxia ischemia in neonates, and there is some evidence of neural damage reduction in the neonatal hypoxic–ischemic model upon PAD inhibition [7,60]. It has been proposed that the calcium-dependent activation of the cysteine protease calpain is required to cleave AIF, that is then released from the mitochondrial membrane [29,61]. Fig. 7. PAD3 expression in hNSCs modulates thapsigargin-induced cell death via AIF trans- location. A) Effect of staurosporine (Staur) and thapsigargin (Thaps) treatments for 4 h on caspase 3 activation (cl-Casp3) and AIF translocation to the nucleus assessed by immuno- cytochemistry and confocal microscopy. Thapsigargin unlike staurosporine does not acti- vate caspase 3, but both induce AIF translocation. B) Confocal images showing translocation of AIF to the nucleus in thapsigargin-treated cells. PAD inhibition by Cl- amidine (Cl-am) blocks AIF translocation. Scale bars in A and B are 25 μm. C) Immunopre- cipitation of hNSC protein extract with the PAD3 antibody (Ly: full lysate; Ft: ﬂow through; w1: wash 1; w3: wash 3; El: eluate). Note that AIF, vimentin (Vim) and actin are co- immunopreciptated only in thapsigargin-treated cells. D) PAD3 is immunoprecipitated under all conditions tested; Con: control. E) Western blot of protein extracted from control, thapsigargin- or Cl-amidine and thapsigargin-treated hNSC. The cleaved form of AIF (tAIF) is detected in thapsigargin-treated extracts but not in extract from cells pre-treated with Cl-amidine. F) Immunoprecipitation of control and thapsigargin-treated hNSC protein extracts with the anti-citrullinated protein antibofdy, F95: AIF and vimentin are co- immunoprecipitated only in thapsigargin-treated cells. 4. Discussion Confocal images of hNSC stained for actin (detected by phalloidin, green) and vimentin (green) and AIF (red) 3 h after treatment; nuclei are counterstained with Hoechst dye (blue). Double-labeling for actin or vimentin and AIF. Scale bars = 25 μm; all images are at the same magniﬁcation. B) High magniﬁcation images showing possible association of vimentin and AIF and disorganization of the cytoskeleton upon thapsigargin treatment. C) Immunoprecipitation of hNSC protein extract with the AIF antibody (Ly: full lysate; Ft: ﬂow through; w1: wash 1; w3: wash 3; El: eluate). Vimentin but not actin is co-immunoprecipitated by AIF. 1169 K.P. U et al. / Biochimica et Biophysica Acta 1843 (2014) 1162 1171 Fig. 8. Thapsigargin induces cytoskeletal disorganization in hNSCs that is reduced by PAD inhibition and affects AIF–vimentin association. Confocal images of hNSC stained for actin (detected by phalloidin, green) and vimentin (green) and AIF (red) 3 h after treatment; nuclei are counterstained with Hoechst dye (blue). Double-labeling for actin or vimentin and AIF. Scale bars = 25 μm; all images are at the same magniﬁcation. B) High magniﬁcation images showing possible association of vimentin and AIF and disorganization of the cytoskeleton upon thapsigargin treatment. C) Immunoprecipitation of hNSC protein extract with the AIF antibody (Ly: full lysate; Ft: ﬂow through; w1: wash 1; w3: wash 3; El: eluate). Vimentin but not actin is co-immunoprecipitated by AIF. factor) citrullination appears to be required for ATP5 mRNA transport to the mitochondrial surface [1]. previous work in animal injury models, our ﬁndings in a human model identify PAD3 pathway targeting as a novel approach to reducing neural tissue loss in human pathologies where increased intracellular Ca2+ greatly contributes to cell damage, such as traumatic and hypoxic/ ischemic insults to the CNS. Extensive investigation will be required to assess the role of the cy- toskeleton in AIF translocation and the precise role of PAD in this pro- cess and in AIF cleavage. Nonetheless, altogether our results clearly position PAD3 as an early player in the cascade of events leading to Ca2+ dependent/caspase 3-independent cell death in hNSCs. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.bbamcr.2014.02.018. Finally, it has been suggested that AIF-mediated neural cell death is of particular importance in neural tissue and in some tumors [56,58,68]. Acknowledgements This work was supported by grants from the Child Research Appeal Trust (grant 08DB04 to PF), the Biotechnology and Biological Sciences Research Council (PF) and the National Institutes of Health (grant GM079357 to PRT). The human embryonic and fetal material was pro- vided by the Human Developmental Biology Resource (http://hdbr. org) jointly funded by the Medical Research Council (grant G070089) and The Wellcome Trust (grant GR082557). We are grateful to J. Ham, JP Brockes and JC Sowden for reading the manuscript. The authors de- clare no competing ﬁnancial interests. 4. Discussion In- formation on PAD3 localization in adult human CNS is lacking, but PAD3 mRNA has been detected in the human cerebellum [21]. Therefore it will be important to establish whether PAD3 expression determines AIF- mediated cell death restriction to speciﬁc neural cell subpopulations in pathologies involving increase in intracellular Ca2+. Future studies on PAD3 expression in normal and pathological human specimen will be crucial to clarifying the role of this isozyme in neural pathologies. 5. Conclusions We have shown that human PAD3 (PADI3) is a key player in hNSC homeostasis, and particularly in caspase 3 independent cell death in- duced by increased intracellular calcium in hNSCs. This to our knowl- edge is the ﬁrst study that demonstrates that PAD3 is an upstream regulator of Ca2+-induced cell death in human neural cells and sheds some light on the mechanisms involved. Furthermore, it highlights dif- ferences in the role of PAD2 and PAD3 in hNSCs. Together with our [1] D. Ding, M. Enriquez-Algeciras, K.R. Dave, M. Perez-Pinzon, S.K. Bhattacharya, The role of deimination in ATP5b mRNA transport in a transgenic mouse model of mul- tiple sclerosis, EMBO Rep. 13 (2012) 230–236. [2] B. Jang, J.K. Jin, Y.C. Jeon, H.J. Cho, A. Ishigami, K.C. Choi, R.I. Carp, N. Maruyama, Y.S. Kim, E.K. Choi, Involvement of peptidylarginine deiminase-mediated post-translational [2] B. Jang, J.K. Jin, Y.C. Jeon, H.J. Cho, A. Ishigami, K.C. Choi, R.I. Carp, N. Maruyama, Y.S. Kim, E.K. Choi, Involvement of peptidylarginine deiminase-mediated post-translational 4. Discussion We have shown that in hNSCs, PAD activation is required for AIF cleavage consistent with AIF co-precipitating with PAD3 in thapsigargin-treated cells and its inhibi- tion by Cl-amidine. This positions PAD signaling upstream of AIF release. It is conceivable that AIF citrullination is required to expose AIF to calpain as suggested for ﬁlaggrin and NSE and this will require extensive investigation [3,62]. Furthermore, vimentin, a well-known PAD target that we have shown here to become associated with PAD3 when cyto- plasmic Ca2+ is increased, has also been reported to be cleaved by calpain and to play a role in cell death [27]. It is tempting to speculate that citrullination of vimentin may play a role in AIF translocation. Several cytoskeletal proteins are known to be PAD targets, and in the oocyte PAD6 is required for lattice formation and regulation of ribosomal component movements [19,63]. Vimentin is involved in several cellular processes, including mitochondria motility and apoptosis [2,27,64–67]. The role of vimentin association with AIF has yet to be elucidated, but it is conceivable that vimentin destabiliza- tion upon citrullination could affect the mitochondria facilitating AIF re- lease from these organelles and its subsequent translocation to the nucleus. A possible role for PAD3 in regulating translocation of a mitochondrial-associated protein to the nucleus is also interesting in the context of recent work showing a role for citrullination in protein trafﬁcking in the opposite direction, where REF (RNA binding export K.P. U et al. / Biochimica et Biophysica Acta 1843 (2014) 1162–1171 1169 Fig. 8. Thapsigargin induces cytoskeletal disorganization in hNSCs that is reduced by PAD inhibition and affects AIF–vimentin association. Confocal images of hNSC stained for actin (detected by phalloidin, green) and vimentin (green) and AIF (red) 3 h after treatment; nuclei are counterstained with Hoechst dye (blue). Double-labeling for actin or vimentin and AIF. Scale bars = 25 μm; all images are at the same magniﬁcation. B) High magniﬁcation images showing possible association of vimentin and AIF and disorganization of the cytoskeleton upon thapsigargin treatment. 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https://openalex.org/W4254104267	https://www.researchsquare.com/article/rs-77379/v1.pdf?c=1602715734000	English	null	Identification of a Novel Epithelial-Mesenchymal Transition Gene Signature Regulated by KEAP1-NRF2 Pathway in Esophageal Carcinoma	Research Square (Research Square)	2,020	cc-by	7,317	Identification of a Novel Epithelial-Mesenchymal Transition Gene Signature Regulated by KEAP1- NRF2 Pathway in Esophageal Carcinoma Mohamed Elshaer Xiu Jun Wang Department of Pharmacology and Cancer Institute, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310009, PR China Xiuwen Tang (  xiuwentang@zju.edu.cn ) Zhejiang University School of Medicine https://orcid.org/0000-0002-6601-1234 Mohamed Elshaer Department of Biochemistry and Department of Thoracic Surgery of the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, PR China Identification of a Novel Epithelial-Mesenchymal Transition Gene Signature Regulated by KEAP1- NRF2 Pathway in Esophageal Carcinoma Mohamed Elshaer Department of Biochemistry and Department of Thoracic Surgery of the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, PR China Ahmed Hammad Department of Biochemistry and Department of Thoracic Surgery of the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, PR China Xiu Jun Wang Department of Pharmacology and Cancer Institute, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310009, PR China Xiuwen Tang (  xiuwentang@zju.edu.cn ) Zhejiang University School of Medicine https://orcid.org/0000-0002-6601-1234 Research Keywords: TCGA, KEAP1, NRF2, epithelial-mesenchymal transition, esophageal cancer, biomarker Posted Date: October 6th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-77379/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Identification of a Novel Epithelial-Mesenchymal Transition Gene Signature Regulated by KEAP1- NRF2 Pathway in Esophageal Carcinoma Mohamed Elshaer Department of Biochemistry and Department of Thoracic Surgery of the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, PR China Ahmed Hammad Department of Biochemistry and Department of Thoracic Surgery of the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, PR China Xiu Jun Wang Department of Pharmacology and Cancer Institute, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310009, PR China Xiuwen Tang (  xiuwentang@zju.edu.cn ) Zhejiang University School of Medicine https://orcid.org/0000-0002-6601-1234 Research Keywords: TCGA, KEAP1, NRF2, epithelial-mesenchymal transition, esophageal cancer, biomarker Posted Date: October 6th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-77379/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Ahmed Hammad Department of Biochemistry and Department of Thoracic Surgery of the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, PR China Xiu Jun Wang Background KEAP1-NRF2 pathway alterations were identified in many cancers including, esophageal cancer (ESCA). Identifying biomarkers that are associated with mutations in this pathway will aid in defining this cancer subset; and hence in supporting precision and personalized medicine. Results We identified 11 epithelial-mesenchymal transition (EMT) genes regulated by the KEAP1-NRF2 pathway in ESCA patients. Five of the 11 genes showed significant over-expression in KEAP1-NRF2-mutated ESCA cell lines. In addition, the over-expression of these five genes was significantly associated with poor survival in 3 independent ESCA datasets, including the TCGA-ESCA dataset. Conclusion Altogether, we identified a novel EMT 5-gene signature regulated by the KEAP1-NRF2 axis and this signature is strongly associated with metastasis and drug resistance in ESCA. These 5-genes are potential biomarkers and therapeutic targets for ESCA patients in whom the KEAP1-NRF2 pathway is altered. Methods In this study, 182 tumor samples from the Cancer Genome Atlas (TCGA)-ESCA RNA-Seq V2 level 3 data were segregated into two groups KEAP1-NRF2-mutated (22) and wild-type (160).The two groups were subjected to differential gene expression analysis, and we performed Gene Set Enrichment Analysis (GSEA) to determine all significantly affected biological pathways. Then, the enriched gene set was integrated with the differentially expressed genes (DEGs) to identify a gene signature regulated by the KEAP1-NRF2 pathway in ESCA. Furthermore, we validated the gene signature using mRNA expression data of ESCA cell lines provided by the Cancer Cell Line Encyclopedia (CCLE). The identified signature was tested in 3 independent ESCA datasets to assess its prognostic value. Research Keywords: TCGA, KEAP1, NRF2, epithelial-mesenchymal transition, esophageal cancer, biomarker Posted Date: October 6th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-77379/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. Read Full License Page 1/19 Page 1/19 Background Esophageal cancer is the sixth most common cause of cancer death and the eighth in incidence worldwide. In fact, it accounts for 4% of cancer diagnoses and for 6% of cancer deaths. The prognosis for esophageal carcinoma is poor, with a 5-year survival rate of 19% and only 0.9% for advanced esophageal carcinoma (1). To maintain oxidative homeostasis, cancer cells increase the transcription of antioxidant genes by acquiring either stabilizing mutations in NFE2L2 (encoding NRF2, the master transcriptional regulator of the cellular antioxidant program) or by selecting for inactivating mutations in its negative regulator, Page 2/19 Page 2/19 KEAP1 (2). KEAP1 is a substrate receptor of the Cul3-RING ubiquitin ligase (CRL3) that, under physiological conditions, constitutively binds and targets NRF2 for degradation. In response to oxidative stress, the KEAP1-NRF2 binding is inhibited and, consequently, NRF2 is stabilized (3). KEAP1 (2). KEAP1 is a substrate receptor of the Cul3-RING ubiquitin ligase (CRL3) that, under physiological conditions, constitutively binds and targets NRF2 for degradation. In response to oxidative stress, the KEAP1-NRF2 binding is inhibited and, consequently, NRF2 is stabilized (3). The TCGA network has revolutionized the cancer research by enriching the cancer research community with a huge amount of cancer-related data. This revolution has enabled researchers to identify cancer driver genes, cancer dependency, prognostic biomarkers and therapeutic targets. In addition, it has enabled researchers to segregate one cancer type into subgroups in order to assist personalized and precision medicine field. Identification of genes and biological processes that are regulated by the KEAP1- NRF2 pathway in different cancers may provide an effective approach for therapy of subset of cancers that harbor KEAP1-NRF2 pathway alterations. Moreover, these gene signatures can be used to predict survival of patients. In previous studies, we identified gene signatures that are regulated by the KEAP1-NRF2 pathway in lung adenocarcinoma (LUAD) and head and neck squamous cancer (HNSC) (4-6). In the current study, we used the genomics, and transcriptomics data of the ESCA cohort from TCGA to identify a gene signature regulated by the KEAP1-NRF2 pathway in ESCA. Overall database selection Overall database selection TCGA RNA-Seq gene expression version 2 level 3 data (Illumina HiSeq platform) for 182 ESCA tissues were downloaded from the Broad GDAC (Global Data Assembly Centers) Firehose website (http://gdac.broadinstitute.org/). All the mutation data used in the present study was obtained from CVCDAP (https://omics.bjcancer.org/cvcdap/home.do) and UCSC Xena Browser (https://xenabrowser.net/) (7, 8). Twelve percent (22 out of 185) of the TCGA-ESCA patients were found to harbor mutations in either KEAP1, NRF2 or both. Then, we segregated these patients into two groups, one had 22 patients with mutations in either KEAP1, NRF2 or both (mutated group) while the other had 160 patients with neither KEAP1 nor NRF2 mutations (wild-type group). Survival analysis For the identification of prognostic biomarkers, Kaplan-Meier curves were generated by using the web- based patients survival analysis tool SurvExpress (10). Log rank test P<0.05 was used as the cutoff for significance. The method of analysis has been discussed in a previous study (5). Identification of NRF2 binding sites by in silico analysis To identify the NRF2 binding sites within the promoter regions of the putative NRF2 regulated genes, we used the transcription factor-binding site finding tool ConTra V3 (9) with stringency core = 0.95 and similarity matrix = 0.85. The search was limited to the -1 kb upstream the transcription start site (TSS). RNA-Seq data analysis TCGA RNA-Seq gene expression version 2 level 3 data (Illumina Hiseq platform) for 182 ESCA tissues were subjected to differential gene expression analysis. Briefly, level 3 transcriptomic data was normalized by the FPKM (Fragments per Kilobase of transcript per Million mapped reads) method. All gene expression values were log-transformed to approximate the data to a normal distribution. In addition, genes with zero values in more than 25% of the patients were excluded. The differentially expressed genes (DEGs) were identified by applying the two-tailed t-test assuming unequal variance. Then, P values were adjusted using the FDR method. DEGs with FDR 0.05 were considered significant. Gene Set Enrichment Analysis (GSEA) Page 3/19 Page 3/19 GSEA (https://omics.bjcancer.org/) was performed to determine all significantly affected biological pathways. GSEA is a computational method that determines whether a defined set of genes shows statistically significant, concordant differences between two biological states. The primary result of the GSEA is the enrichment score (ES), which reflects the degree to which a gene set is overrepresented at the top or bottom of a ranked list of genes. The gene list metric was built using ratio between classes (biological states). A positive ES indicates gene set enrichment at the top of the ranked list (up-regulated); a negative ES indicates gene set enrichment at the bottom of the ranked list (down-regulated). GSEA (https://omics.bjcancer.org/) was performed to determine all significantly affected biological pathways. GSEA is a computational method that determines whether a defined set of genes shows statistically significant, concordant differences between two biological states. The primary result of the GSEA is the enrichment score (ES), which reflects the degree to which a gene set is overrepresented at the top or bottom of a ranked list of genes. The gene list metric was built using ratio between classes (biological states). A positive ES indicates gene set enrichment at the top of the ranked list (up-regulated); a negative ES indicates gene set enrichment at the bottom of the ranked list (down-regulated). Cancer Cell Line Encyclopedia (CCLE) data analysis RNA-seq gene expression data for 1019 cell lines were downloaded from CCLE https://portals.broadinstitute.org/ccle/data. We identified ESCA cell lines that harbor mutations in either KEAP1, NRF2 or both using the COSMIC data base https://cancer.sanger.ac.uk/cell_lines. Then, we divided the ESCA cell lines into two groups mutated and wild-type. Differential gene expression analysis was performed as described above. Overview of the ESCA mutational landscape Overview of the ESCA mutational landscape The lack of prognostic biormarkers for the ESCA subgroup with KEAP1-NRF2 mutations motivated us to focus on identifying prognostic biormakers that is regulated by KEAP1-NRF2 in ESCA. First, we investigated the different driver mutations in the TCGA-ESCA patients and we found that TP53 was the most frequently mutated gene in ESCA as it was found mutated in 86% of patients (Fig.2B). 42% of ESCA patients harbored TTN mutations which made TTN in the second place. MUC16, SYNE1, CSMD3, FLAG, DNAH5, HMCN1, LRP1B, PCLO and RYR2 were mutated in 23%, 20%, 20%, 18%, 16%, 16%, 16%, 15% and 12% of ESCA patients, respectively. KEAP1-NRF2 was found mutated in 12% of ESCA patients. The TCGA-ESCA cohort included 182 patient samples. We segregated the cohort into two groups: KEAP1- NRF2-mutated (22 patients) and wild-type (160 patients). In order to ensure that the differences between the two groups were due to KEAP1-NRF2 mutations, we first investigated the driver mutations in the KEAP1-NRF2-mutated group. We found that TP53 was mutated in 95% of patients in this group, NRF2 was found mutated in 85% patients, TTN, KMTD2, MUC13, KEAP1, PATCH, and SACS were found mutated in 41%, 32%, 27%, 23%, 23% and 23%, respectively (Fig.3A). Then, we performed differential gene mutation analysis between the two groups to investigate the percentages of mutations of these driver genes in the two groups. Only NRF2 and KEAP1 weren't mutated in wild-type group while the other driver genes were found mutated in both the KEAP1-NRF2-mutated and wild-type groups, with similar percentages (Fig.3B). Therefore, none of these driver genes can be considered as variables that contribute to differences between the two groups. In order to better understand the mutational landscape of KEAP1-NRF2 in ESCA, we used the USCS Xena browser to examine the types of mutations and their positions in the domain structure of KEAP1 and NRF2 proteins. As noted earlier, we found that 2.19% of TCGA-ESCA patient samples had KEAP1 mutations while NRF2 was mutated in 9.34% and both were mutated in 0.54%. All the detected KEAP1 mutations were missense mutations while 77.8% (14/18) of NRF2 mutation were missense mutations, 11.1% were intron (2/18), 5.6% (1/18) were in-frame-deletions and 5.6% (1/18) were in-frame-insertions. Overview of the mutational landscape of the KEAP1-NRF2 pathway in cancer Overview of the mutational landscape of the KEAP1-NRF2 pathway in cancer First, we investigated the mutational landscape of the KEAP1-NRF2 pathway in cancer by analyzing 11,079 TCGA samples from 33 different cancer types using the CVCDAP database. As shown in (Fig.2A), we found that the KEAP1-NRF2 pathway was altered in many cancers, however it was altered with a percentage that was higher than 10% in only five cancer types. Lung Squamous Cell Cancer (LUSC) was the cancer type that harbor the highest percentage of KEAP1-NRF2 pathway alterations with mutations in 24.2% (KEAP1, 9.92%; NRF2, 13.69%; both, 0.59%) of samples. LUAD came in the second place with KEAP1-NRF2 pathway mutations in 20.82% (KEAP1, 17.68%; NRF2, 2.97%; both, 0.17%) of LUAD patients. In addition, the KEAP1-NRF2 pathway was altered in 12.07% ESCA patients (KEAP1, 2.19%; NRF2, 9.34%; both, 0.54%), in 11.7% of uterine corpus endometrial carcinoma (UCEC) patients (KEAP1, 3.19%; NRF2, 7.04%; both, 1.48%) and in 10.1% of HNSC (KEAP1, 3.95%; NRF2, 5.49%; both, 0.659%). Page 4/19 Page 4/19 Overview of the ESCA mutational landscape KEAP1 consists of 605 amino-acids, and 3 main domains with two mutations were detected in the BTB (broad-complex, tramtrack, and bric-a-brac) domain, three in the IVR (intervening region), and one in the Kelch domain, which is essential for the binding of NRF2 (Fig.3C). In the case of NRF2 structure, the majority of mutations (17) occurred in the crucial KEAP1-binding domain Neh2, and only one was found in the Neh1 domain. Identification of genes regulated by the KEAP1-NRF2 pathway in ESCA Epithelial-mesenchymal transition is regulated by the KEAP1-NRF2 pathway in ESCA The expression signatures of the hallmark gene sets, each containing 50 specific gene sets, were derived by concentrating multiple gene sets from the Molecular Signatures Database to represent well-defined biological statuses or courses. GSEA was performed to determine whether the identified gene sets showed statistically notable differences between the KEAP1-NRF2-mutated and their wild-type counterparts groups (Additional file 2: Table S2). Interestingly, four gene sets were up-regulated in the KEAP1-NRF2-mutated ESCA, namely, estrogen response late, hypoxia, reactive oxygen species pathway and EMT, the 4 gene sets were greatly enriched, with FDR < 0.05 (Fig.5). The gene set with the lowest FDR, namely, EMT (FDR = 0.001), which contained 194 genes was selected for further analysis. In order to specifically identify EMT genes that is associated with the KEAP1-NRF2 pathway in ESCA, we integrated the DEGs between KEAP1-NRF2-mutated and wild-type ESCA patient samples (log FC> \|1.5\|, FDR < 0.05) with the set of 194 EMT-enriched genes (Fig.6A) using Venny 2.1 web-based tool (http://bioinfogp.cnb.csic.es/tools/venny/index.html). Intriguingly, we found 11 common genes: SPP1, PTHLH, WNT5A, COL11A1, COL7A1, GPC1, SNAI2, ADAM12, FBN2, PFN2, and IGFBP3 (Fig.6B). (http://bioinfogp.cnb.csic.es/tools/venny/index.html). Intriguingly, we found 11 common genes: SPP1, PTHLH, WNT5A, COL11A1, COL7A1, GPC1, SNAI2, ADAM12, FBN2, PFN2, and IGFBP3 (Fig.6B). Epithelial-mesenchymal transition genes were validated using ESCA cell line Epithelial-mesenchymal transition genes were validated using ESCA cell lines In order to validate these 11 EMT-related genes as potential NRF2 targets, we used CCLE to download RNA-seq mRNA expression data of 1019 cell lines. Then, using the COSMIC data base we identified human ESCA cell lines that harbors NRF2 and/or KEAP1 mutations. We selected three cell lines (TE6, TE11 and KYSE180) as the KEAP1-NRF2-mutated group. In addition, we selected another three human ESCA cell lines that have neither NRF2 nor KEAP1 mutations (TE5, TE9 and KYSE150) as the wild-type group. Then, we carried out differential gene expression analysis between the two groups. As shown in GSTM3, AKR1C1 and TXNRD1 (well-known NRF2 targets) showed significant up-regulation in the mutated group compared to the wild-type counterpart, which ensures KEAP1-NRF2 pathway alteration in the group (Fig.6C). Furthermore, we investigated the expression of these 11 EMT-related genes between the two groups. Interestingly, five of the 11 genes (SPP1, WNT5A, PTHLH, PNF2, and GPC1) were significantly up-regulated in the mutated ESCA cell lines (Fig.6D). This finding suggests that these five genes are potential NRF2 targets. dentification of genes regulated by the KEAP1-NRF2 pathway in ESCA In order to identify genes that are regulated by the KEAP1-NRF2 pathway in ESCA, we subjected the TCGA-ESCA data set (22 KEAP1-NRF2-mutated versus 160 wild-type tumor samples) to differential gene expression analysis. We identified 896 DEGs with log FC >\|1\| (p < 0.05 with FDR adjustment) (Fig.4A). Of these DEGs, 403 were up-regulated and 493 were down-regulated (Additional file1: Table S1). Since the ultimate effect of changes in the KEAP1-NRF2 pathway is increased activity of NRF2; and hence the over- expression of its target genes, it was not surprising that several bonafide NRF2 target genes were among Page 5/19 Page 5/19 the up-regulated genes including, AKR1C1, AKR1C2, AKR1B10, GSTM2, UGT1A6, AKR1C3, G6PD, GCLC, GCLM, GSTM3, GPX2, ABCC1, OSGIN1, SRXN1, and TXNRD1. The gene expression profiles of 22 KEAP1- NRF2-mutated and 160 wild-type ESCA patient samples were visualized on a heatmap produced by unsupervised hierarchical clustering, and major differences between the gene expression patterns enabled cluster analysis to discriminate between sample types. Significant differences or trends between KEAP1-NRF2-mutated and wild-type ESCA patient samples were detectable for DEGs with log FC> \|1\| (Fig.4B). CES1, AKR1C1, ADH7, ALDH3A, and CYP4F11 were the top five up-regulated genes in KEAP1- NRF2-mutated ESCA patient samples (Fig.4C), while PIGR, MUC13, TASPAN8, LGALS4, and OLFM4 were the top five down-regulated genes (Fig.4D). Epithelial-mesenchymal transition is regulated by the KEAP1-NRF2 pathway in ESCA Epithelial-mesenchymal transition is regulated by the KEAP1-NRF2 pathway in ESCA For further evidence, we investigated the presence of the putative and known antioxidant responsive elements (AREs), the NRF2 binding site, (Fig.6E) in the promoter region of Page 6/19 Page 6/19 these five genes by using ConTra V3 web tool. We performed insilico analysis within the –1 kb upstream the transcription start site (TSS) of the 5 genes. Interestingly, we identified highly conserved NRF2 binding sites (AREs) in the promoter regions of human PTHLH (positions: -71 and -916), WNT5A (position: -434), SPP1 (positions: -545,-605 and -870), PFN2 (positions:-737 and -864) and GPC1 (position: -458) (Fig.6F). Evaluation of prognostic power of EMT gene-signature regulated by the KEAP1-NRF2 pathway in ESCA In order to evaluate the prognostic power of these 5 genes (SPP1, WNT5A, PTHLH, PFN2, and GPC1) as an EMT-derived signature for KEAP1-NRF2 pathway alterations in ESCA, we first analyzed overall survival in the TCGA-ESCA cohort using the SurvExpress database. A total of 184 patient samples were divided into high-risk (n = 127) and low-risk groups (n = 57) based on their expression patterns (Fig.7A). The separation of risk groups was optimized using the ‘maximize risk group’ option provided in the SurvExpress database. The survival probability estimates in the two risk groups were visualized as Kaplan-Meier plots. Strikingly, overall survival analysis revealed that the patients in the high-risk group had poorer survival (HR = 1.67 (CI: 1.01-2.78); Log-Rank p = 0.04443) than the low-risk group (Figure.7B). Moreover, the Rao Giddings (GSE11595) cohort (34 ESCA patient samples) showed that the expression of SPP1, WNT5A, PTHLH, PFN2, and GPC1 in the high-risk group (n=17) was associated with poorer survival (HR = 6.84 (CI: 2.36 - 19.8); Log-Rank p = 3.072×e-5) than the low-risk group (n=17) (Fig.7C). In addition, we analyzed the overall survival in the Peters C.Fitzgerald (GSE19417) cohort available in the SurvExpress database. After optimized risk group separation, a total of 70 ESCA patient samples were divided into high-risk (n = 27) and low-risk groups (n = 43) based on their expression patterns (Fig.7D). The survival probability estimates in the two risk groups were represented as Kaplan-Meier plots. Similarly, overall survival analysis showed that the patients in the high-risk group had poorer survival (HR = 2.25 (CI: 1.34 - 3.79); Log-Rank p = 0.001659) than the low-risk group. As shown in Fig.5, The 5 genes were significantly over-expressed in the high risk patients compared to the low risk group. Epithelial-mesenchymal transition is regulated by the KEAP1-NRF2 pathway in ESCA Moreover, the expression of SPP1, WNT5A, PTHLH, PFN2, and GPC1 successfully discriminated the survival of the ESCA high risk group from that of the low risk group in three ESCA cohorts (288 patients). These findings indicated that over-expression of the 5 genes is associated with a poor prognosis of ESCA and presents SPP1, WNT5A, PTHLH, PFN2, and GPC1 as an EMT signature based on changes in the KEAP1-NRF2 pathway in ESCA. In order to evaluate the prognostic power of these 5 genes (SPP1, WNT5A, PTHLH, PFN2, and GPC1) as an EMT-derived signature for KEAP1-NRF2 pathway alterations in ESCA, we first analyzed overall survival in the TCGA-ESCA cohort using the SurvExpress database. A total of 184 patient samples were divided into high-risk (n = 127) and low-risk groups (n = 57) based on their expression patterns (Fig.7A). The separation of risk groups was optimized using the ‘maximize risk group’ option provided in the SurvExpress database. The survival probability estimates in the two risk groups were visualized as Kaplan-Meier plots. Strikingly, overall survival analysis revealed that the patients in the high-risk group had poorer survival (HR = 1.67 (CI: 1.01-2.78); Log-Rank p = 0.04443) than the low-risk group (Figure.7B). Moreover, the Rao Giddings (GSE11595) cohort (34 ESCA patient samples) showed that the expression of SPP1, WNT5A, PTHLH, PFN2, and GPC1 in the high-risk group (n=17) was associated with poorer survival (HR = 6.84 (CI: 2.36 - 19.8); Log-Rank p = 3.072×e-5) than the low-risk group (n=17) (Fig.7C). In addition, we analyzed the overall survival in the Peters C.Fitzgerald (GSE19417) cohort available in the SurvExpress database. After optimized risk group separation, a total of 70 ESCA patient samples were divided into high-risk (n = 27) and low-risk groups (n = 43) based on their expression patterns (Fig.7D). The survival probability estimates in the two risk groups were represented as Kaplan-Meier plots. Si il l ll i l l i h d h h i i h hi h i k h d i l (HR Discussion The key role of KEAP1-NRF2 pathway alterations in developing drug- and radio-resistance in ESCA is well- established. The lack of specific biomarkers for KEAP1-NRF2 pathway alterations in ESCA motivated us to analyze TCGA-ESCA data in order to identify different biological processes that are regulated by KEAP1-NRF2 in ESCA; hence identifying new therapeutic targets and biomarkers to predict prognosis of ESCA patients. We found that the KEAP1-NRF2 pathway was altered in 12% of TCGA-ESCA patients. Swada et al., performed whole-exome sequence analysis of tumor and nontumor esophageal tissues collected from 144 patients with ESCA (11). They found that NRF2 was mutated in 16.7% of the patients and it was one of the most frequently mutated gene in their cohort while KEAP1 was mutated in almost Page 7/19 Page 7/19 5% of patients. We performed GSEA to detect gene sets that showed statistically-notable differences between KEAP1- NRF2-mutated samples and their wild-type counterparts groups. Interestingly, 4 gene sets, namely, estrogen response late, hypoxia, reactive oxygen species pathway and epithelial mesenchymal transition, were greatly enriched, with FDR < 0.05. Since the ultimate effect of KEAP1-NRF2 pathway alterations is the stabilization of NRF2, the master transcriptional regulator of the cells antioxidant program(12), it was not surprising to find the reactive oxygen species pathway among the top pathways that show statistically notable differences between the KEAP1- NRF2-mutated and wild-type groups. Surprisingly, pathways such as EMT and hypoxia were greatly enriched. As EMT was more enriched, we selected EMT pathway to identify a NRF2-KEAP1 pathway signature in ESCA. EMT, an evolutionarily conserved developmental program, has been implicated in carcinogenesis and confers metastatic properties upon cancer cells by enhancing mobility, invasion, and resistance to apoptotic stimuli. Furthermore, EMT-derived tumor cells acquire stem cell properties and exhibit marked therapeutic resistance (13). Given these attributes, the complex biological process of the EMT has been heralded as a key hallmark of carcinogenesis, and targeting EMT pathways constitutes an attractive strategy for cancer treatment (14). Recently, it has been suggested that NRF2 contributes to malignant transformation of pancreatic duct epithelium through distinct EMT-related mechanisms accounting for an invasive phenotype (15). Furthermore, the expression of NRF2 is correlated with the lymph node metastasis of esophageal squamous cell carcinoma and blockage of NRF2 enhances the expression of E-cadherin , the well-known marker of epithelial cell polarity (16). Discussion In order to identify an EMT-derived signature for the KEAP1-NRF2 pathway in ESCA, we integrated the EMT gene list obtained from GSEA with the DEGs between the mutated and wild-type groups (log FC> \|1.5\|, FDR < 0.05). Interestingly, 11 genes were identified (SPP1, PTHLH, WNT5A, COL11A1, COL7A1, GPC1, SNAI2, ADAM12, FBN2, PFN2, and IGFBP3). Then, we validated these 11 genes by subjecting the KEAP1-NRF2-muatated and wild-type ESAC cell lines to differential gene expression analysis. Intriguingly, only 5 of the 11 genes showed significant up- regulation between the two groups (SPP1, WNT5A, PTHLH, PFN2, and GPC1). Further, we evaluated the prognostic power of these 5 genes using three ESCA cohorts (288 patients), and we found that the over- expression of these five genes were associated with a poor prognosis in ESCA. In agreement with our analysis, SPP1 and EMT have been shown by bioinformatics analysis to have a close association in colorectal cancer (17). Moreover, Osteopontin, encoded by SPP1 promotes the EMT in hepatocellular carcinoma through regulating vimentin (18), and high SPP1 expression in hepatocellular carcinoma is associated with poor survival outcome (19). Additionally, up-regulation of WNT5A has been suggested to promote EMT and metastasis in pancreatic cancer models, which involves activation of β- catenin-dependent canonical Wnt signaling(20). Furthermore, it has been illustrated that WNT5A promotes EMT and metastasis in non-small-cell lung cancer (NSCLC), and high WNT5A expression is associated with poor prognosis in NSCLC patients (21). In addition, parathyroid hormone related-protein, encoded by PTHLH has been found to promote EMT in prostate and pancreatic cancers (22, 23). It has been indicated that PTHLH is a poor prognosis marker and promotes cell growth of HNSC. Besides, it has been illustrated that inhibition of PFN2 hinders cell invasion and migration, as well as induces an EMT phenotype, including increased expression of epithelial marker E-cadherin, decreased mesenchymal Page 8/19 Page 8/19 marker Vimentin, Snail, Slug and ZEB1, and morphological changes in ESCA cells in vitro (24). High PFN2 expression independently predicts poor overall survival in primary HNSC and ESCA (24,25). Moreover, Over-expression of GPC1 activates EMT which then increases invasion and migration in colorectal cancer and ESCA (26-28). Additionally, it has been suggested that GPC1 plays an important role in regulating TGF-β-mediated EMT and stemness, and could be a potential future therapeutic target to prevent progression of gastric cancer (29). Discussion It has been pointed out that GPC1 is over-expressed and implies a poor prognosis in several cancers including, ESCA, uterine cervical cancer, pancreatic cancer, and methothelioma (30-34). Altogether, the above evidence suggests an oncogenic role of the 5-gene signature in many cancers. Abbreviations Page 9/19 KEAP1 Kelch‑like ECH‑associated protein 1 NRF2 Nuclear factor erythroid 2‑related 2 LUAD Lung adenocarcinoma NSCLC Non-small-cell lung cancer TCGA The cancer genome atlas ARE Antioxidant response element ESCA ESE Esophageal cancer EMT Epithelial-mesenchymal transition CCLE Cancer cell line encyclopedia DEGs Differentially expressed genes PTHLH Parathyroid hormone like hormone FDR False discovery rate GSEA Gene set enrichment analysis Declarations Ethics approval and consent to participate Page 9/19 KEAP1 Kelch‑like ECH‑associated protein 1 NRF2 Nuclear factor erythroid 2‑related 2 LUAD Lung adenocarcinoma NSCLC Non-small-cell lung cancer TCGA The cancer genome atlas ARE Antioxidant response element ESCA ESE Esophageal cancer EMT Epithelial-mesenchymal transition CCLE Cancer cell line encyclopedia DEGs Differentially expressed genes PTHLH Parathyroid hormone like hormone FDR False discovery rate GSEA Gene set enrichment analysis Declarations Ethics approval and consent to participate KEAP1 Kelch‑like ECH‑associated protein 1 NRF2 Nuclear factor erythroid 2‑related 2 LUAD Lung adenocarcinoma NSCLC Non-small-cell lung cancer TCGA The cancer genome atlas ARE Antioxidant response element ESCA ESE Esophageal cancer EMT Epithelial-mesenchymal transition CCLE Cancer cell line encyclopedia DEGs Differentially expressed genes PTHLH Parathyroid hormone like hormone FDR False discovery rate GSEA Gene set enrichment analysis Conclusion Our study identified an EMT-derived gene signature regulated by the KEAP1-NRF2 pathway that is strongly associated with tumorigenesis, metastasis, and drug resistance in ESCA. This 5-gene signature provides potential biomarkers and therapeutic targets for ESCA patients in whom KEAP1-NRF2 pathway is activated. Acknowledgements Not applicable Contributions ME conducted the study. XT supervised the study. AH and XJW assisted in data interpretation. ME and XT wrote the manuscript with assistance from all authors. All authors read and approved the manuscript. Ethics approval and consent to participate Not applicable Funding This work was supported by the National Natural Science Foundation of China (31571476, 31971188, and 31370772). Corresponding author Correspondenceto Xiuwen Tang Availability of data and materials The datasets used in this study are publicly available as noted in the text. Consent for publication Not applicable Competing interests The authors declare that they have no competing interests. References 1. Testa U, Castelli G, Pelosi E. Esophageal Cancer: Genomic and Molecular Characterization, Stem Cell Compartment and Clonal Evolution. 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Figure 1 Schematic diagram showing the analysis overflow that was followed in this study. Page 13/19 Figure 2 General mutational landscape A) Bar chart representing TCGA-pan cancer analysis of KEAP1-NRF2 pathway alterations in diffrent cancers. B) Landscape of genetic alterations across the 185 ESCA samples. The samples are sorted by mutation rates (top bars), while genes are sorted by the proportion of altered samples (left bars). Figure 2 General mutational landscape A) Bar chart representing TCGA-pan cancer analysis of KEAP1-NRF2 pathway alterations in diffrent cancers. B) Landscape of genetic alterations across the 185 ESCA samples. The samples are sorted by mutation rates (top bars), while genes are sorted by the proportion of altered samples (left bars). Page 14/19 Page 14/19 Figure 3 Mutational landscape of ESCA samples with KEAP1 and/or NRF2 mutations. A) Landscape of genetic alterations across the 22 ESCA samples with KEAP1 and/or NRF2 mutations. The samples are sorted by mutation rates (top bars), while genes are sorted by the proportion of altered samples (left bars). B) Differential mutational analysis between KEAP1-NRF2-mutated and wild-type ESCA samples. C) Lollipop plot showing the locations of mutations in the functional domains of NRF2 protein. D) Lollipop plot showing the locations of mutations in the functional domains of KEAP1 protein. The lollipops show the locations of the mutations as identified by whole-exon sequencing. Figure 3 Figure 3 Mutational landscape of ESCA samples with KEAP1 and/or NRF2 mutations. A) Landscape of genetic alterations across the 22 ESCA samples with KEAP1 and/or NRF2 mutations. The samples are sorted by mutation rates (top bars), while genes are sorted by the proportion of altered samples (left bars). B) Differential mutational analysis between KEAP1-NRF2-mutated and wild-type ESCA samples. C) Lollipop plot showing the locations of mutations in the functional domains of NRF2 protein. D) Lollipop plot showing the locations of mutations in the functional domains of KEAP1 protein. The lollipops show the locations of the mutations as identified by whole-exon sequencing. Mutational landscape of ESCA samples with KEAP1 and/or NRF2 mutations. A) Landscape of genetic alterations across the 22 ESCA samples with KEAP1 and/or NRF2 mutations. The samples are sorted by mutation rates (top bars), while genes are sorted by the proportion of altered samples (left bars). B) Differential mutational analysis between KEAP1-NRF2-mutated and wild-type ESCA samples. C) Lollipop plot showing the locations of mutations in the functional domains of NRF2 protein. D) Lollipop plot showing the locations of mutations in the functional domains of KEAP1 protein. The lollipops show the locations of the mutations as identified by whole-exon sequencing. Page 15/19 Figure 4 Differential gene expression analysis. A) Volcano plot showing the distribution of DEGs between KEAP NRF2-mutated and wild-type ESCA patient samples based on significance and fold change. B) Heatma showing the top DEGs between KEAP1- NRF2 mutated and wild-type ESCA patient samples with Log FC>\|1\| and FDR <0.05. C) Box plots showing the top 5 overexpressed genes between KEAP1- NRF2- mutated and wild-type ESCA patient samples. D) Box plots showing the top 5 down-regulated genes between KEAP1-NRF2-mutated and wild-type ESCA patient samples. Center lines show the medians; bo limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times th interquartile range from the 25th and 75th percentiles; outliers are represented by dots. Figure 4 Differential gene expression analysis. A) Volcano plot showing the distribution of DEGs between KEAP1- NRF2-mutated and wild-type ESCA patient samples based on significance and fold change. B) Heatmap showing the top DEGs between KEAP1- NRF2 mutated and wild-type ESCA patient samples with Log FC>\|1\| and FDR <0.05. C) Box plots showing the top 5 overexpressed genes between KEAP1- NRF2- mutated and wild-type ESCA patient samples. D) Box plots showing the top 5 down-regulated genes between KEAP1-NRF2-mutated and wild-type ESCA patient samples. Center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles; outliers are represented by dots. Page 16/19 Figure 5 Gene set enrichment analysis. Enrichment plots of four gene sets that are importantly differentiated between KEAP1-NRF2-mutated and wild-type ESCA samples. Figure 5 Gene set enrichment analysis. Enrichment plots of four gene sets that are importantly differentiated between KEAP1-NRF2-mutated and wild-type ESCA samples. Page 17/19 Page 17/19 Figure 6 Figure 6 Identification of the EMT signature of ESCA patients with altered KEAP1-NRF2 pathway. A) Venny diagram showing the overlapping between the EMT-enriched gene set and DEGs between KEAP1-NRF2- mutated and wild-type ESCA patient samples. B) Box plots showing the differential expression of 11 overlapped EMT genes between KEAP1- NRF2-mutated and wild-type ESCA patient samples. C) Bar chart showing differential mRNA expression of some well-known NRF2 targets between ESCA cell lines with KEAP1 and/or NRF2 mutations and their wild-type counterparts. D) Bar chart showing 5 EMT genes that were significantly differentially expressed between ESCA cell lines with KEAP1 and/or NRF2 mutations and their wild-type counterparts. (p < 0.05, * p < 0.01, *** p < 0.001). E) The NRF2 binding motif as provided by JASPER. (F) Schematic representation of the locations of insilico-predicted NRF2 binding sites (AREs) in the promoter regions of the human SPP1, WNT5A, PTHLH, PFN2, and GPC1genes. Figure 7 Five-gene signature predicts poor survival in three independent cohorts. (A) Box plots showing the expression differences of the 5-gene signature in low (green) and high (red) risk groups of TCGA–ESCA patients (y-axis, gene expression value of each gene).(B) Kaplan-Meier survival plots showing that high expression of the 5-gene signature is associated with poor survival in TCGA–ESCA patients. (C) The Rao Giddings (GSE11595) cohort. D) The Peters C.Fitzgerald (GSE19417) cohort. Red, high-risk group; green, low-risk group; top right corner inset, numbers of high- and low-risk samples (x-axis, time; y-axis, overall survival probability; HR, hazard ratio; CI, confidence interval). Figure 7 Figure 8 Schematic diagram summarizing the findings of this study. Figure 7 Five-gene signature predicts poor survival in three independent cohorts. (A) Box plots showing the expression differences of the 5-gene signature in low (green) and high (red) risk groups of TCGA–ESCA patients (y-axis, gene expression value of each gene).(B) Kaplan-Meier survival plots showing that high expression of the 5-gene signature is associated with poor survival in TCGA–ESCA patients. (C) The Rao Giddings (GSE11595) cohort. D) The Peters C.Fitzgerald (GSE19417) cohort. Red, high-risk group; green, low-risk group; top right corner inset, numbers of high- and low-risk samples (x-axis, time; y-axis, overall survival probability; HR, hazard ratio; CI, confidence interval). Page 18/19 Figure 8 Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Additionalfile2.xlsx Additionalfile1.xlsx Page 19/19
https://openalex.org/W4310794358	https://www.researchsquare.com/article/rs-2272045/latest.pdf	English	null	Are we really targeting and stimulating DLPFC by placing tES electrodes over F3/F4?	Research Square (Research Square)	2,022	cc-by	12,795	Hamed Ekhtiari (  hekhtiari@laureateinstitute.org ) Hamed Ekhtiari (  hekhtiari@laureateinstitute.org ) Are we really targeting and stimulating DLPFC by placing tES electrodes over F3/F4? Hamed Ekhtiari (  hekhtiari@laureateinstitute.org ) Laureate Institute for Brain Research https://orcid.org/0000-0001-6902-8798 Ghazaleh Soleimani Rayus Kuplicki Laureate Institute for Brain Research https://orcid.org/0000-0003-2954-6421 Jazmin Camchong Alexander Opitz Martin Paulus Laureate Institute for Brain Research, Tulsa, USA https://orcid.org/0000-0002-0825-3606 Kelvin Lim University of Minnesota https://orcid.org/0000-0002-2390-7268 Article Keywords: Transcranial electrical stimulation, transcranial direct current stimulation (tDCS), bipolar montage, dorsolateral prefrontal cortex (DLPFC), frontopolar cortex, computational head models, electric field (EF) Posted Date: December 6th, 2022 DOI: https://doi.org/10.21203/rs.3.rs-2272045/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Are we really targeting and stimulating DLPFC by placing tES electrodes over F3/F4? Hamed Ekhtiari (  hekhtiari@laureateinstitute.org ) Laureate Institute for Brain Research https://orcid.org/0000-0001-6902-8798 Ghazaleh Soleimani Rayus Kuplicki Laureate Institute for Brain Research https://orcid.org/0000-0003-2954-6421 Jazmin Camchong Alexander Opitz Martin Paulus Laureate Institute for Brain Research, Tulsa, USA https://orcid.org/0000-0002-0825-3606 Kelvin Lim University of Minnesota https://orcid.org/0000-0002-2390-7268 Article Keywords: Transcranial electrical stimulation, transcranial direct current stimulation (tDCS), bipolar montage, dorsolateral prefrontal cortex (DLPFC), frontopolar cortex, computational head models, electric field (EF) Posted Date: December 6th, 2022 DOI: https://doi.org/10.21203/rs.3.rs-2272045/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Are we really targeting and stimulating DLPFC by placing tES electrodes over F3/F4? Hamed Ekhtiari (  hekhtiari@laureateinstitute.org ) Laureate Institute for Brain Research https://orcid.org/0000-0001-6902-8798 Ghazaleh Soleimani Rayus Kuplicki Laureate Institute for Brain Research https://orcid.org/0000-0003-2954-6421 Jazmin Camchong Alexander Opitz Martin Paulus Laureate Institute for Brain Research, Tulsa, USA https://orcid.org/0000-0002-0825-3606 Kelvin Lim University of Minnesota https://orcid.org/0000-0002-2390-7268 Article Keywords: Transcranial electrical stimulation, transcranial direct current stimulation (tDCS), bipolar montage, dorsolateral prefrontal cortex (DLPFC), frontopolar cortex, computational head models, elect field (EF) Posted Date: December 6th, 2022 DOI: https://doi.org/10.21203/rs.3.rs-2272045/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Highlights In most frequently used DLPFC tES montages, EF peaks were not located under the electrodes The EF peak in DLPFC montages is located in the medial frontopolar area An independent clinical population showed slight between-group differences There is a large interindividual variation in both location and strength of the EF peak In most frequently used DLPFC tES montages, EF peaks were not located under the electrodes The EF peak in DLPFC montages is located in the medial frontopolar area In most frequently used DLPFC tES montages, EF peaks were not located under the electrodes In most frequently used DLPFC tES montages, EF peaks were not located The EF peak in DLPFC montages is located in the medial frontopolar are An independent clinical population showed slight between-group differences An independent clinical population showed slight between-group differen There is a large interindividual variation in both location and strength of the EF peak There is a large interindividual variation in both location and strength of DOI: https://doi.org/10.21203/rs.3.rs-2272045/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 Page 1/23 Page 1/23 Page 1/23 Abstract Background: Most transcranial electrical stimulation (tES) clinical trials place target electrodes over DLPFC based on the assumption that it would mainly stimulate the underlying brain region. Here, we assessed delivered electric fields (EF) using a symmetric and asymmetric DLPFC stimulation montage to identify additional prefrontal regions that are inadvertently targeted beyond DLPFC. Methods: Head models were generated from the human connectome project database's T1+T2-weighted MRIs of 80 healthy adults. Two common DLPFC montages (symmetric: F4/F3, asymmetric: F4/Fp1 with 5×7cm electrodes, 2mA intensity) were simulated. Averaged EF was extracted from (1) the center of the target electrode (F4), and (2) the top 1% of voxels that showed the strongest EF in individualized EF maps. Inter-individual variabilities were quantified with standard deviation (SD) of EF peak location and value. These steps were replicated with 66 participants with methamphetamine use disorder (MUD) as an independent clinical population. Results: In the healthy adults, EFs in the frontopolar area were significantly higher than EF “under” the target electrode in both symmetric (peak:0.41±0.06, F4:0.22±0.04) and asymmetric (peak:0.38±0.04, F4:0.2±0.04) montages (Heges’g>0.7). Group-level location for EF peaks in MNI space was located in the medial-frontopolar cortex, such that individualized EF peaks were placed in a cube with a volume of symmetric/asymmetric: 29cm3/46cm3. Similar results (with slight between-group differences) were found for MUDs that highlighted the role of the medial frontopolar cortex in both healthy and clinical populations. Conclusions: We highlighted that in common DLPFC tES montages, DLPFC was not maximally targeted and the frontopolar area was the area that received the highest EFs. Considering inter-individual and inter- groups variability, we specifically recommended that the frontopolar role should be considered as a potential mechanism underlying the clinical efficacy of DLPFC stimulation. 1. Introduction The dorsolateral prefrontal cortex (DLPFC) is important for many neurocognitive processes [1, 2]. Impairment of neuroplasticity in the DLPFC has also been reported in different neuropsychiatric diseases such as depression [3], schizophrenia [4], and substance use disorders (SUDs) [5]. In this regard, DLPFC is Page 2/23 Page 2/23 commonly used in non-invasive brain stimulation methods including transcranial magnetic (TMS) or electrical (tES) stimulation as a promising intervention target [6, 7]. Recent advancements in TMS coil placement (e.g., neuro-navigation systems [8] or fMRI-informed target selection [9]) provide optimized and individualized approaches for DLPFC targeting [10]. However, in tES studies, the 10–20 standard system is widely used for targeting and placing the electrodes over DLPFC [11]. This system proposes an appropriate method to link the external scalp locations to the underlying cortex which is typically used in electroencephalography (EEG) electrode placement [12]. With respect to the previous meta-analyses, in tES protocols, stimulating electrodes are typically placed over the F4/F3 locations to modulate the right/left DLPFC [13]. It has been shown that F4/F3 coordinates over the scalp correspond with DLPFC locations in the cortex [14]. Most clinical trials place stimulation electrodes over the scalp based on the assumption that the target electrode (e.g., over F4/F3) will mainly stimulate the underlying brain region (e.g., DLPFC) as the main target. However, using gyri-precise head models, it has been shown that tES electrodes produce diffuse current flow and maximal electric fields (EFs), corresponding to areas of maximal stimulation, can fall outside the target electrodes rather than underneath them as commonly assumed [15–17]. Despite the diffusivity of the current [18], the effects of placing electrodes over F4/F3 have been attributed to modulation of the right/left DLPFC even in recent publications [19, 20] and less attention has been paid to the possibility that a strong EF in other regions can contribute to the observed clinical/behavioral outcomes of DLPFC-targeting protocols. EF modeling and delineation of different cortical regions (e.g., by defining spheres around a specific coordinate or using atlas-based parcellation), may help provide a useful quantitative comparison of the electrical dose delivered to different targeted or non-targeted brain areas. Based on the analysis of both normal and tangential components of the EFs in modulating prefrontal cortex, Csifcsak et al reported that strong stimulation can be found not only in DLPFC but also in the medial prefrontal cortex (MPFC) in bipolar montages which are commonly used for depression [21]. 1. Introduction Their study supported previous modeling studies that have suggested the medial prefrontal cortex as a new target for depression in TMS studies [22, 23]. In another example of atlas- based parcellation of the computational head models, four different electrode arrangements were simulated for a single standard subject (ICBME152) and 22 cortical areas were extracted per hemisphere [22]. That study demonstrated that maximum values of tangential and normal components of EFs were located in the orbital and frontopolar cortices when using an F3-Fp2 montage [24]. Previous works pointed out the importance of inter-individual variability of the EF and submaximal stimulation dose at the intended target [25], suggesting the need for a systematic analysis of dosage in targeted and non-targeted regions. EF variation has been linked to substantial differences in morphological features (e.g., skull thickness, cortex morphology, and gyrification [26, 27]). These differences may explain why cortical EF is not restricted to a region “under” the electrode; understanding the distribution of the peak current density and its cortical location in a large sample may help to design more optimal protocols and better interpret stimulation outcomes at both individual and group-levels. Page 3/23 Page 3/23 In this context, the primary goal of this study is to evaluate the site/strength of the maximum tES-induced EFs in the prefrontal cortex at a group-level while considering two of the most used electrode montages for DLPFC stimulation (anode/cathode over F4/F3 and F4/Fp1) with 2 mA stimulation intensity in both clinical and healthy populations. This work highlights the importance of considering tES-induced EFs on brain regions that are not located underneath the stimulating electrodes and support a model-driven approach for tES target determination. 2.1. Participants Unprocessed T1 and T2-weighted structural MRIs from 80 (44 female) randomly selected healthy adults (age (year) between 31–35 (n = 32, 15 female), between 26–30 (n = 34, 21 female), between 22–25 (n = 14, 8 female) (the exact age of participants are not reported in the database) were obtained from the freely available Human Connectome Project database (HCP, http://www.humanconnectomeproject.org/data/ ) with deidentified anatomical scans. Structural MRI data in the HCP data archive were collected with a Siemens MAGNETOM 3T scanner with 32 channel head coil and the following parameters for T1-weighted MRIs: TR/TE = 2400/2.14, flip angle = 8, the field of view = 224 x 224 x 180 mm3 and voxel size = 0.7 mm3, and T2-weighted MRIs: TR/TE = 3200/565. More details on imaging parameters and data acquisition can be found in the HCP database, Appendix I: Structural Session Scan Protocol. Unprocessed T1 and T2-weighted structural MRIs from 80 (44 female) randomly selected healthy adults (age (year) between 31–35 (n = 32, 15 female), between 26–30 (n = 34, 21 female), between 22–25 (n = 14, 8 female) (the exact age of participants are not reported in the database) were obtained from the freely available Human Connectome Project database (HCP, http://www.humanconnectomeproject.org/data/ ) with deidentified anatomical scans. Structural MRI data in the HCP data archive were collected with a Siemens MAGNETOM 3T scanner with 32 channel head coil and the following parameters for T1-weighted MRIs: TR/TE = 2400/2.14, flip angle = 8, the field of view = 224 x 224 x 180 mm3 and voxel size = 0.7 mm3, and T2-weighted MRIs: TR/TE = 3200/565. More details on imaging parameters and data acquisition can be found in the HCP database, Appendix I: 2.2. Creation of head models and EF simulations Individualized computational head models were generated from a combination of high-resolution T1 and T2-weighted MRIs. Head models were created for all 80 subjects using the standard SimNIBS 3.2 pipeline [28]. Briefly, T1 and T2-weighted images were segmented into six tissue types, including white matter (WM), gray matter (GM), cerebrospinal fluid (CSF), skull, scalp, and eyeballs, using an automated tissue segmentation approach in SPM 12 based on the “headreco” function. Segmentation results were evaluated carefully slice by slice to ensure proper tissue classification. Tetrahedral volume meshes with about 3×106 elements were created for each head model and visualized using Gmsh and MATLAB. Three subjects were removed due to problems with the head generation process. Gmsh failed to mesh one or more surfaces in 3 attempts for two subjects. In another subject, mesh generation failed in decoupling between white matter and gray matter ventricles. All results were reported based on EFs for 77 healthy subjects. Page 4/23 Two of the most commonly used conventional tES montages for targeting DLPFC were simulated for all subjects by placing 5×7 cm electrodes with 1 mm thickness over (1) F4/F3 (symmetric montage) and (2) F4/Fp1 (asymmetric montage). A standard EEG cap (EEG10-10-UI-Jurak-2007) was used for placing electrodes over the scalp. In the symmetric montage, centers of the target electrodes were located over F4 and F3 locations with the long axis of the pads pointing towards the vertex of the head. In the asymmetric montage, the forehead electrode (Fp1) was positioned over the left eyebrow with the long axis of the pad parallel to the horizontal plane (as shown in Fig. 1, panel b). Here we focused on 35 cm2 rectangular electrodes since this size/shape was by far the most commonly used in previously published studies (e.g., in our updated systematic review in the field of addiction medicine, among a total of 67 published studies, only 4 studies used circular while 38 studies used two 5x7 electrode [29]). However, the orientation of non-circular electrodes can affect the strength and direction (normal component) of the EFs over the cortex. Therefore, the locations and orientations of the electrodes were precisely checked in Gmsh to guarantee consistency after automatic placement using a modified MATLAB code in SimNIBS. Through the SimNIBS pipeline, the finite element method (FEM) was used to simulate EFs for head meshes. 2.2. Creation of head models and EF simulations Linear and isotropic electrical conductivities were assumed based on previously established conductivity values for each tissue type [17]: white matter = 0.126, gray matter = 0.275, cerebrospinal fluid = 1.654, skull = 0.010, skin = 0.465, and eyeballs = 0.5 all in Siemens per meter (S/m). The absolute and normal components of the EFs were calculated for each montage and each individual. Surface-based head models were then transformed from individualized space to the “fsaverage” standard space (http://surfer.nmr.mgh.harvard.edu) to make the results comparable across the population in terms of brain coordinates. Although normalization to the standard space can affect EF distribution patterns, our previous study showed no statistically significant difference between the group-level results obtained from standard space and subject space [25]. 2.3. Data analysis methods Averaged EF strength was extracted from the main regions of interest (RO (please see 2.4) for each individual. Peak EF was defined as the 99th perc and both location and strength of the peak were extracted at the individua subject variations were quantified using the relative standard deviation (S locations. Numerical statistical analyses were performed using the R pack normality showed our database is normally distributed, t-tests were used t differences between EFs in two separate brain regions. All data reported a were reported based on P values and Hedges’ g factors to explain the leve sizes. Averaged EF strength was extracted from the main regions of interest (ROIs) in the prefrontal cortex (please see 2.4) for each individual. Peak EF was defined as the 99th percentile of the EF over the cortex and both location and strength of the peak were extracted at the individual and group levels. Between- subject variations were quantified using the relative standard deviation (SD) of the EFs strength and locations. Numerical statistical analyses were performed using the R package. As the Shapiro-Wilk test of normality showed our database is normally distributed, t-tests were used to examine significant differences between EFs in two separate brain regions. All data reported as mean ± SD. Statistical results were reported based on P values and Hedges’ g factors to explain the level of significance and effect sizes. p 2.5. Replication of the results with a sample of the clinical population Based on our updated systematic review on transcranial electrical stimulation trials (tES) in substance use disorders (SUDs), by May 2022, 66 out of 75 tES electrode montages (88%) in SUDs with successful outcomes in modulating drug craving and drug consumption used bipolar DLPFC stimulation with symmetric or asymmetric montages [29]. However, less attention has been paid to the distribution of tES- induced EFs in the field of addiction medicine. In order to determine the replicability of the results in a clinical population, all previous steps were repeated for 66 participants (age (year) between 18–60, all male) with methamphetamine use disorders (MUDs) as a representative example of a clinical population (more details about participants, T1 and T2 MRIs, and data collection protocols can be found in our previously published paper [25]). 2.4. Defining regions of interest (ROI) In order to compare EF strength over DLPFC and around peaks, two individualized 10 mm spheres were defined: (1) around the center of the target electrode (F4) location, and (2) around the 99th percentile of the EFs for each individual. Spheres were combined with the MNI mask to ensure analyses did not include EFs from non-brain or white matter voxels. The Brainnetome atlas (which has a fine-grained parcellation of the cortex as a multimodal parcellation atlas based on structural MRI, diffusion tensor imaging, and resting-state fMRI connectivity) was used for the parcellation of the computational head models [30]. Inspired by [31], ROIs were placed in 9 main subregions in the Brainnetome atlas; Superior frontal gyrus: A9l, lateral area [13, 48, 40], A9m, medial area [6, 38, 35], A10m: medial area [8, 58, 13]. Middle frontal gyrus: A9/46d, dorsal area [30, 37, 36], A9/46v, Page 5/23 ventral area [42, 44, 14], A46 [28, 55, 17], A10l, lateral area [25, 61,–4], and Orbital gyrus: A11l, lateral area [23, 36,–18], A11m, medial area [6, 57,–16]. Averaged EF strength was calculated for each of the 9 prefrontal subregions. Differences between subregions were calculated using ANOVA and post hoc pairwise t-test. 2.5. Replication of the results with a sample of the clinical population ventral area [42, 44, 14], A46 [28, 55, 17], A10l, lateral area [25, 61,–4], and Orbital gyrus: A11l, lateral area [23, 36,–18], A11m, medial area [6, 57,–16]. Averaged EF strength was calculated for each of the 9 prefrontal subregions. Differences between subregions were calculated using ANOVA and post hoc 3.1. Comparing EFs in the center of target electrodes and hot spots Individual and group-level analysis of personalized head models showed that the 99th percentile of the EFs (peaks) is not located underneath the stimulating electrodes (F4; [40.5, 41.4, 27] coordinate over the cortex in MNI space). Averaged locations for the peak EFs across the population were [-1.21, 57.66, 18.67] for symmetric and [-2.62, 49.00, -4.54] for asymmetric montages (please see section 3.3 for more details on inter-individual variations in terms of locations and EF values). With respect to the brain anatomical subregions, EF peaks were located within the frontopolar area; a region occupying the anterior portion of the brain’s frontal lobe (dominantly corresponding to Brodmann’s area 10) which is distinct from DLPFC (which is located on the lateral and dorsal part of the medial convexity of the frontal lobe [32]) where the stimulating electrode was placed (dominantly comprises Brodmann’s areas 9 and 46) [2]. In both electrode montages, averaged EFs in a 10 mm sphere around the peak EFs (symmetric: 0.32 ± 0.07, asymmetric: 0.24 ± 0.05; mean ± SD in V/m) was significantly (P value for: symmetric = 3.01x10^-4, asymmetric: 4.52x10^-6) higher than a 10 mm sphere around F4 location over the cortex in which the center of the target electrode was placed (symmetric: 0.22 ± 0.05, asymmetric: 0.19 ± 0.05 in V/m) with large effect sizes (Hedges’g for: symmetric = 0.8598 with 95% CI (0.53,1.19) and asymmetric = 0.7630 with 95% CI (0.43,1.09)). Our results also showed that, in both locations, underneath the electrode and averaged peaks the symmetric montage produced significantly (P = 0.00015) higher EFs compared to asymmetric electrode arrangement with medium effect sizes (Hedges’g for: symmetric = 0.6234 with 95% CI (0.95, 0.30) and asymmetric = 0.7965 with 95% CI (0.47, 1.13)). Page 6/23 Page 6/23 As shown in Fig. 1, EFs were also extracted in the coordinates related to the individualized 99th percentile and center of the stimulating electrode (without averaging within 10 mm spheres) and similar results were found. EF strength in the coordinate related to the 99th percentile (symmetric = 0.41 ± 0.06, asymmetric = 0.38 ± 0.05) was significantly (P value = 2.2x10^-16 for both montages) higher than the coordinate related to the center of stimulating electrode (symmetric = 0.23 ± 0.05, asymmetric = 0.20 ± 0.04) with large effect sizes (Hedges’g for: symmetric = 3.2091 with 95% CI (2.73, 3.69) and asymmetric = 3.8339 with 95% CI (3.30, 4.37)). 3.2. Inter-individual variabilities Inter-individual variability in terms of peaks’ locations and strength was compared across the population. Variations across the population are visualized in Fig. 2. In the symmetric DLPFC stimulation, the mean location for the peaks across the population in MNI space was [-1.21, 57.66, 18.67] with [6.43, 4.37, 8.08] as SD, and all peak locations were placed in a cube with a volume of 29 cm3 (X = 3.1, Y = 1.9, Z = 4.9). Group-level mean value for the EF strength was also 0.41 ± 0.06 [V/m] (ranges from 0.31 to 0.68). In the asymmetric DLPFC stimulation, the mean location for the peaks in MNI space was [-2.62, 49.00, -4.54] with [7.05, 7.71, 8.94] as SD, and all peak locations were placed in a cube with a volume of 46 cm3 (X = 3.3, Y = 3.4, Z = 4.1). Group-level mean value for the EF strength was also 0.38 ± 0.04 [V/m] (ranges from 0.28 to 0.59). 3.1. Comparing EFs in the center of target electrodes and hot spots EFs were also significantly higher in the symmetric montage compared to asymmetric (P value for: 99th percentile = 0.004, the center of target electrode = 1.7x10^-5) with small effect sizes for 99th percentile individualized coordinate (Hedges’g = 0.4724 with 95% CI (0.15, 0.79)) and medium effect size for the coordinate related to the center of target electrode (Hedges’g = 0.7114 with 95% CI (0.34, 1.04)). [Insert Fig. 1 around here] 3.3. Atlas-based parcellation results In addition to the spherical ROIs around the center of target electrode (DLPFC) and peaks (medial frontopolar), averaged EFs were also extracted from the main regions of the prefrontal cortex including superior frontal gyrus (SFG), middle frontal gyrus (MFG), and orbital gyrus (OrG) in the Brainnetome atlas (areas related to the Brodmann area 9, 10, 11, and 46 as defined in the method section); SFG: A9l, A9m, A10m, MFG: A9/46d, A9/46v, A46, A10l, OrG: A11m, and A11l. Averaged EFs in each subregion in both left and right hemispheres along with the representation of the regions over the cortex can be found in the supplementary materials (Figure S1). Cumulative EF strength with a symmetric montage in the right frontopolar (A10m + A10l) was significantly (P < 0.01 with Hedges’ g = 0.3412) higher than right DLPFC (A9/46v + A9/46d). Additionally, symmetric montage induced slightly higher EFs compared to the asymmetric montage in almost all subregions but differences were significant only in the A10m in both hemispheres (P < 0.001). 3.4. Replication results in a clinical population Replication results in a clinical population In the asymmetric montage averaged location of the EF peaks in MNI space was [-3.14, 56.90, 6.33] with [7.37, 5.86, 9.74] as SD, and all peaks were placed in a cube with a volume of 45.9 cm3. Our EF strength results (Figure S2 in supplementary materials) showed that EF strength in the frontopolar area (symmetric: 0.31 ± 0.07, asymmetric: 0.32 ± 0.07) where EF peaks were placed is significantly (P < 0.001) higher than DLPFC (symmetric: 0.18 ± 0.05, asymmetric: 0.19 ± 0.05) underneath the target electrode with large effect sizes (Hedges’g for symmetric = 1.6190 with 95% CI (1.19, 2.04) and asymmetric = 1.3599 with 95% CI (0.95, 1.77)). However, no significant difference (P > 0.1) was found between the EFs strength in symmetric and asymmetric montages in this population such that effect sizes were negligible (for peak EFs Hedges’g = 0.13 with 95% CI (-0.24, 0.50) and around F4 location Hedges’g = -0.02 with 95% CI (-0.39, 0.34)). Additionally, by considering inter-individual variability (Figure S3 in supplementary materials), in atlas- based parcellation of the head models, results (Figure S4 in supplementary materials) showed that the frontopolar area (A10l + A10m) received a significantly higher EF strength compared to DLPFC (A9/46v + A9/46d) in both symmetric and asymmetric montages (P < 0.001). Additionally, by considering inter-individual variability (Figure S3 in supplementary materials), in atlas- based parcellation of the head models, results (Figure S4 in supplementary materials) showed that the frontopolar area (A10l + A10m) received a significantly higher EF strength compared to DLPFC (A9/46v + A9/46d) in both symmetric and asymmetric montages (P < 0.001). Our results showed that averaged peak location in symmetric (healthy: [-1.21, 57. 66, 18.67], MUDs: [1.88, 60.34, 19.12] with maximum 4.12 mm3 Euclidean distance from F4 location over the cortex) and asymmetric montages (healthy: [-2.62, 49, -4.54], MUDs: [-3.14, 56.9, 6.33] with maximum 13.45 mm3 Euclidean distance from F4 location over the cortex) have a substantial overlap between the two groups such that in both groups peaks were located within the frontopolar area (Fig. 3). Furthermore, using an unpaired t-test for between-group comparison in terms of EF strength in spherical regions around peaks, a significantly higher EF strength in the asymmetric montage in the MUD (0.31 ± 0.07) was found compared to the healthy group (0.23 ± 0.05) with a medium effect size (Hedges’ g = 0.6172 with 95% CI (0.27,0.97)). Replication results in a clinical population A significant difference in the symmetric montage was also found around the F4 location with a higher EF strength in the healthy group (0.23 ± 0.05) compared to MUDs (0.18 ± 0.04) with a large effect size (Hedges’ g = -0.9059 with 95% CI (-1.27,-0.55)). [Insert Fig. 3 around here] Replication results in a clinical population Page 7/23 Page 7/23 All the above-mentioned steps were replicated for a group of participants with methamphetamine use disorder (MUD) as an independent clinical population. Similar to the healthy subjects, inter-individual variability was found in both location and strength of the EFs across the population. In the symmetric montage averaged location for the EF peaks in MNI space was [1.88, 60.34, 19.12] with [5.71, 3.34, 6.72] as SD, and all peaks were placed inside a cube with a volume of 21.7 cm3. In the asymmetric montage averaged location of the EF peaks in MNI space was [-3.14, 56.90, 6.33] with [7.37, 5.86, 9.74] as SD, and all peaks were placed in a cube with a volume of 45.9 cm3. Our EF strength results (Figure S2 in supplementary materials) showed that EF strength in the frontopolar area (symmetric: 0.31 ± 0.07, asymmetric: 0.32 ± 0.07) where EF peaks were placed is significantly (P < 0.001) higher than DLPFC (symmetric: 0.18 ± 0.05, asymmetric: 0.19 ± 0.05) underneath the target electrode with large effect sizes (Hedges’g for symmetric = 1.6190 with 95% CI (1.19, 2.04) and asymmetric = 1.3599 with 95% CI (0.95, 1.77)). However, no significant difference (P > 0.1) was found between the EFs strength in symmetric and asymmetric montages in this population such that effect sizes were negligible (for peak EFs Hedges’g = 0.13 with 95% CI (-0.24, 0.50) and around F4 location Hedges’g = -0.02 with 95% CI (-0.39, 0.34)). Additionally, by considering inter-individual variability (Figure S3 in supplementary materials), in atlas- based parcellation of the head models, results (Figure S4 in supplementary materials) showed that the frontopolar area (A10l + A10m) received a significantly higher EF strength compared to DLPFC (A9/46v + A9/46d) in both symmetric and asymmetric montages (P < 0.001). All the above-mentioned steps were replicated for a group of participants with methamphetamine use disorder (MUD) as an independent clinical population. Similar to the healthy subjects, inter-individual variability was found in both location and strength of the EFs across the population. In the symmetric montage averaged location for the EF peaks in MNI space was [1.88, 60.34, 19.12] with [5.71, 3.34, 6.72] as SD, and all peaks were placed inside a cube with a volume of 21.7 cm3. 4. Discussion Page 8/23 In this study, we modeled the target selectivity of two of the most frequently used electrode montages for modulating DLPFC; symmetric (anode/cathode: F4/F3) and asymmetric (anode/cathode: F4/Fp1) montages to explore whether and to which extent DLPFC could be the main target compared to other brain regions that are not intentionally targeted but received strong electric field (EF). Individualized computational head models were generated for two groups of participants; healthy participants and people with methamphetamine use disorders (MUDs). Specifically, this investigation yielded four main Page 8/23 results. First, the peaks of EF are located far (45.54 Euclidian distance in MNI space) from DLPFC (F4 location as the center of target electrode). Second, group-level EF peaks were dominantly placed near the medial frontopolar cortex in both montages and both healthy participants and people with methamphetamine use disorder (MUD) groups, such that peak EF (99th percentile) received significantly higher EFs compared to the cortical area under the center of the target electrodes; DLPFC. Third, variations were found within and between groups in terms of EF peaks’ location and strength. 4.1. Importance of peak EFs in brain stimulation studies Therefore, if the main goal of the stimulation is reaching the brain area beneath the target electrode (F3/F4), it is recommended to define an ROI in the targeted brain region and extract averaged value from no-threshold EF maps. 4.1. Importance of peak EFs in brain stimulation studies Our computational approach with a substantial sample size underlies the utility of head models for uncovering potentially stimulated brain regions based on EF peaks. As investigated in previous brain stimulation studies [33], the efficacy of the stimulation protocol may vary depending on the brain region being modulated. However, systematic analysis of EF distribution patterns and finding the association between tES-induced EFs and stimulation outcomes requires a decision about which EF measures (e.g., maximum (99th percentile to remove outliers), mean, median, or a binary thresholded EF map) are appropriate to use. Here, inspired by previously published studies in the field [34, 35], we focused on EF peaks to provide an indicator of the location at which the target activity is most likely to be perturbed [36]. Then, peak EF location and strength in the peak location were compared with the cortical area under the center of target electrodes as the main “intended” anatomical target. In the application of tES with large electrode pads (conventional tES), it has been repeatedly reported that maximal EF is not underneath the electrodes but rather between them and outside the “area under the electrode” [15, 37, 38]. With respect to the diffuse current flow, it’s not surprising that we found EF peaks out of DLPFC; similar to previous studies on motor cortex stimulation that reported maximum EFs between two electrodes [39]. However, our data suggest that the top 1% of voxels with the highest EF are located farther from the intended cortical target (DLPFC). Therefore, if the main goal of the stimulation is reaching the brain area beneath the target electrode (F3/F4), it is recommended to define an ROI in the targeted brain region and extract averaged value from no-threshold EF maps. In the application of tES with large electrode pads (conventional tES), it has been repeatedly reported that maximal EF is not underneath the electrodes but rather between them and outside the “area under the electrode” [15, 37, 38]. With respect to the diffuse current flow, it’s not surprising that we found EF peaks out of DLPFC; similar to previous studies on motor cortex stimulation that reported maximum EFs between two electrodes [39]. However, our data suggest that the top 1% of voxels with the highest EF are located farther from the intended cortical target (DLPFC). 4.2. Inter-individual variability Our results showed significant inter-individual variability in the location and strength of the peak EF in directly/indirectly targeted brain areas which are in line with previous studies that emphasize the importance of considering personalized head models [26, 37, 38]. The main issue with the inter-individual variability of EFs over the cortex is that the potential effect of tES is limited by small effect sizes and it would be difficult to detect intervention effects at the group-level [40–42]. Different sources of variability including anatomical parameters (e.g., fat thickness, skull thickness, and amount of CSF that affect current flow through the brain) affect EF distribution patterns [26, 43–45]. The variability of the simulation results indicates that personalized electrode montages should be considered to have similar cortical stimulation doses inside a target area across a population. Our results showed significant inter-individual variability in the location an directly/indirectly targeted brain areas which are in line with previous stud importance of considering personalized head models [26, 37, 38]. The mai variability of EFs over the cortex is that the potential effect of tES is limited would be difficult to detect intervention effects at the group-level [40–42]. including anatomical parameters (e.g., fat thickness, skull thickness, and a current flow through the brain) affect EF distribution patterns [26, 43–45]. simulation results indicates that personalized electrode montages should cortical stimulation doses inside a target area across a population. Page 9/23 Our atlas-based parcellation results further support the results obtained from the ROI definition based on considering the peak and center of target electrode locations such that EF strength in the right frontopolar was higher than right DLPFC. Although the most common approach for exploratory ROI analysis (e.g., in fMRI analysis) is to create small spheres around the peaks’ locations (e.g., peaks of activation clusters), atlas-based parcellation may help to simply explore and localize informative regions (e.g., subregions of the prefrontal cortex involved in DLPFC stimulation) based on the spatial distribution patterns of the EFs. However, as suggested by [46], the specific brain parcellation of each atlas impacts the spatial accuracy of the extracted brain regions especially when differences in the observed EF distribution patterns are small. Hence, in accordance with recent research [47], the Brainnetome atlas, with fine-grained parcellation of the prefrontal cortex, was used to accurately extract how EF was distributed through the subregions of the prefrontal cortex. 4.2. Inter-individual variability Nonetheless, regions specified with atlas-based parcellation could be large (e.g., entire SFG), and even if the region contains highly modulated nodes, strong EFs may only occur in a small proportion of vertices in the ROI. This suggests that by simply averaging across a parcel spatial detail about the EF will be lost. Focusing on individualized brain EF maps and defining ROIs based on personalized EF distribution patterns or using a fine-grained atlas (e.g., Schaefer atlas [48]) might be a better approach compared to averaging through a large parcellated brain area. 4.4. DLPFC montages make peak EF in frontopolar areas 4.4. DLPFC montages make peak EF in frontopolar areas There is converging evidence highlighting the role of brain regions with strong EFs like frontopolar as a novel stimulation target for non-invasive brain stimulation for designing new clinical trials [49–52]. In this context, our findings about strong stimulation strength within the frontopolar cortex in commonly used electrode montages over DLPFC are in line with a previous modeling study that reported strong EF intensity within the medial prefrontal cortex (MPFC) rather than DLPFC in all commonly used bipolar DLPFC electrode montages in depression [21]. This study suggested that symptom improvement in DLPFC tES trials in depression might not necessarily be specifically related to the DLPFC and other brain areas with strong EF (e.g, MPFC) may also contribute to the tES treatment outcomes. Furthermore, preliminary evidence from clinical trials confirmed associations between EF strength and behavioral changes in depression [31]. For instance, in a DLPFC stimulation study with electrodes over F5/F6 locations in a group of participants with major depressive disorder (MDD), a significant positive correlation between EFs within the MPFC-dorsal area (A9/46d) and behavioral outcomes were reported. While the correlation between EFs in DLPFC (A9 and A46) and the same behavioral outcome (negative affect) was negative (greater score reductions were associated with lower EF strength). Taken together, a brain region with higher EFs might be linked to behavioral outcomes through several psychophysiological mechanisms and neural substrates. Therefore, based on our simulations, we argue that conventional electrodes over DLPFC also stimulated the frontopolar area. We recommend taking into account the cognitive process associated with the frontopolar region in the interpretation of the results in conventional tES studies over DLPFC [26, 43–45]. 4.5. Dose-response relationship Page 10/23 Here, we only focused on highly modulated brain regions with respect to the strong EF over the cortex using high-resolution structural MRI data. It has been assumed that brain regions with higher EFs contribute to greater alteration in brain responses and may have stronger effects on stimulation outcomes (e.g., higher EFs followed by greater functional alterations) [53]. Under the assumption that EF strength over the cortex relates to the tES responses at the functional level, the association between stimulation dose and brain responses in a cortical target could explain dose-response relationships in tES studies. Recent advancements in neuroimaging (like fMRI) and neurophysiology (like TMS) suggest a complicated non-linear and state-dependent dose-response relationship in tES studies. 4.4. DLPFC montages make peak EF in frontopolar areas However, there are still some supporting evidence that highlight the role of brain regions with strong EFs in physiological/neural response to tES that may help to determine the role of DLPFC and frontopolar area and the importance of brain regions with higher EFs in symmetric/asymmetric DLPFC stimulation studies. One of the earliest studies in which both EFs and fMRI data were discussed was by Halko et al 2011. The single-subject case study of tDCS with combined visual rehabilitation training after stroke revealed that EF strength was correlated with task-based fMRI activation in some predefined ROIs; higher EF was linearly related to stronger functional activation [54]. In a group of participants with left-sided glioma, averaged EF strength was extracted from the left and right M1 ROIs, and a significant correlation between averaged EFs in the right M1 and changes in global resting-state connectivity from the right M1 was reported [55]. Antonenko et al. also assessed the relationship between tES-induced EFs and neurophysiological outcomes and significant negative/positive correlations were reported between the tangential/normal component of the EF and resting-state functional connectivity in the sensorimotor cortex [56]. Additionally, Jamil et al investigated whether cerebral blood flow (CBF) activations across the cortex agree respectively with EFs obtained from head models. Using a voxel-wise rank correlation, a significant correlation between averaged EF distribution patterns in MNI space and the group-level T- contrast images was reported at the voxel level in both cathodal and anodal stimulation [57]. Recently it has also been shown that current density in the left DLPFC positively correlated with changes in functional connectivity between two predefined ROIs (left dorsolateral and left ventrolateral PFC) during a working memory task based on using psychophysiological interaction analysis and simulating precise computational head models [58]. All previous dose-response relationships highlight the importance of EF strength and its effects on stimulation outcomes that shed light on how modulated brain areas with stronger EFs may affect the response to stimulation. A similar investigation approach (which has not been investigated in healthy subjects) is needed to assess dose-response relationships in the frontopolar area while DLPFC is targeted. In addition to correlational methods, testing for the causal role of the frontopolar in DLPFC stimulation studies can help to confirm the importance of the frontopolar cortex in the regulation of stimulation outcomes. l ( ) d 4.6. Bipolar montages (asymmetric vs. symmetric) induce different EF patterns in the frontal lobe 4.6. Bipolar montages (asymmetric vs. symmetric) induce different EF patterns in the frontal lobe Page 11/23 Another finding in this study was significant differences between symmetric and asymmetric montages in terms of EF strength in healthy subjects. As we discussed in our previous publication with a network- based perspective [25], cathode location significantly affects EFs strength in targeted or non-targeted brain areas [38, 59]. Here, we found that symmetric DLPFC stimulation induced significantly higher EFs in both DLPFC and peaks compared to asymmetric montage. Lower EF strength in asymmetric montages could be related to a lower distance between the electrodes over the scalp compared to the symmetric montage [60]. It can be attributed to more current shunting in asymmetric montage compared to symmetric since electrodes are in closer proximity [61]. More significant changes in behavioral outcomes using symmetric DLPFC stimulation might be related to the stronger cortical EFs compared to asymmetric montages. For instance, previous studies showed that symmetric montages over DLPFC could diminish risk-taking behavior during a Balloon Analogue Risk Task (BART) [62, 63], while asymmetric DLPFC stimulation shows no such effect [64]. Between montage difference in terms of peaks’ strength only in the healthy group (not MUDs) suggest that generalization of the results obtained from EF distribution patterns fundamentally depends on the population of interest and highlights the importance of head modeling in tES protocol optimization in both individual and group levels [65, 66]. 4.7. Optimized tES targeting: Converging evidence from HD- tES, neuroimaging, and TMS studies In this study, we only focused on conventional large electrode pads and electrode positioning based on EEG standard system because clinical tES typically uses conventional electrodes and scalp-based landmarks to target the DLPFC. Conforming with previous computational head modeling studies, our results revealed that placing stimulating electrodes directly above the cortical target does not guarantee its optimal stimulation dose under the electrode. More focal electrode montages could help to focus on the stimulation target. With respect to the previous systematic reviews [67], there is a lack of evidence for using focal stimulations over the predefined ROI using precisely targeted high-definition (HD) montages for the treatment of neuropsychiatric disorders [67]. In order to modulate DLPFC as the main target, instead of large pads, HD electrodes could be placed over this brain region to be focally stimulated. Our results suggested that in commonly used montages for targeting DLPFC, DLPFC did not receive the highest EF intensity. An optimization algorithm could be used to find an electrode montage to maximally target DLPFC while minimizing EF in non-targeted brain areas. With HD electrodes/optimized montage placed over DLPFC, the frontopolar area could receive much less stimulation. If desired, the frontopolar region could be stimulated using a separate set of HD electrodes. However, its efficiency and tolerability (by fixing small electrodes over the forehead) should be further assessed in experimental trials [11]. Additionally, fMRI-informed montage selection is a promising step towards more efficient and precise brain stimulation protocols which has recently started to gain momentum, especially in the field of TMS [68–70], and potentially provides valuable information about functional localization to guide EF peak locations based on modulating active neural circuits or functional networks [71, 72]. Closing the loop between brain-state and stimulation parameters may also help to increase the efficiency of the optimization algorithm using concurrent tES-fMRI. In this context, stimulation dose (e.g., current strength, Page 12/23 Page 12/23 electrode location, frequency, and phase difference) could be optimized at an individual level to maximally stimulate the ongoing brain state [73]. 4.9. Substance use disorders as a sample clinical population 4.9. Substance use disorders as a sample clinical population Here, we considered a group of participants with MUDs as an independent clinical population sample and highlighted the role of the frontopolar cortex. Despite the growing interest in non-invasive brain stimulation technologies for addiction treatment, the ideal location for intervention is still elusive. Much of the initial attention in this field was focused on the DLPFC (88% of total tES trials) with more promising results with symmetric compared to asymmetric montages [29]. The importance of frontopolar areas was also reported in previous dose-response relationship research for people with SUDs. Here, we considered a group of participants with MUDs as an independent clinical population sample and highlighted the role of the frontopolar cortex. Despite the growing interest in non-invasive brain stimulation technologies for addiction treatment, the ideal location for intervention is still elusive. Much of the initial attention in this field was focused on the DLPFC (88% of total tES trials) with more promising results with symmetric compared to asymmetric montages [29]. The importance of frontopolar areas was also reported in previous dose-response relationship research for people with SUDs. Esmaeilpour et al used atlas-based parcellation for defining ROIs over the head models and fMRI data during a standard drug cue reactivity task were collected immediately before and after stimulation in a group of participants with MUDs. Their results showed a significant correlation between changes in brain activation and averaged EF strength only in the frontopolar cortex as the area that received maximum EF compared to other predefined regions [46]. Similar results were also found in a larger cohort of participants with MUDs; a significant correlation between the normal component of the EF and changes in functional activity in the frontopolar cortex was reported during a drug cue reactivity task. Esmaeilpour et al used atlas-based parcellation for defining ROIs over the head models and fMRI data during a standard drug cue reactivity task were collected immediately before and after stimulation in a group of participants with MUDs. Their results showed a significant correlation between changes in brain activation and averaged EF strength only in the frontopolar cortex as the area that received maximum EF compared to other predefined regions [46]. 4.8. Considering network-based modulation Using atlas-based parcellation of the head models, we argued that placing the electrodes over DLPFC modulated multiple nodes in the prefrontal cortex which has the potential to act synergistically to enhance stimulation outcomes through excitatory/inhibitory pathways between two anatomically distinct brain regions. It has been reported that the contribution of brain regions in response to tES-induced EFs through brain networks would be more effective than modulating a single network node [74]. In this regard, the distribution of the EFs and peaks’ location could be optimized based on the interaction between main network nodes. Previous tES-fMRI studies suggest that network-based electrode montages led to a better outcome compared to conventional stimulation montages [72]. However, there is subtle nuance in the application of tES with a network-based approach, especially with conventional electrodes [75]. Considering HD or multi-array electrodes may be more efficient for network-based targeting. 4.9. Substance use disorders as a sample clinical population Similar results were also found in a larger cohort of participants with MUDs; a significant correlation between the normal component of the EF and changes in functional activity in the frontopolar cortex was reported during a drug cue reactivity task. Our suggestion about the importance of frontopolar areas in tES for addiction conforms with recent evidence obtained from lesion-based studies that reported the frontopolar cortex as a key region in trans- diagnostically relevant neural circuits contributing to addiction [76]. Lesion-based fMRI data revealed that a higher likelihood of decreasing substance use was associated with brain injury to areas that had negative connectivity to the frontopolar areas and recommended frontopolar as an ideal neuromodulation treatment target for addiction in future studies [76]. In addition to within-group variability, our between-group results suggest that EF distribution patterns cannot be simply transferred from healthy to clinical populations and more research is still needed to pinpoint the optimal target, Page 13/23 stimulation dose over the targeted region, and ultimately maximize clinical benefits at individual/group- levels. 4.10. Limitations and future directions Our study has some limitations that could be addressed in future research. The main limitation is that we only focused on the structural MRI and simulation approaches. We did not consider other behavioral/neural outcomes to determine the role of EFs in each targeted and non-targeted brain region. Our findings are specific to stimulation alone. The context of the received dose over the cortex also guides the induced neuroplasticity (e.g., if stimulation is delivered while participants are performing a specific cognitive task, the engaged brain regions for task performance will also experience plasticity). Furthermore, in our clinical population, we did not examine the relationship between EFs and clinical response (e.g., drug consumption/craving). Open questions remain about how to relate EFs with behavioral or neurophysiological changes in both healthy and clinical populations. The relationship between EF peaks and stimulation outcomes is not a trivial matter and there is still no clear understanding of the underlying neural substrates. More neuroimaging/neurophysiological assessments are needed to determine the causal role of medial frontopolar cortices in response to bipolar DLPFC stimulation. Furthermore, since we did not consider age/sex-matched clinical population and because the imaging protocols were different between the two groups, we did not directly compare our results between MUDs and healthy subjects as conducted by [47]. Because it has been reported that EF distribution patterns in brain stimulation studies may be different in the field of addiction compared to healthy subjects with respect to the brain anatomical alterations [65, 66] and because sex differences in skull thickness, for example, would lead to differences in peak EF location/value, comparing EF peaks between SUDs and healthy subjects in age and sex-matched case-control cohorts could be further investigated in future studies. For instance, our results showed that the center of averaged EF peak location is deeper in the brain of our clinical population compared to healthy subjects. The main reason could be investigated in future studies. 5. Conclusion Here, we discussed that, in two of the most frequently used electrode montages in tES studies which are commonly intended for modulating DLPFC, DLPFC was not maximally targeted by placing large electrodes over this region and other parts of the prefrontal cortex also received strong electric field strength. Considering inter-individual variability, our results highlighted the crucial role of the frontopolar area in tES over DLPFC. Based on individual and group-level analysis of EF peaks, in both healthy subjects and MUDs, we recommend avoiding attributing F3/F4 tES results (with symmetric or asymmetric configuration) only to DLPFC. Other brain regions in medial prefrontal areas like medial frontopolar should be considered in upcoming trials placing large electrode pads over DLPFC. Conflict of interest The authors declare no competing interests. The authors declare no competing interests. The authors declare no competing interests. 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(a) Bars show mean values and error bars show standard deviations (SD) of the EF strength in volts per meter ([V/m]) across 77 healthy subjects in individualized 99th percentile of the EF (peak EF, in red) and center of the target electrode (F4, in blue) over the cortex across the population for symmetric (target/return electrodes over F4/F3 in dark colors) and asymmetric (target/return electrodes over F4/Fp1 in light colors) montages. (b) Electrode configurations over the scalp for DLPFC stimulation are visualized with the target (in green)/return (in black) electrodes over F4/F3 in symmetric and F4/Fp1 in asymmetric montages. EF 73. Soleimani, G., et al., Closing the loop between brain and electrical stimulation: Towards precision neuromodulation treatments. 2022. Figure 2 Inter-individual variability of electric field (EF) strength and locations. I. Scatter plot (for location in MNI space) colored based on EF strength (hot colors represent strong EF strength) for visualizing inter- individual variability in peaks (99th percentile) of the EFs in (a) symmetric and (b) asymmetric DLPFC Figure 1 Comparing EF (electric field) in the center of the target electrode vs. peak EF. (a) Bars show mean values and error bars show standard deviations (SD) of the EF strength in volts per meter ([V/m]) across 77 healthy subjects in individualized 99th percentile of the EF (peak EF, in red) and center of the target electrode (F4, in blue) over the cortex across the population for symmetric (target/return electrodes over F4/F3 in dark colors) and asymmetric (target/return electrodes over F4/Fp1 in light colors) montages. (b) Electrode configurations over the scalp for DLPFC stimulation are visualized with the target (in green)/return (in black) electrodes over F4/F3 in symmetric and F4/Fp1 in asymmetric montages. EF Comparing EF (electric field) in the center of the target electrode vs. peak EF. (a) Bars show mean values and error bars show standard deviations (SD) of the EF strength in volts per meter ([V/m]) across 77 healthy subjects in individualized 99th percentile of the EF (peak EF, in red) and center of the target electrode (F4, in blue) over the cortex across the population for symmetric (target/return electrodes over F4/F3 in dark colors) and asymmetric (target/return electrodes over F4/Fp1 in light colors) montages. (b) Electrode configurations over the scalp for DLPFC stimulation are visualized with the target (in green)/return (in black) electrodes over F4/F3 in symmetric and F4/Fp1 in asymmetric montages. EF the center of the target electrode vs. peak EF. (a) Bars show mean values Page 20/23 Page 20/23 distribution patterns at the group-level (spatial mean values across the population) are visualized over the cortex in superior view. (c)Locations of the 10 mm spheres around F4 (in blue) and averaged location of the peak EF across the population (in red) are visualized over the standard brain in fsaverage space. In each panel, the left side corresponds to the symmetric montage (F4-F3) and the right side corresponds to the asymmetric (F4-Fp1) montage. Figure 2 Inter-individual variability of electric field (EF) strength and locations. I. Scatter plot (for location in MNI space) colored based on EF strength (hot colors represent strong EF strength) for visualizing inter- individual variability in peaks (99th percentile) of the EFs in (a) symmetric and (b) asymmetric DLPFC Inter-individual variability of electric field (EF) strength and locations. I. Scatter plot (for location in MNI space) colored based on EF strength (hot colors represent strong EF strength) for visualizing inter- individual variability in peaks (99th percentile) of the EFs in (a) symmetric and (b) asymmetric DLPFC Page 21/23 Page 21/23 montages. II.Visualizing the location of the 99th percentile of the EF over the standard fsaverage space (red dots over the cortex represent each individual). Distribution plots represent the distribution of the EF strength within the 10 mm spheres around F4 and 99th percentile (blue and red spheres over the cortex in anterior view) for (a) symmetric and (b) asymmetric montages. Boxplots showing differences between EFs in the coordinates related to individualized peaks and F4 in [V/m]. Dots and spaghetti lines over the boxplots represent the data for individual subjects. Abbreviation: EF: electric field. Page 22/23 boxplots represent the data for individual subjects. Abbreviation: EF: electric field. Figure 3 Comparison between healthy subjects and methamphetamine users in terms of location and strength of the peaks at both individual and group levels. Overlap between 10 mm spheres around mean EF peaks in healthy subjects (in brown) and a group of participants with methamphetamine use disorders (in purple) compared to the center of the target electrode F4 (in blue) in both symmetric (left panel) and asymmetric (right panel) montages. Small dots represent each individual in each group and big circles represent 10 mm spheres around averaged peaks’ locations at the group-level. MNI coordinates for F4 = [40.5, 41.4, 27], averaged peak location in the symmetric montage (F4/F3): healthy subjects = [-1.21, 57.66, 18.67] and methamphetamine users = [1.88, 60.34, 19.12] with 4.12 mm3 Euclidean distance, asymmetric Figure 3 Figure 3 Comparison between healthy subjects and methamphetamine users in terms of location and strength of the peaks at both individual and group levels. Overlap between 10 mm spheres around mean EF peaks in healthy subjects (in brown) and a group of participants with methamphetamine use disorders (in purple) compared to the center of the target electrode F4 (in blue) in both symmetric (left panel) and asymmetric (right panel) montages. Small dots represent each individual in each group and big circles represent 10 mm spheres around averaged peaks’ locations at the group-level. MNI coordinates for F4 = [40.5, 41.4, 27], averaged peak location in the symmetric montage (F4/F3): healthy subjects = [-1.21, 57.66, 18.67] and methamphetamine users = [1.88, 60.34, 19.12] with 4.12 mm3 Euclidean distance, asymmetric Page 22/23 montage (F4/Fp1): healthy subjects = [-2.62, 49, -4.54], methamphetamine users = [-3.14, 56.90, 6.32] with 13.45 mm3 Euclidean distance. montage (F4/Fp1): healthy subjects = [-2.62, 49, -4.54], methamphetamine users = [-3.14, 56.90, 6.32] with 13.45 mm3 Euclidean distance. SupplementaryMaterials.docx Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. SupplementaryMaterials.docx Page 23/23
https://openalex.org/W4282838557	https://www.frontiersin.org/articles/10.3389/fphar.2022.907695/pdf	English	null	Phosphatase and Tensin Homology Deleted on Chromosome 10 Inhibitors Promote Neural Stem Cell Proliferation and Differentiation	Frontiers in pharmacology	2,022	cc-by	4,888	Citation: Liu X, Cui Y, Li J, Guan C, Cai S, Ding J, Shen J and Guan Y (2022) Phosphatase and Tensin Homology Deleted on Chromosome 10 Inhibitors Promote Neural Stem Cell Proliferation and Differentiation. Phosphatase and Tensin Homology Deleted on Chromosome 10 Inhibitors Promote Neural Stem Cell Proliferation and Differentiation Xiaojiang Liu 1†, Yiqiu Cui 1†, Jun Li 1, Cheng Guan 1, Shu Cai 1, Jinrong Ding 1, Jianhong Shen 2 and Yixiang Guan 1* 1Department of Neurosurgery, Afﬁliated Haian Hospital of Nantong University, Nantong, China, 2Department of Neurosurgery, Afﬁliated Hospital of Nantong University, Nantong, China 1Department of Neurosurgery, Afﬁliated Haian Hospital of Nantong University, Nantong, China, 2Department of Neurosurgery, Afﬁliated Hospital of Nantong University, Nantong, China Phosphatase and tensin homology deleted on chromosome 10 (PTEN) is a tumor suppressor gene. Its encoded protein has phosphatase and lipid phosphatase activities, which regulate the growth, differentiation, migration, and apoptosis of cells. The catalytic activity of PTEN is crucial for controlling cell growth under physiological and pathological conditions. It not only affects the survival and proliferation of tumor cells, but also inhibits a variety of cell regeneration processes. The use of PTEN inhibitors is being explored as a potentially beneﬁcial therapeutic intervention for the repair of injuries to the central nervous system. PTEN inﬂuences the proliferation and differentiation of NSCs by regulating the expression and phosphorylation of downstream molecular protein kinase B (Akt) and the mammalian target of rapamycin (mTOR). However, the role of PTEN inhibitors in the Akt/mTOR signaling pathway in NSC proliferation and differentiation is unclear. Dipotassium bisperoxo (picolinoto) oxovanadate (V) [bpv(pic)] is a biologically active vanadium compound that blocks PTEN dephosphorylation and suppresses its activity, and has been used as a PTEN lipid phosphatase inhibitor. Here, bpv(pic) intervention was found to signiﬁcantly increase the number of rat NSCs, as determined by bromodeoxyuridine staining and the cell counting kit-8, and to increase the percentage of neurons undergoing differentiation, as shown by immunoﬂuorescence staining. Bpv(pic) intervention also signiﬁcantly increased PTEN and mTOR expression, as shown by real-time PCR analysis and western blotting. In conclusion, PTEN inhibitor bpv(pic) promotes the proliferation and differentiation of NSCs into neurons. Edited by: Anwen Shao, Zhejiang University, China Reviewed by: Jin Hu, Fudan University, China Chiyuan Ma, Nanjing General Hospital of Nanjing Military Command, China Correspondence: Yixiang Guan haianswgyx@163.com †These authors have contributed equally to this work Edited by: Anwen Shao, Zhejiang University, China Reviewed by: Jin Hu, Fudan University, China Chiyuan Ma, Nanjing General Hospital of Nanjing Military Command, China Correspondence: Yixiang Guan haianswgyx@163.com Correspondence: Yixiang Guan haianswgyx@163.com †These authors have contributed equally to this work Specialty section: This article was submitted to Neuropharmacology, a section of the journal Frontiers in Pharmacology Received: 30 March 2022 Accepted: 27 May 2022 Published: 14 June 2022 Specialty section: This article was submitted to Neuropharmacology, a section of the journal Frontiers in Pharmacology Received: 30 March 2022 Accepted: 27 May 2022 Published: 14 June 2022 Keywords: Pten, mTOR, neural stem cells, proliferation, differentiation Keywords: Pten, mTOR, neural stem cells, proliferation, differentiation ORIGINAL RESEARCH published: 14 June 2022 doi: 10.3389/fphar.2022.907695 ORIGINAL RESEARCH published: 14 June 2022 doi: 10.3389/fphar.2022.907695 INTRODUCTION As an anti-oncogene with dual speciﬁc phosphatase activity, phosphatase and tensin homology deleted on chromosome 10 (PTEN) has become a research hotspot in recent years. It plays an important role in a variety of diseases, including cancer, liver disease (Ikeda et al., 2020; Chen et al., 2021), and diabetes (Lu et al., 2021), where it is involved in cell migration, proliferation, Front. Pharmacol. 13:907695. doi: 10.3389/fphar.2022.907695 Front. Pharmacol. 13:907695. doi: 10.3389/fphar.2022.907695 June 2022 \| Volume 13 \| Article 907695 Frontiers in Pharmacology \| www.frontiersin.org 1 Liu et al. Neural Stem Cell PTEN expression decreased neuronal proliferation and differentiation through the activation of PI3K/Akt/mTOR signaling. Our ﬁndings enhance our understanding of the mechanism of NSC differentiation during neurogenesis. differentiation, apoptosis, and metabolism (Yamada and Araki, 2001; Hamada et al., 2005; Salmena et al., 2008; Chow and Salmena, 2020). PTEN mainly catalyzes the conversion of phosphatidylinositol trisphosphate (PIP3) to phosphatidylinositol biphosphate (PIP2) by inhibiting the classical phosphatidylinositol 3 kinase (PI3K)- serine/threonine kinase (Akt) signaling pathway (Song et al., 2005). When PI3K receives signals from tyrosine kinase and G protein-coupled receptors, activated PI3K converts PIP2 to PIP3, and reduces PIP3 to PIP2. PIP3 then binds to the N-PI3KPH domain of downstream Akt, which is transferred from the cytoplasm to the cell membrane (Park et al., 2010). Cell Lines and Reagents g Sixteen-day-old pregnant SD rats (Guan et al., 2015) were provided by the Laboratory Animal Center of Nantong University. This study was conducted in accordance with the recommendations of the National Institutes of Health Laboratory Animal Care and Use Guidelines. The isolated fetal rat cerebral cortex was removed under aseptic conditions, meninges were stripped in Dulbecco’s modiﬁed Eagle medium (DMEM) containing 0.25% trypsin for 10 min, and the cell suspension was obtained in DMEM containing 5% horse serum and 10% fetal bovine serum (Gibco, Grand Island, NY, United States) at a density of 1 × 106 cells/ml. Cells were then cultured at 37°C with 5% CO2 in DMEM supplemented with neurobasal neuron- speciﬁc medium (Gibco) containing 1% B-27 supplement and 0.25% L-Glutamine. y p With the assistance of 3-phosphoinositol-dependent protein kinase 1, PIP3 activates Akt by phosphorylating its threonine phosphorylation site (Thr308) or serine phosphorylation site (Ser473). Activated Akt then activates mammalian target of rapamycin (mTOR). The PI3K/Akt/mTOR signaling pathway activates and regulates cell proliferation, differentiation, and migration (Jung et al., 2021). The pathway is also involved in the repair and regeneration of central nerve injuries, as shown by PTEN gene knockout using a PTEN inhibitor or small interfering RNA which accelerated the growth of axons at the injured site (Lu et al., 2020). Although PTEN is not required to determine cell fate in the central nervous system (CNS), it was shown to function in NSC differentiation, where its expression changes dynamically. PTEN expression begins in the late stages of mouse CNS development and peaks in adulthood. It is widely expressed in the brain of adult mice, especially in neurons (Li et al., 2020; Yu et al., 2020). Proliferation of NSCs After bpv(pic) Intervention After harvesting, the second generation of NSCs was seeded into 24-well plates at a density of 5 × 104 cells/mL. Bpv(pic) (ATCC, Manassas, VA, United States) was added to the intervention group at a ﬁnal concentration of 200 nmol/L (Thellung et al., 2019). NSCs were cultured for 5–7 days at 37°C with 5% CO2, then the number of cells was determined using the cell counting kit-8 (CCK-8; Abcam) and compared between the two groups. Brieﬂy, cell proliferation was measured by adding 100 µL DMEM/ F-12 and 10 µL CCK-8 reagent to each plate, and incubating for 8 h at 37°C with 5% CO2. The absorbance at 425 nm was then measured using the Multiskan MK33 microplate reader (Thermo Electron Corporation, Shanghai, China). Bromodeoxyuridine (BrdU) solution was also added to the intervention group at a ﬁnal concentration of 5 μmol/L to stain proliferating neonatal neurons which were observed using an Olympus IX71 microscope. mTOR is an important signaling molecule in the PTEN signaling pathway, which regulates pentameric neuronal ASH2-like, histone lysine methyltransferase complex subunit at the transcriptional level (Nguyen and Anderson, 2018). Consequently, it affects neuronal differentiation and directional axonal outgrowth (Jia et al., 2021). PI3K/AKT/ mTOR signaling was shown to regulate neuronal cell maturation and differentiation, while Park (Park et al., 2008) reported regeneration of the optic nerve after PTEN knockdown following the reactivation of PI3K/Akt/mTOR signaling. PTEN also regulates neuronal apoptosis, proliferation, renewal, and differentiation, and inhibits neuronal regeneration by inhibiting transduction of the PI3K/AKT signaling pathway. Thus, inhibiting PTEN promotes the survival and differentiation of NSCs. Frontiers in Pharmacology \| www.frontiersin.org Bpv(pic) Promoted NSC Proliferation p (p ) BrdU staining showed that the number of NSCs in the intervention group (65 ± 6 cells) was signiﬁcantly higher than in the control group (42 ± 5 cells) (p < 0.05) (Figure 2A). Absorbance values were 0.997 ± 0.085 and 0.788 ± 0.083 for the intervention and control groups, respectively. The CCK-8 assay found that bpv(pic) signiﬁcantly inhibited the proliferation of the intervention group compared with the control (p < 0.05). These data together suggest that bpv(pic) promoted the proliferation of NSCs (Figure 2B). Statistical Analysis ll f d anti-rabbit IgG H&L (Alexa Fluor® 594) (diluted 1:1000; Abcam) secondary antibodies at 20°C for 2 h. DNA was stained by immediately incubating the slides in 4′,6-diamidino-2- phenylindole (0.2 mg/ml) for 2 min. Slides were stored in the dark at 4°C, then six ﬁelds of view per slide were randomly selected. The percentage of positively staining cells in each ﬁeld was calculated under an Olympus IX71 microscope, and the average value was compared between control and intervention groups. All assays were performed in duplicate a total of three times. Data are expressed as the mean ± SEM, and were analyzed by the Student’s t-test and one-way analysis of variance. SPSS v. 17.0 statistical software was used for analysis, and p values ≤ 0.05 were considered statistically signiﬁcant. NSC Differentiation Vanadium and vanadium peroxide compounds are widely used as general inhibitors of protein tyrosine phosphatase, especially bisperoxovanadium compounds which include dipotassium bisperoxo (picolinoto) oxovanadate (V) [bpV(pic)] (vanadium diperoxys 5-hydroxypyridine). Bpv(pic) is a speciﬁc inhibitor of PTEN that promotes neural stem cell (NSC) proliferation and differentiation in vitro and in vivo, with no signiﬁcant effect on cell survival (Guan et al., 2021). Together, these ﬁndings suggest that PTEN plays an important role not only in peripheral nerve damage but also in the repair and regeneration of central nerve injury. NSCs were inoculated at a density of 5 × 104 cells/ml into 24-well plates with polylysine-coated glass slides in differentiation solution (DMEM/F-12 supplemented with 1% fetal bovine serum) which was changed after 2 h (Guan et al., 2015). Bpv(pic) was added to the intervention group at a ﬁnal concentration of 200 nmol/L, and all cells were cultured for a further 7 days. Cells were then incubated with the following primary antibodies at 4°C for 16 h: rabbit anti-rat βIII tubulin antibody (diluted 1:1000; Abcam), mouse anti-rat glial ﬁbrillary acidic protein (GFAP) antibody (diluted 1:1000; Abcam), and rabbit anti-rat receptor interacting protein (RIP) antibody (diluted 1:1000; Abcam). They were then incubated with goat In this study, we examined the role of a PTEN inhibitor in NSC proliferation and differentiation. We found that inhibiting June 2022 \| Volume 13 \| Article 907695 Frontiers in Pharmacology \| www.frontiersin.org 2 Liu et al. Neural Stem Cell FIGURE 1 \| NSC single cell cloning. (A) Single cell culture. (B) After 3 days culture. (C) After 5 days culture. (D) Proliferation to form NSCs after 7 days subculture. Scale bar: 100 µm. FIGURE 1 \| NSC single cell cloning. (A) Single cell culture. (B) After 3 days culture. (C) After 5 days culture. (D) Proliferation to fo Scale bar: 100 µm. ng. (A) Single cell culture. (B) After 3 days culture. (C) After 5 days culture. (D) Proliferation to form NSCs after 7 days subculture. FIGURE 1 \| NSC single cell cloning. (A) Single cell culture. (B) After 3 days culture. (C) After 5 days culture. (D) Proliferation to form NSCs after 7 days subculture. Scale bar: 100 µm. Real-Time PCR Analysis NSCs Self-Renewed and Proliferated Single-cell cloning experiments showed that individual NSCs (Figure 1A) divided after 3 days (d) of culture (Figure 1B), exhibited colonies of 15–28 cells after 5 days (Figure 1C), and proliferated to form a colony of around 50 cells after 7 days (Figure 1D). This suggests that colony formation occurred through the self-renewal and proliferation of NSCs rather than the aggregation of individual NSCs. Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, United States), then reverse-transcribed into cDNA using the Omniscript RT Kit (Qiagen) according to the manufacturer’s instructions. PCR was carried out using the following conditions: 95°C for 2 min, then 30 cycles of 95°C for 15 s, 54°C for 30 s, and 72°C for 1 min (Guan et al., 2015). Primer sequences were: mTOR-F: 5′-AGGAGGGACGTTTGC TCAGA-3′ and mTOR-R: 5′-TCCCTCACTGAACACAGCAG- 3′; PTEN-F: 5′-ACCAGGACCAGAGGAAACCT-3′ and PTEN- R: 5′-TTTGTCAGGGTGAGCACAAG-3′; and β-actin-F: 5′- AGGCATCCTGACCCTGAAGTAC-3′ and β-actin-R: 5′-TCT TCATGAGGTAGTCTGTCAG-3′. Western Blotting FIGURE 2 \| Cell assessments 5 days after bpv(pic) intervention. (A) Cells after staining with BrdU. Scale bar: 100 µm. (B) Cell proliferation as detected by the CCK-8 assay.p < 0.05. FIGURE 2 \| Cell assessments 5 days after bpv(pic) intervention. (A) Cells after staining with BrdU. Scale bar: 100 µm. (B) Cell proliferation as detected by the CCK-8 assay.p < 0.05. FIGURE 3 \| Bpv(pic) modulates the differentiation of NSCs. (A) Immunoﬂuorescence using anti-β-Tubulin III, anti-GFAP, and anti-RIP antibodies after bpv(pic) intervention. Scale bar: 100 µm. (B) Statistical analysis of immunoﬂuorescence. p < 0.05. Bpv(pic) Promoted the Differentiation of NSCs Into Neurons and Inhibited Their Differentiation Into Glial Cells Immunoﬂuorescence staining (Figure 3A) with anti-βIII tubulin, anti-GFAP, and anti-RIP antibodies showed that the percentage of NSCs differentiating (Figure 3B) into neurons was signiﬁcantly higher in the intervention group (27.860 ± 1.927%) than in the control group (13.120 ± 1.130%) (p < 0.05). Moreover, the percentage of differentiated glial cells was signiﬁcantly lower in the intervention group (61.900 ± 1.840%) than in the control group (77.520 ± 1.035%) (p < 0.05). Some NSCs differentiated into oligodendrocytes, but there was no signiﬁcant difference in the percentage of these between the two groups (p > 0.05). Bpv(pic) Enhanced the Expression of mTOR and PTEN in NSCs Bpv(pic) Enhanced the Expression of mTOR and PTEN in NSCs RT-PCR (Figure 4A) and western blotting (Figure 4B) were used to detect the expression of mTOR and PTEN at mRNA and protein levels, respectively. We observed signiﬁcantly increased expression of mTOR and PTEN in the intervention group compared with the control group (p < 0.05), with a greater increase seen in mTOR expression. Western Blotting Membranes were incubated with primary antibodies against β- actin (diluted 1:3000; Abcam), PTEN (diluted 1:1000; Abcam), and mTOR (diluted 1:1000; Abcam). June 2022 \| Volume 13 \| Article 907695 Frontiers in Pharmacology \| www.frontiersin.org 3 Liu et al. Neural Stem Cell Bpv(pic) Promoted the Differentiation of NSCs Into Neurons and Inhibited Their Differentiation Into Glial Cells Immunoﬂuorescence staining (Figure 3A) with anti-βIII tubulin, anti-GFAP, and anti-RIP antibodies showed that the percentage of NSCs differentiating (Figure 3B) into neurons was signiﬁcantly higher in the intervention group (27.860 ± 1.927%) than in the control group (13.120 ± 1.130%) (p < 0.05). Moreover, the percentage of differentiated glial cells was signiﬁcantly lower in the intervention group (61.900 ± 1.840%) than in the control group (77.520 ± 1.035%) (p < 0.05). Some NSCs differentiated into oligodendrocytes, but there was no signiﬁcant difference in the percentage of these between the two groups (p > 0.05). Bpv(pic) Enhanced the Expression of mTOR and PTEN in NSCs RT-PCR (Figure 4A) and western blotting (Figure 4B) were used to detect the expression of mTOR and PTEN at mRNA and protein levels, respectively. We observed signiﬁcantly increased expression of mTOR and PTEN in the intervention group compared with the control group (p < 0.05), with a greater increase seen in mTOR expression. FIGURE 2 \| Cell assessments 5 days after bpv(pic) intervention. (A) Cells after staining with BrdU. Scale bar: 100 µm. (B) Cell proliferation as detected by the CCK-8 assay.p < 0.05. FIGURE 3 \| Bpv(pic) modulates the differentiation of NSCs. (A) Immunoﬂuorescence using anti-β-Tubulin III, anti-GFAP, and anti-RIP antibodies after bpv(pic) intervention. Scale bar: 100 µm. (B) Statistical analysis of immunoﬂuorescence. p < 0.05. FIGURE 3 \| Bpv(pic) modulates the differentiation of NSCs. (A) Immunoﬂuorescence using anti-β-Tubulin III, anti-GFAP, and anti-RIP antibodies after bpv(pic) intervention. Scale bar: 100 µm. (B) Statistical analysis of immunoﬂuorescence. p < 0.05. FIGURE 2 \| Cell assessments 5 days after bpv(pic) intervention. (A) Cells after staining with BrdU. Scale bar: 100 µm. (B) Cell proliferation as detected FIGURE 3 \| Bpv(pic) modulates the differentiation of NSCs. (A) Immunoﬂuorescence using anti-β-Tubulin III, anti-GFAP, and anti-RIP antibodies after bpv(pic) intervention. Scale bar: 100 µm. (B) Statistical analysis of immunoﬂuorescence. p < 0.05. FIGURE 2 \| Cell assessments 5 days after bpv(pic) intervention. (A) Cells after staining with BrdU. Scale bar: 100 µm. (B) Cell proliferation as detected by the CCK-8 assay.p < 0.05. DISCUSSION investigate its effects on NSC proliferation and differentiation (Mak et al., 2010; Zhang et al., 2017). Bpv(pic) was previously shown to signiﬁcantly enhance NSC proliferation using a mechanism involving activation of the Akt/mTOR signaling pathway (Zeng and Zhou, 2008; Li et al., 2009). In the present study, we observed a signiﬁcantly higher number of NSCs after bpv(pic) treatment compared with the control. Additionally, we detected signiﬁcantly increased expression of PTEN and mTOR in NSCs treated with bpv(pic). This increase in mTOR reﬂects inhibition of the action of PTEN and an increase, rather than a corresponding decrease, in PTEN expression itself. Because bpv(pic) did not interfere with PTEN expression at the molecular level, bpv(pic) combined with downstream molecules of PTEN, leading to positive feedback that increased PTEN expression (Que et al., 2007; Winbanks et al., 2007). After bpv(pic) treatment, downstream pathways were activated to increase the expression level of mTOR and affect cell proliferation and differentiation. In nerve cells, the function of mTOR must be maintained within a certain range to promote cell differentiation. However, there is currently no consensus on whether inhibiting PTEN to increase mTOR expression Chappell et al., 2011 promotes or inhibits cell differentiation. Nerve regeneration and repair play important roles in nerve function recovery. NSCs are key cells in these processes because of their potential for self-renewal and multidirectional differentiation (Rueger and Androutsellis- Theotokis, 2013), although further research is needed to fully understand their involvement (Saha et al., 2012). Inhibiting PTEN expression was shown to increase the survival and proliferation of mesenchymal stem cells in myocardial infarction (Feng et al., 2020), while the proliferation of NSCs and neural progenitor cells is signiﬁcantly increased following PTEN deletion. Thus, the study of molecular mechanisms that affect NSC proliferation and differentiation is crucial to promoting the repair of neural function. PTEN is the ﬁrst tumor suppressor gene known to encode a protein with phosphatase activity. It plays an important role in a variety of diseases by affecting cell proliferation, differentiation, apoptosis, and metabolism, and achieves its physiological effects by interacting with a series of downstream effector molecules (Kuchay et al., 2017). mTOR is one such signal molecule in the PTEN signaling pathway, which is activated through phosphorylation and mediates a series of downstream molecules to promote the synthesis of cellular proteins and cell growth (Yoon and Chen, 2008). Bpv(pic) Enhanced the Expression of mTOR and PTEN in NSCs RT-PCR (Figure 4A) and western blotting (Figure 4B) were used to detect the expression of mTOR and PTEN at mRNA and protein levels, respectively. We observed signiﬁcantly increased expression of mTOR and PTEN in the intervention group compared with the control group (p < 0.05), with a greater increase seen in mTOR expression. June 2022 \| Volume 13 \| Article 907695 Frontiers in Pharmacology \| www.frontiersin.org 4 Liu et al. Neural Stem Cell FIGURE 4 \| mTOR and PTEN expression in NSCs 5 days after bpv (pic) intervention. (A) mRNA expression of mTOR and PTEN in rat NSCs. (B) Protein expression of mTOR and PTEN in rat NSCs. β-actin was used as a loading control. p < 0.05. FIGURE 4 \| mTOR and PTEN expression in NSCs 5 days after bpv (pic) intervention. (A) mRNA expression of mTOR and PTEN in rat NSCs. (B) Protein expression of mTOR and PTEN in rat NSCs. β-actin was used as a loading control. *p < 0.05. 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FUNDING This study was supported by the Scientiﬁc Research Program of Nantong (JCZ21028), the Scientiﬁc Research Program of Health and Planning Commission of Jiangsu (Z2019033), and the Scientiﬁc Research Program of Jiangsu Health Vocational College (JKC202009). CONCLUSION Guidelines. Appropriate measures were taken to minimize the use of animals as well as their suffering. Guidelines. Appropriate measures were taken to minimize the use of animals as well as their suffering. In summary, the PTEN inhibitor bpv(pic) promoted the proliferation of NSCs and their differentiation into neurons to some extent. This demonstrates the potential of bpv(pic) to be used in the recovery and treatment of CNS injuries. In summary, the PTEN inhibitor bpv(pic) promoted the proliferation of NSCs and their differentiation into neurons to some extent. This demonstrates the potential of bpv(pic) to be used in the recovery and treatment of CNS injuries. AUTHOR CONTRIBUTIONS XJL and YXG wrote the paper and conceived of and designed the experiments. YQC; JL and JHS analyzed the data. CG; SC and JRD collected and provided the samples for this study. All authors have read and agreed to the published version of the manuscript. ETHICS STATEMENT Animal experiments were approved by the Experimental Animal Ethics Committee of the Afﬁliated Haian Hospital of Nantong University. All animal experiments were performed in accordance with the recommendations of the National Institutes of Health Laboratory Animal Care and Use DISCUSSION Our ﬁndings suggest that bpv(pic) inhibits the expression of PTEN and promotes the migration and differentiation of NSC into neurons, thus enhancing the repair of central nervous system injuries. This should be explored in future work to investigate potential treatments of central nerve injury. 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https://openalex.org/W2916569432	https://europepmc.org/articles/pmc6459007?pdf=render	English	null	Learning Promotes Subfield-Specific Synaptic Diversity in Hippocampal CA1 Neurons	Cerebral cortex	2,019	cc-by	11,670	Learning Promotes Subﬁeld-Speciﬁc Synaptic Diversity in Hippocampal CA1 Neurons Y. Sakimoto1, J. Mizuno2, H. Kida1, Y. Kamiya4, Y. Ono5 and D. Mitsushima 1,2,3 Y. Sakimoto1, J. Mizuno2, H. Kida1, Y. Kamiya4, Y. Ono5 and D. Mitsushima 1,2,3 1Department of Physiology, Yamaguchi University Graduate School of Medicine, Yamaguchi 755-8505, Japan, 2Department of Physiology and Neuroscience, Kanagawa Dental University, Kanagawa 238-8580, Japan, 3The Research Institute for Time Studies, Yamaguchi University, Yamaguchi 753-8511, Japan, 4Uonuma Institute of Community Medicine, Niigata University Medical Hospital, Niigata 951-8520, Japan and 5Department of Electronics and Bioinformatics, Meiji University School of Science and Technology, Tokyo 101-8301, Japan Address correspondence to Dai Mitsushima, Department of Physiology, Yamaguchi University School of Medicine, 1-1-1 Minami-Kogushi Ube, Yamaguchi 755-8505, Japan. Email: mitsu@yamaguchi-u.ac.jp orcid.org/0000-0002-6931-7714 Address correspondence to Dai Mitsushima, Department of Physiology, Yamaguchi University School of Yamaguchi 755-8505, Japan. Email: mitsu@yamaguchi-u.ac.jp orcid.org/0000-0002-6931-7714 Y. Sakimoto and J. Mizuno equally contributed each other Y. Sakimoto and J. Mizuno equally contributed each other Abstract The hippocampus is functionally heterogeneous between the dorsal and ventral subﬁelds with left–right asymmetry. To determine the possible location of contextual memory, we performed an inhibitory avoidance task to analyze synaptic plasticity using slice patch-clamp technique. The training bilaterally increased the AMPA/NMDA ratio at dorsal CA3–CA1 synapses, whereas the training did not affect the ratio at ventral CA3–CA1 synapses regardless of the hemisphere. Moreover, sequential recording of miniature excitatory postsynaptic currents and miniature inhibitory postsynaptic currents from the same CA1 neuron clearly showed learning-induced synaptic plasticity. In dorsal CA1 neurons, the training dramatically strengthened both excitatory and inhibitory postsynaptic responses in both hemispheres, whereas the training did not promote the plasticity in either hemisphere in ventral CA1 neurons. Nonstationary ﬂuctuation analysis further revealed that the training bilaterally increased the number of AMPA or GABAA receptor channels at dorsal CA1 synapses, but not at ventral CA1 synapses, suggesting functional heterogeneity of learning-induced receptor mobility. Finally, the performance clearly impaired by the bilateral microinjection of plasticity blockers in dorsal, but not ventral CA1 subﬁelds, suggesting a crucial role for contextual learning. The quantiﬁcation of synaptic diversity in speciﬁed CA1 subﬁelds may help us to diagnose and evaluate cognitive disorders at the information level. Key words: AMPA receptor, contextual learning, GABAA receptor, self-entropy, synaptic plasticity © The Author(s) 2019. Published by Oxford University Press. 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 reuse, distribution, and reproduction in any medium, provided the original work is properly cited. O R I G I N A L A R T I C L E O R I G I N A L A R T I C L E doi: 10.1093/cercor/bhz022 Advance Access Publication Date: 23 February 2019 Original Article doi: 10.1093/cercor/bhz022 Advance Access Publication Date: 23 February 2019 Original Article doi: 10.1093/cercor/bhz022 Advance Access Publication Date: 23 February 2019 Original Article Inhibitory Avoidance Task First, we analyzed learning-induced synaptic plasticity in 4 CA1 subﬁelds to analyze the learning-induced synaptic plastic- ity. Second, we sequentially recorded miniature EPSCs (mEPSCs) and miniature IPSCs (mIPSCs) in the same neuron to specify the CA1 subﬁeld of learning-created synaptic diversity. Considering that each presynaptic vesicle contains approxi- mately 2000 glutamate (Ryan et al. 1993; Hori and Takahashi 2012) or 2500 GABA molecules (Telgkamp et al. 2004; Pugh and Raman 2005), the mE(I)PSC analysis allows for the quantiﬁca- tion of postsynaptic currents and plasticity. Nonstationary ﬂuc- tuation analysis further revealed subﬁeld-speciﬁc evidence of learning at a single-channel level. Moreover, by analyzing the appearance probability of the synaptic strength in each neuron, we proposed a new approach to quantify learning-induced syn- aptic diversity as self-entropy increases after the training. The learning clearly increased the cell-speciﬁc self-entropy levels in dorsal, but not ventral CA1 subﬁelds, and local blockade of the synaptic plasticity blocked the learning in dorsal but not ventral CA1 subﬁelds, suggesting contributory CA1 subﬁelds at the information level. Since learning is known to modulate both excitatory and inhibitory synaptic plasticity in key brain areas such as hippocampus (Mitsushima et al. 2013), amyg- dala (Lin et al. 2011; Ganea et al. 2015), or cortical areas (Ghosh et al. 2015, 2016; Kida et al. 2016), this approach may help us to diagnose and evaluate cognitive disorders in mul- tiple brain regions. The IA training apparatus (length: 33 cm, width: 58 cm, height: 33 cm) was a 2-chambered box consisting of a lighted safe side and a dark shock side separated by a trap door (Fig. 1A; Mitsushima et al. 2011, 2013). For training, rats were placed in the light side of the box facing a corner opposite the door. After the trap door was opened, the rats could enter the dark box at will. The latency before entering the novel dark box was mea- sured as a behavioral parameter (latency before IA learning). Soon after the animals entered the dark side, we closed the door and applied a scrambled electrical foot-shock (2 s, 1.6 mA) via electriﬁed steel rods in the ﬂoor of the box. The rats were kept in the dark compartment for 10 s before being returned to their home cage. Untrained control rats were not moved from their home cages. Thirty minutes after the procedure described above, the rats were placed in the light side. Inhibitory Avoidance Task The latency before entering the dark box was measured as an indicator of learning perfor- mance (latency after IA learning). Introduction 1998). Moreover, the acquisition of some hippocampal- dependent tasks seems to require the left–right asymmetry of the hippocampal circuit (Goto et al. 2010). Using an inhibitory avoidance (IA) task with a hippocampus-dependent contextual learning paradigm (Izquierdo et al. 1998), we previously found that contextual learning requires synaptic plasticity for both The hippocampus is functionally heterogeneous between the dorsal and ventral subﬁelds (Fanselow and Dong 2010), with left–right asymmetry (Shinohara et al. 2013). Dorsal subﬁelds seem to serve cognitive functions, whereas ventral subﬁelds correspond to the affective hippocampus (Moser and Moser © The Author(s) 2019. Published by Oxford University Press. 2184 \| Cerebral Cortex, 2019, Vol. 29, No. 5 2184 the National Institute of Health (NIH Publication No. 85-23, revised 1996). excitatory and inhibitory inputs at CA1 synapses (Mitsushima et al. 2011, 2013). However, there is no synaptic evidence to prove the location of encoded memory within a broad CA1 area. Drug Injection Under sodium pentobarbital anesthesia (30–50 mg/kg, i.p.), a stainless-steel guide cannula (outer diameter, 0.51 mm) was implanted stereotaxically into the just above the target region of the dorsal or ventral hippocampus. The experiment was per- formed 1–3 days after the implantation. After cannula implan- tation, a stylet was inserted into the guide until drug injection. Materials and Methods On the day of the experiment, the stylet was replaced with 1.0 mm longer injector without restraint animals in their home cage (outer diameter 0.31 mm). The coordinates of dorsal CA1 were 3.0 mm posterior to bregma, 2.0 mm lateral to the midline, and 3.8 mm below the surface of the skull. The coordinates of ventral CA1 were 4.2 mm posterior to bregma, 5.5 mm lateral to the midline, and 5.5 mm below the surface of the skull. Animals Young male Sprague-Dawley rats (postnatal 28–31 days of age) were used. After weaning, same sex groups of 2–3 rats were housed in plastic cages (length 25 cm, width 40 cm, height 25 cm) at a constant temperature of 23 ± 1 °C under a constant cycle of light and dark (light on: 8:00 A.M. to 8:00 P.M.). But, the rats were individually housed at least 24 h prior to the experi- ment to avoid any episodic experience. Food (MF, Oriental Yeast Co. Ltd, Tokyo Japan) and tap water were available ad libitum in all experimental periods. All animal housing and sur- gical procedures followed the guidelines of the Institutional Animal Care and Use Committee of Kanagawa Dental University and Yamaguchi University. The guidelines comply with the Guide for the Care and Use of Laboratory Animals published by Approximately 20 min before the IA learning procedure, saline, NMDA receptor antagonist (3 μg/μL per side, d-AP5, Sigma Chemical Co., St. Louis, MO), or nicotinic α7 receptor antagonist (40 μg/μL per side, methyllycaconitine citrate, Sigma) was directly injected into the CA1 through ﬁne ﬂexible silicone tubing (o.d. 0.5 mm, Kaneka Medix Co. Osaka, Japan) without restraining the animals. We used AP5 to block AMPA receptor-mediated plastic- ity at excitatory synapses (Morris et al. 1986; Park et al. 2004), and also used Mla to block GABAA receptor-mediated plasticity Figure 1. Inhibitory avoidance (IA) task. (A) IA training apparatus. (B) Foot-shock increased the latency to entering the dark box. The number of rats in each group is shown at the bottom of each bar. Error bars indicate + standard error of the mean (SEM). P < 0.01 versus before the training. ratus. (B) Foot-shock increased the latency to entering the dark box. The number of rats in each group is ard error of the mean (SEM) P < 0 01 versus before the training Miniature Postsynaptic Current Recordings Miniature excitatory postsynaptic currents (mEPSCs) are thought to correspond to the responses elicited by the presynaptic release of a single vesicle of glutamate. In contrast, miniature inhibitory postsynaptic currents (mIPSCs) are thought to correspond of GABA. Increase in the amplitudes of mEPSCs and mIPSCs reﬂect postsynaptic transmission strengthening, while increase in the event frequency reﬂects increases in the number of func- tional synapses or the presynaptic release probability. One hour after the paired foot-shock, rats were anesthetized with pentobarbital and acute brain slices prepared (Mitsushima et al. 2011, 2013). We used naïve rats for untrained group, which were injected with the same dose of anesthesia in their home cage. The results in unpaired or walk-through controls were reported previously (Mitsushima et al. 2013). For the whole-cell recordings (Kida et al. 2016), the brains were quickly perfused with ice-cold dissection buffer (25.0 mM NaHCO3, 1.25 mM NaH2PO4, 2.5 mM KCl, 0.5 mM CaCl2, 7.0 mM MgCl2, 25.0 mM glucose, 90 mM choline chloride, 11.6 mM ascorbic acid, 3.1 mM pyruvic acid) and gassed with 5% CO2/95% O2. Coronal (target CA1 area: AP −3.8 mm, DV 2.5 mm, LM ± 2.0 mm) or horizontal brain slices (target CA1 area: AP −5.2 mm, DV 7.7 mm, LM ± 5.8 mm) were cut (350 μm, Leica vibratome, VT-1200) in dissection buffer and transferred to physiological solution (22–25 °C, 114.6 mM NaCl, 2.5 mM KCl, 26 mM NaHCO3, 1 mM NaH2PO4, 10 mM glucose, 4 mM MgCl2, 4 mM CaCl2, pH 7.4, gassed with 5% CO2/95% O2). The recording chamber was perfused with physiological solution containing 0.1 mM picro- toxin and 4 μM 2-chloroadenosine at 22–25 °C. For the mEPSC and mIPSC recordings, we used the physiological solution con- taining 0.5 μM TTX to block Na+ channels. For the miniature recordings, the mEPSCs (−60 mV holding potential) and mIPSCs (0 mV holding potential) were recorded sequentially for 5 min in the same CA1 neuron. The miniature events were detected using the software, and the events above 10 pA were used for the analysis. We recorded at least for 5 min to determine the events frequency of mEPSCs or mIPSCs. The amplitudes of the events were averaged to obtain the mean amplitude. Bath application of an AMPA receptor blocker (CNQX, 10 μM) or GABAA receptor blocker (bicuculline methio- dide, 10 μM) consistently blocked the mEPSC or mIPSC events, respectively. Nonstationary Fluctuation Analysis AMPA receptor-mediated evoked EPSCs and GABAA receptor- mediated mIPSCs were analyzed by nonstationary ﬂuctuation analysis (Ghosh et al. 2015; Ono et al. 2016). To isolate ﬂuctua- tions in current decay due to stochastic channel gating, the mean waveform was scaled to the peak of individual E(I)PSCs. The requirements for such analysis include a stable current decay time course throughout the recording and an absence of any correlation between the decay time course and peak ampli- tude. The relationship between the peak-scaled variance and the mean current is given by the following equation: Patch recording pipettes (4–7 MΩ) were ﬁlled with intracellu- lar solution (127.5 mM cesium methanesulfonate, 7.5 mM CsCl, 10 mM Hepes, 2.5 mM MgCl2, 4 mM Na2ATP, 0.4 mM Na3GTP, 10 mM sodium phosphocreatine, 0.6 mM EGTA at pH 7.25). Whole-cell recordings were obtained from CA1 pyramidal neu- rons from the rat hippocampus using an Axopatch 700 A ampli- ﬁer (Axon Instruments). The whole-cell patch-clamp data were collected with Clampex 10.4, and the data were analyzed using Clampﬁt 10.4 software (Axon Instruments). σ = − + iI I N b / l 2 2 The AMPA/NMDA Ratio where σ2 is the variance, I is the mean current, N is the number of channels activated at the peak of the mean current, i is the unitary conductance, and bl is the background variance. In our experiments, 31–69 EPSCs and 14–133 IPSCs were analyzed from selected epochs in each of the cells in which there was no correlation between current decay (63% decay time) and peak amplitude (P > 0.05, Spearman’s rank-order correlation test). The weighted mean channel current can be estimated by ﬁtting the full parabola or initial slope of the relationship. All the analysis was done using MATLAB software (MathWorks, MA, USA). The number of channels was further divided by the cor- responding value of mean E(I)PSC amplitude to obtain the sin- gle channel current. The AMPA/NMDA ratio is conventional way to evaluate post- synaptic plasticity at glutamatergic excitatory synapses. Since concomitant increases in both components may not change the ratio, further analysis of AMPA evoked responses is neces- sary to elucidate the receptor-speciﬁc plasticity (i.e., I/O curve for evoked EPSCs, amplitude of miniature AMPA receptor- mediated current, or further ﬂuctuation analysis of the cur- rent). The recording chamber was perfused with physiological solution bubbled with the gas mixture and maintain the tem- perature at 22–25 °C. Then, we added 0.1 mM picrotoxin to the solution to block the GABAA-mediated response. We also added 4 μM 2-chloroadenosine to stabilize the evoked neural response. The patch recording pipettes were ﬁlled with the intracellular solution for voltage-clamp recordings. The resistance of the recording pipette in the aCSF was between 4 and 7 MΩ. Electrophysiological Recordings We recently published detailed technical protocol of slice-patch clamp technique for analyzing learning-induced synaptic plas- ticity with a short demonstration movie (Kida et al. 2017). Using the technique, we examined learning-induced synaptic plastic- ity in dorsal or ventral CA1 neurons. Figure 1. Inhibitory avoidance (IA) task. (A) IA training apparatus. (B) Foot-shock increased the latency to entering the dark box. The number of rats in each group is shown at the bottom of each bar. Error bars indicate + standard error of the mean (SEM). *P < 0.01 versus before the training. Learning Promotes Synaptic Diversity in CA1 Sakimoto et al. 2185 cells. The electrically evoked EPSC amplitudes were measured from the peak of the postsynaptic current to the basal current level immediately before electrical stimulation. The stimulus intensity was increased until a synaptic response with an amplitude >−10 pA was recorded. AMPA/NMDA ratios were cal- culated as the ratio of the peak current at −60 mV to the current at +40 mV 150 ms after stimulus onset (40–60 traces averaged for each holding potential). at inhibitory synapses. Mla is known to block acetylcholine- induced strengthen of GABAA receptor-mediated postsynaptic current in CA1 pyramidal neurons (Mitsushima et al. 2013; Townsend et al. 2016). Miniature Postsynaptic Currents in Dorsal CA1 Neurons • Similarly, probability distribution of N data of the parameter (neuron N) was calculated using the formula [=EXP(−(((data of neuron N −any point)/h)^2/2))/SQRT(2 PI())]. To further analyze the learning-dependent synaptic plasticity, we recorded mEPSC or mIPSC in the presence of 0.5 μM TTX on both sides of the dorsal hippocampus (Fig. 3A). By changing the mem- brane potential, we sequentially recorded mEPSCs (at −60 mV) and mIPSCs (at 0 mV) from the same neuron as reported previously (Mitsushima et al. 2013). The postsynaptic currents are thought to correspond to the response elicited by a single vesicle of glutamate or GABA. In contrast, the number of synapses is known to affect the frequency of the events (Pinheiro and Mulle 2008). • Sum all probability distribution from neuron 1 to N, and the integral value was normalized to 1. Based on the probability distribution, we calculated individual appearance probability of all recorded neurons. Then, the appear- ance probability of the neuron was converted to the self-entropy using Shannon’s formula (= −LOG [appearance probability of the neuron, 2]) (Fig. 3F). For graphic expression, the distribution was visualized 2-dimensionally in the R software environment (R Foundation for Statistical Computing, Vienna, Austria) (Figs 3C, G,L,P and 4B,F,J,N). At dorsal CA1 synapses, the strength of AMPA receptor- mediated excitatory inputs versus GABAA receptor-mediated inhibitory inputs was measured in each neuron and plotted 2-dimensionally (Fig. 3B). The Kernel analysis revealed the distribution of appearance probability (Fig. 3C). Although untrained rats exhibited low and narrow distribution range, IA-trained rats had a broad distribution suggesting a diversity of synaptic input onto CA1 neurons. For mEPSCs, 2-way ANOVA revealed a signiﬁcant main effect of training (F1,104 = 18.780, P < 0.0001), but the main effect of laterality (F1,104 = 2.237, P = 0.14) or interaction (F1,104 = 0.998, P = 0.32) was not signiﬁcant (Fig. 3D). For mIPSCs, 2-way ANOVA revealed a signiﬁcant main effect of training (F1,104 = 44.627, P < 0.0001), but the main effect of Inhibitory Avoidance Task ⎜ ⎟ ⎛ ⎝ ⎞ ⎠ ∑ ( ) = − = P x nh K x X h 1 n i n i 1 To investigate a possible location of the contextual memory in 4 CA1 subﬁelds, rats were subjected to an IA task (Fig. 1A; Izquierdo et al. 1998; Mitsushima et al. 2011, 2013). In this learn- ing paradigm, rats were allowed to cross from a light box to a dark box, where an electric foot-shock (1.6 mA, 2 s) was deliv- ered. Half an hour after the IA task, we measured the latency in the illuminated box as contextual learning performance. With paired foot-shock, the latency was much longer after training than before the training (t11 = 14.0, P < 0.0001). where K is a smooth function called the Gaussian kernel func- tion and h > 0 is the smoothing bandwidth that controls the amount of smoothing. We chose Silverman’s reference band- width or Silverman’s rule of thumb (Silverman 1986; Sheather 2004). It is given by the following equation: = − h An 0.9 1/5 where A = min (standard deviation, interquartile range/1.34). By normalizing integral value in untrained controls, we found the distribution of appearance probability at any point. Then, we calculated the appearance probability at selected points. All data points for probability in untrained and trained rats were converted to self-entropy (bit) using the Shannon entropy con- cept, deﬁned from the Information Theory (Shannon 1948). AMPA/NMDA Ratio To specify the subﬁelds where the contextual learning drives AMPA receptors into CA3–CA1 synapses, we measured the AMPA- to NMDA-type glutamate receptor response ratio in the dorsal or ventral hippocampus of both hemispheres. Figure 2A shows experimental design and the recording location of dorsal CA1 neurons. At dorsal CA3–CA1 synapses, 2-way ANOVA revealed a signiﬁcant main effect of training (F1,44 = 12.637, P = 0.0009), but the main effect of laterality (F1,44 = 0.007, P = 0.93) or interaction (F1,44 = 0.156, P = 0.70) was not signiﬁcant (Fig. 2B,C). Figure 2D shows experimental design and the recording location of ventral CA1 neurons. At ventral CA3–CA1 synapses, the main effects of training (F1,39 = 0.960, P = 0.22), laterality (F1,39 = 1.641, P = 0.21), and interaction (F1,39 = 0.022, P = 0.88) were not signiﬁcant (Fig. 2E,F). These results suggest that the training bilaterally strengthened AMPA receptor-mediated CA3–CA1 synapses in dorsal CA1 neurons, regardless of the hemisphere, but not in ventral CA1 neurons. To calculate using spreadsheet software, the data of 4 mini- ature parameters were summarized in 4 different sheet, and we obtained the bandwidth (h) of individual parameter in untrained group using a formula [=0.9 STDEV (neuron 1, neuron 2,,, neuron N)/COUNT (neuron 1, neuron 2,,, neuron N) ^ (1/5)]. Then, using the data of untrained group, we calculated the dis- tribution of appearance probability as follows: • Probability distribution of ﬁrst data of a parameter (neuron 1) was calculated using a formula [=EXP(−(((data of neuron 1 − any point)/h)^2/2))/SQRT (2 * PI())]. • Also, probability distribution of second data of the parameter (neuron 2) was calculated using the formula [=EXP(−(((data of neuron 2 −any point)/h)^2/2))/SQRT(2 * PI())]. Self-Entropy Analysis We used standard spreadsheet software (Excel 2010, Microsoft Co., Redmond, WA, USA) to calculate the self-entropy per neu- ron. First, we obtained 4 miniature parameters (mean mEPSC To analyze the function of CA3–CA1 synapses, bipolar tung- sten stimulating electrodes (Unique Medical Co., Ltd., Tokyo, Japan) were placed in CA1 ~200–300 μm lateral from recorded Cerebral Cortex, 2019, Vol. 29, No. 5 2186 amplitude, mean mIPSC amplitude, mean mEPSC frequency, and mean mIPSC frequency) in individual CA1 pyramidal neu- rons. Then, we determined the distribution of appearance probability of 4 miniature parameters separately using 1- dimensional Kernel density analysis. Geometric/topographic feature of the appearance probability was calculated using Kernel density analysis. Let X1, X2,…, Xn denote a sample of size n from real observations. The Kernel density estimate of P at the point x is given by the following equations: to evaluate the difference in miniature responses between dorsal and ventral synapses. The Shapiro–Wilk test and F-test were used for normality and equality of variance, respectively. Because the self-entropy data had large variations within a group, we per- formed log (1 + x) transformation prior to the analysis (Mitsushima et al. 1994). P < 0.05 was considered signiﬁcant. Statistical Analysis We used the paired t test to analyze IA latency and unpaired t test to analyze estimated open channel numbers. The AMPA/ NMDA ratio, mEPSC, mIPSC, and self-entropy were analyzed using 2-way factorial ANOVA in which the between-group factors were laterality and training. We used one-way factorial ANOVA Learning Promotes Synaptic Diversity in CA1 Sakimoto et al. \| 2187 laterality (F1,104 = 0.724, P = 0.40) or interaction (F1,104 = 0.299, P = P = 0.96) was not signiﬁcant (Fig. 3H). Thus, the training did Figure 2. AMPA/NMDA ratio in 4 CA1 subﬁelds. (A) Experimental design and coronal section of dorsal CA1 for patch-clamp analysis. (B) The AMPA/NMDA ratio and cumulative distribution in dorsal left and (C) right CA1 neurons. The trained rats had signiﬁcantly higher ratios at dorsal CA3–CA1 synapses than untrained rats. (D Experimental design and horizontal section of ventral CA1 for patch-clamp analysis. (E) The AMPA/NMDA ratio and distribution in ventral left and (F) right CA1 neu rons. IA training did not affect the ratio of ventral CA3–CA1 synapses. Green squares indicate the recorded CA1 subﬁelds in the dorsal and ventral hippocampus Upper insets show representative traces. The number of cells in each group is shown at the bottom of each bar. Vertical bar = 40 pA; horizontal bar = 50 ms. Error bars indicate + SEM. P < 0.05, P < 0.01 versus untrained. Figure 2. AMPA/NMDA ratio in 4 CA1 subﬁelds. (A) Experimental design and coronal section of dorsal CA1 for patch-clamp analysis. (B) The AMPA/NMDA ratio and cumulative distribution in dorsal left and (C) right CA1 neurons. The trained rats had signiﬁcantly higher ratios at dorsal CA3–CA1 synapses than untrained rats. (D) Experimental design and horizontal section of ventral CA1 for patch-clamp analysis. (E) The AMPA/NMDA ratio and distribution in ventral left and (F) right CA1 neu- rons. IA training did not affect the ratio of ventral CA3–CA1 synapses. Green squares indicate the recorded CA1 subﬁelds in the dorsal and ventral hippocampus. Upper insets show representative traces. The number of cells in each group is shown at the bottom of each bar. Vertical bar = 40 pA; horizontal bar = 50 ms. Error bars indicate + SEM. P < 0.05, *P < 0.01 versus untrained. Figure 2. AMPA/NMDA ratio in 4 CA1 subﬁelds. (A) Experimental design and coronal section of dorsal CA1 for patch-clamp analysis. Statistical Analysis (B) The AMPA/NMDA ratio and cumulative distribution in dorsal left and (C) right CA1 neurons. The trained rats had signiﬁcantly higher ratios at dorsal CA3–CA1 synapses than untrained rats. (D) Experimental design and horizontal section of ventral CA1 for patch-clamp analysis. (E) The AMPA/NMDA ratio and distribution in ventral left and (F) right CA1 neu- rons. IA training did not affect the ratio of ventral CA3–CA1 synapses. Green squares indicate the recorded CA1 subﬁelds in the dorsal and ventral hippocampus. Upper insets show representative traces. The number of cells in each group is shown at the bottom of each bar. Vertical bar = 40 pA; horizontal bar = 50 ms. Error bars indicate + SEM. P < 0.05, *P < 0.01 versus untrained. P = 0.96) was not signiﬁcant (Fig. 3H). Thus, the training did not affect the balance of mEPSC versus mIPSC amplitudes, suggesting the balance of excitatory versus inhibitory input strength onto dorsal CA1 neurons. laterality (F1,104 = 0.724, P = 0.40) or interaction (F1,104 = 0.299, P = 0.59) was not signiﬁcant (Fig. 3E). These results suggest that the training bilaterally strengthened both excitatory and inhibitory synapses onto dorsal CA1 neurons, regardless of the hemisphere. laterality (F1,104 = 0.724, P = 0.40) or interaction (F1,104 = 0.299, P = 0.59) was not signiﬁcant (Fig. 3E). These results suggest that the training bilaterally strengthened both excitatory and inhibitory synapses onto dorsal CA1 neurons, regardless of the hemisphere. The balance of excitatory/inhibitory (E/I) inputs was obtained by dividing the mean mEPSC amplitude by the mean mIPSC amplitude of the same neuron. For the E/I balance of miniature amplitudes, the main effect of training (F1,104 = 0.203, P = 0.65), laterality (F1,104 = 2.469, P = 0.12), or interaction (F1,104 = 0.002, The balance of excitatory/inhibitory (E/I) inputs was obtained by dividing the mean mEPSC amplitude by the mean mIPSC amplitude of the same neuron. For the E/I balance of miniature amplitudes, the main effect of training (F1,104 = 0.203, P = 0.65), laterality (F1,104 = 2.469, P = 0.12), or interaction (F1,104 = 0.002, Self-Entropy in Dorsal CA1 Neurons Based on the information theory of Shannon (1948), we calcu- lated appearance probability of the mean amplitudes of 2188 \| Cerebral Cortex, 2019, Vol. 29, No. 5 2188 Figure 3. Diversity of mEPSC/mIPSC amplitudes and self-entropy per neuron after training. (A) Representative traces of mEPSCs and mIPSCs in dorsal left and right CA1 neurons. mEPSCs and mIPSCs were sequentially recorded from the same CA1 neuron in the presence of tetrodotoxin (0.5 μM). (B) 2-Dimensional plot of the amplitudes of mean mEPSC and mIPSC. The circle or square plot indicates the data from a right or left CA1 neuron, respectively. (C) Kernel density analysis visualized the distribu- tion of appearance probability at any point. (D) Mean amplitudes of mEPSCs and (E) mIPSCs in dorsal CA1 neurons. IA training signiﬁcantly increased both amplitudes in both hemispheres. (F) The self-entropy of each dot and (G) visualized density in dorsal CA1 neurons. (H) E/I balance of miniature amplitudes and (I) mean self-entropy per dorsal CA1 neuron. (J) Representative traces in ventral left and right CA1 neurons. (K) 2-Dimensional plot of the mean mEPSC and mIPSC amplitudes in ventral CA1 neurons and (L) and visualized distribution of appearance probability at any point. (M) Mean amplitudes of the mEPSCs and (N) mIPSCs in ventral CA1 neurons. IA train- ing affected neither of them, regardless of the hemisphere. (O) The self-entropy of each dot and (P) visualized density in ventral CA1 neurons. (Q) E/I balance of miniature amplitudes and (R) mean self-entropy per ventral CA1 neuron. E = mEPSC; I = mIPSC, vertical bar = 20 pA; horizontal bar = 200 ms. Gray indicates untrained groups and black is trained groups. The number of cells in each group is shown at the bottom of each bar. Error bars indicate ± SEM. P < 0.05, *P < 0.01 versus untrained. i i i f / li d d lf f i i ( ) i f d i d l l f d i h Figure 3. Diversity of mEPSC/mIPSC amplitudes and self-entropy per neuron after training. (A) Representative traces of mEPSCs and mIPSCs in dorsal left and right CA1 neurons. mEPSCs and mIPSCs were sequentially recorded from the same CA1 neuron in the presence of tetrodotoxin (0.5 μM). (B) 2-Dimensional plot of the amplitudes of mean mEPSC and mIPSC. The circle or square plot indicates the data from a right or left CA1 neuron, respectively. Self-Entropy in Dorsal CA1 Neurons (C) Kernel density analysis visualized the distribu- tion of appearance probability at any point. (D) Mean amplitudes of mEPSCs and (E) mIPSCs in dorsal CA1 neurons. IA training signiﬁcantly increased both amplitudes in both hemispheres. (F) The self-entropy of each dot and (G) visualized density in dorsal CA1 neurons. (H) E/I balance of miniature amplitudes and (I) mean self-entropy per dorsal CA1 neuron. (J) Representative traces in ventral left and right CA1 neurons. (K) 2-Dimensional plot of the mean mEPSC and mIPSC amplitudes in ventral CA1 neurons and (L) and visualized distribution of appearance probability at any point. (M) Mean amplitudes of the mEPSCs and (N) mIPSCs in ventral CA1 neurons. IA train- ing affected neither of them, regardless of the hemisphere. (O) The self-entropy of each dot and (P) visualized density in ventral CA1 neurons. (Q) E/I balance of miniature amplitudes and (R) mean self-entropy per ventral CA1 neuron. E = mEPSC; I = mIPSC, vertical bar = 20 pA; horizontal bar = 200 ms. Gray indicates untrained groups and black is trained groups. The number of cells in each group is shown at the bottom of each bar. Error bars indicate ± SEM. P < 0.05, *P < 0.01 versus untrained. neuron was calculated as the self-entropy and plotted 2-dimensionally (Fig. 3F). For example, a point with high appearance probability (around the mean level of mE(I)PSC amplitude) indicates low self-entropy, whereas a point with mEPSCs and mIPSCs. First, we found the distribution of appearance probability in untrained controls (Fig 3C, left), and then we analyzed the appearance probability of all recorded neurons one-by-one. Each probability of single Learning Promotes Synaptic Diversity in CA1 Sakimoto et al. \| 21 2189 the frequency of mEPSC versus mIPSC events was measured in each neuron and plotted 2-dimensionally (Fig. 4A). The Kernel analysis revealed the distribution of appearance probability (Fig. 4B). Although untrained rats exhibited low and narrow dis- tribution range, IA-trained rats had a broad distribution sug- gesting a diversity of the number of functional synapses onto a single CA1 neuron. For mEPSCs, 2-way ANOVA revealed a sig- niﬁcant main effect of training (F1,104 = 6.942, P = 0.0097), but the main effect of laterality (F1,104 = 0.023, P = 0.88) or interac- tion (F1,104 = 0.035, P = 0.85) was not signiﬁcant (Fig. 4C). Miniature Postsynaptic Currents in Ventral CA1 Neurons Conversely in ventral CA1 neurons, IA training did not affect the miniature responses (Fig. 3K). For mEPSCs, the main effects of training (F1,103 = 0.002, P = 0.96), laterality (F1,103 = 0.0002, P = 0.99), and interaction (F1,103 = 1.748, P = 0.19) were not signiﬁ- cant (Fig. 3M). For mIPSCs, the main effects of training (F1,103 = 0.052, P = 0.82), laterality (F1,103 = 0.833, P = 0.36), and interac- tion (F1,103 = 1.025, P = 0.31) were not signiﬁcant (Fig. 3N). The Kernel analysis visualized the distribution of appearance prob- ability (Fig. 3L). Thus, the training did not affect the amplitudes in either hemisphere. These results suggest that the training strengthened neither excitatory nor inhibitory synapses onto ventral CA1 neurons, regardless of the hemisphere. Self-Entropy of the Frequency in Dorsal CA1 Neurons Using the distribution appearance probability in untrained con- trols (Fig. 4B, left), we analyzed the appearance probability at selected points. The probabilities at all data points were calcu- lated as the self-entropy and plotted 2-dimensionally (Fig. 4E). Although all recorded neurons exhibited different self-entropy each other, the Kernel analysis further visualized the density distribution (Fig. 4F). IA training dramatically increased the amount of information per dorsal CA1 neurons (Fig. 4H). For the E/I balance of miniature amplitudes, the main effects of training (F1,103 = 0.244, P = 0.62), laterality (F1,103 = 0.069, P = 0.79), and interaction (F1,103 = 2.287, P = 0.13) were not signiﬁcant (Fig. 3Q). The training did not affect the balance of mEPSC versus mIPSC amplitudes, suggesting the balance of excitatory versus inhibitory input strength onto ventral CA1 neurons. Self-entropy in the mEPSC frequency exhibited a signiﬁcant main effect of training (F1,104 = 7.944, P = 0.0058), but the main effect of laterality (F1,104 = 0.019, P = 0.89) or interaction (F1,104 = 0.067, P = 0.80) was not signiﬁcant (Fig. 4E). Similarly, self- entropy in the mIPSC frequency exhibited a signiﬁcant main effect of training (F1,104 = 6.753, P = 0.0107), but the main effect of laterality (F1,104 = 0.061, P = 0.81) or interaction (F1,104 = 0.001, P = 0.97) was not signiﬁcant (Fig. 4E). Thus, the training clearly increased the self-entropy of dorsal CA1 neurons in both hemi- spheres, and the Kernel analysis further visualized the density distribution (Fig. 4F). The average level was 11.5 ± 0.2 bits in untrained rats, whereas the trained rats showed 54.5 ± 21.4 bits per single CA1 neuron (Fig. 4H). Self-Entropy in Dorsal CA1 Neurons For mIPSCs, 2-way ANOVA revealed a signiﬁcant main effect of training (F1,104 = 13.893, P = 0.0003), but the main effect of later- ality (F1,104 = 1.760, P = 0.19) or interaction (F1,104 = 0.054, P = 0.82) was not signiﬁcant (Fig. 4D). These results suggest that the training increased in the number of excitatory and inhibitory synapses onto dorsal CA1 neurons in both hemispheres. very rare probability (a deviated point of mE(I)PSC ampli- tude) indicates high self-entropy. We found that all recorded neurons exhibited different self- entropy each other (Fig. 3F). In the dorsal CA1, self-entropy in the excitatory synapse exhibited a signiﬁcant main effect of training (F1,104 = 9.322, P = 0.0029), but the main effect of lateral- ity (F1,104 = 1.229, P = 0.27) or interaction (F1,104 = 0.879, P = 0.35) was not signiﬁcant (Fig. 3F). Similarly, self-entropy in the inhib- itory synapse exhibited a signiﬁcant main effect of training (F1,104 = 21.393, P < 0.0001), but the main effect of laterality (F1,104 = 1.205, P = 0.27) or interaction (F1,104 = 0.007, P = 0.93) was not signiﬁcant (Fig. 3F). The Kernel analysis further visual- ized the density distribution (Fig. 3G). Thus, the training clearly increased the self-entropy of dorsal CA1 neurons in both hemi- spheres. The average level was 13.4 ± 0.2 bits in untrained rats, whereas IA-trained rats showed 30.3 ± 8.0 bits per single CA1 neuron (Fig. 3I). The balance of excitatory/inhibitory (E/I) frequency was obtained by dividing the mean mEPSC frequency by the mean mIPSC frequency of the same neuron. For the E/I balance of min- iature frequency, the main effect of training (F1,104 = 0.198, P = 0.66), laterality (F1,104 = 0.149, P = 0.70), or interaction (F1,104 = 0.78, P = 0.38) was not signiﬁcant (Fig. 4G). Thus, the training did not affect the balance of mEPSC versus mIPSC frequency, sug- gesting the balance of the number of excitatory versus inhibitory synapses onto dorsal CA1 neurons. Self-Entropy in Ventral CA1 Neurons Using the distribution of appearance probability in untrained controls (Fig 3L, left), we calculated the self-entropy of all recorded neurons one-by-one (Fig. 3O). We found all recorded neurons exhibited different self-entropy each other. In ventral CA1 neurons, self-entropy in the excitatory synapse did not exhibit a signiﬁcant main effect of training (F1,103 = 0.001, P = 0.97), laterality (F1,103 = 0.356, P = 0.55), or interaction (F1,103 = 0.930, P = 0.34). Similarly, self-entropy in the inhibitory synapse did not exhibit a signiﬁcant main effect of training (F1,103 = 1.284, P = 0.26), laterality (F1,103 = 1.158, P = 0.28), or interaction (F1,103 = 2.023, P = 0.16). Thus, the training did not affect the self-entropy in either hemisphere, and the visualized density distribution was shown in Figure 3P. The average levels of self- entropy were 15.0 ± 0.2 bits (untrained) and 14.9 ± 0.3 bits (IA- trained) per single CA1 neuron (Fig. 3R). Frequencies of the mE(I)PSC Events in Ventral CA1 Neurons IA training did not affect the frequency in ventral CA1 neu- rons. The frequency of mEPSC versus mIPSC events was mea- sured in each neuron and plotted 2-dimensionally (Fig. 4I). Ventral CA1 neurons exhibited relatively wide distribution range in both untrained and IA-trained rats, and the Kernel analysis showed the distribution of appearance probability (Fig. 4J). For mEPSCs, the main effects of training (F1,103 = 0.017, P = 0.90), laterality (F1,103 = 2.144, P = 0.15), and interac- tion (F1,103 = 0.22, P = 0.64) were not signiﬁcant (Fig. 4K). For mIPSCs, the main effects of training (F1,103 = 0.866, P = 0.35), Frequencies of the mE(I)PSC Events in Dorsal CA1 Neurons The number of functional synapses is known to affect the fre- quency of the mEPSC/mIPSC events. At dorsal CA1 synapses, 2190 \| Cerebral Cortex, 2019, Vol. 29, No. 5 2190 Figure 4. Diversity of mEPSC/mIPSC frequency and self-entropy per neuron after training. (A) 2-Dimensional plot of the frequency of mEPSCs and mIPSCs in dorsal CA1 neurons. The circle or square plot indicates the data from a right or left CA1 neuron, respectively. (B) Kernel density analysis visualized the distribution of appearance prob- ability. (C) Mean frequencies of mEPSCs and (D) mIPSCs in dorsal CA1 neurons. IA training signiﬁcantly increased both frequencies in both hemispheres. (E) The self- entropy of each dot and (F) visualized density in dorsal CA1 neurons. (G) E/I balance of miniature frequencies and (H) mean self-entropy per dorsal CA1 neuron. (I) 2- Dimensional plot of the self-entropy of the frequencies in ventral CA1 neurons. The circle or square plot indicates the data from right or left CA1 neuron, respectively. (J) Kernel density analysis visualized the distribution of appearance probability. (K) Mean frequencies of mEPSCs and (L) mIPSCs in ventral CA1 neurons. Right side exhibited signiﬁcantly wider variation of mEPSC frequency than left side. IA training affected neither of them, regardless of the hemisphere. (M) The self-entropy of each dot and (N) visualized density in ventral CA1 neurons. (O) E/I balance of miniature frequencies and (P) mean self-entropy per ventral CA1 neuron. E = mEPSC; I = mIPSC. Gray indicates untrained groups and black is trained groups. The number of cells in each group is shown at the bottom of each bar. Error bars indicate ± SEM. P < 0.05, *P < 0.01 versus untrained. Figure 4. Diversity of mEPSC/mIPSC frequency and self-entropy per neuron after training. (A) 2-Dimensional plot of the frequency of mEPSCs and mIPSCs in dorsal CA1 neurons. The circle or square plot indicates the data from a right or left CA1 neuron, respectively. (B) Kernel density analysis visualized the distribution of appearance prob- ability. (C) Mean frequencies of mEPSCs and (D) mIPSCs in dorsal CA1 neurons. IA training signiﬁcantly increased both frequencies in both hemispheres. (E) The self- entropy of each dot and (F) visualized density in dorsal CA1 neurons. (G) E/I balance of miniature frequencies and (H) mean self-entropy per dorsal CA1 neuron. (I) 2- Dimensional plot of the self-entropy of the frequencies in ventral CA1 neurons. Frequencies of the mE(I)PSC Events in Dorsal CA1 Neurons The circle or square plot indicates the data from right or left CA1 neuron, respectively. (J) Kernel density analysis visualized the distribution of appearance probability. (K) Mean frequencies of mEPSCs and (L) mIPSCs in ventral CA1 neurons. Right side exhibited signiﬁcantly wider variation of mEPSC frequency than left side. IA training affected neither of them, regardless of the hemisphere. (M) The self-entropy of each dot and (N) visualized density in ventral CA1 neurons. (O) E/I balance of miniature frequencies and (P) mean self-entropy per ventral CA1 neuron. E = mEPSC; I = mIPSC. Gray indicates untrained groups and black is trained groups. The number of cells in each group is shown at the bottom of each bar. Error bars indicate ± SEM. P < 0.05, *P < 0.01 versus untrained. balance of mEPSC versus mIPSC frequency, suggesting the bal- ance of the number of excitatory versus inhibitory synapses onto ventral CA1 neurons. laterality (F1,103 = 0.922, P = 0.34), and interaction (F1,103 = 0.273, P = 0.60) were not signiﬁcant (Fig. 4L). Thus, the train- ing affected frequency of neither mEPSC nor mIPSC regard- less of the hemispheres. These results suggest that the training did not affect the number of excitatory and inhibi- tory synapses onto ventral CA1 neurons, regardless of the hemisphere. Self-Entropy of the Frequency in Ventral CA1 Neurons Although the recorded neurons exhibited different self-entropy each other, self-entropy in the mEPSC frequency did not exhibit a signiﬁcant main effect of training (F1,103 = 0.055, P = 0.82), laterality (F1,103 = 2.838, P = 0.095), or interaction (F1,103 = 0.147, P = 0.70, Fig. 4M). Similarly, self-entropy in the mIPSC frequency did not exhibit a signiﬁcant main effect of training (F1,103 = 0.519, P = 0.47), laterality (F1,103 = 0.538, P = 0.47), or interaction (F1,103 = 0.342, P = 0.56, Fig. 4M). Thus, the training affected nei- ther self-entropy regardless of the hemispheres. The Kernel For the E/I balance of miniature frequency, the main effects of training (F1,103 = 0.75, P = 0.39) and interaction (F1,103 = 0.086, P = 0.77) were not signiﬁcant (Fig. 4O), but right side of CA1 neurons exhibited higher E/I balance of the frequency than left side (F1,103 = 4.865, P = 0.03). The results suggest that CA1 neurons receive more excitatory inputs in the right hemi- sphere than in the left hemisphere, providing a synaptic evi- dence of laterality. Meanwhile, the training did not affect the Learning Promotes Synaptic Diversity in CA1 Sakimoto et al. \| 2191 2191 Figure 5. Estimated number of open channels and single channel current. (A) An example of nonstationary ﬂuctuation analysis for AMPA receptor current. (B) Mean number of open Na+ channels at dorsal and ventral CA1 synapses. IA training signiﬁcantly increased the number of open channels only at dorsal synapses. (C) Ventral synapses exhibited greater single-channel current than dorsal synapses, although the training did not affect the current. (D) An example of nonstationary ﬂuctuation analysis for GABAA receptor current. (E) Mean number of open Cl−channels at dorsal and ventral CA1 synapses. IA training signiﬁcantly increased the number of open channels only at dorsal synapses. Ventral GABAergic synapses possessed more channels than dorsal synapses. (F) Neither training nor CA1 region affected the single-channel current. The number of cells in each group is shown at the bottom of each bar. Error bars indicate + SEM. P < 0.05 versus untrained. #P < 0.05 versus dorsal. Figure 5. Estimated number of open channels and single channel current. (A) An example of nonstationary ﬂuctuation analysis for AMPA receptor current. (B) Mean number of open Na+ channels at dorsal and ventral CA1 synapses. IA training signiﬁcantly increased the number of open channels only at dorsal synapses. The Number of Postsynaptic AMPA Receptor Channels The Number of Postsynaptic AMPA Receptor Channels Bilateral Microinjection of Plasticity Blockers To examine whether IA alters the number of AMPA receptors, we used evoked EPSC responses to calculate the number of opening AMPA receptors at dorsal or ventral CA3–CA1 synapses using nonstationary ﬂuctuation analysis (Fig. 5A). The number of open AMPA receptors was signiﬁcantly larger in IA-trained rats than untrained rats at dorsal CA3–CA1 synapses (t33 = 2.28, P = 0.029), but not at ventral CA3–CA1 synapses (t18 = 0.32, P = 0.75, Fig. 5B). Although the single-channel current was signiﬁ- cantly greater at ventral synapses than dorsal synapses (F1,53 = 6.02, P = 0.017), the training did not affect the single-channel current (Fig. 5C). These results suggest that the training pro- motes postsynaptic plasticity by increasing the number of AMPA receptor-channels at dorsal but not ventral CA3–CA1 synapses. To examine the physiological role of plastic changes affecting the IA learning, saline, AP5, or Mla was bilaterally microinjected into the dorsal or ventral CA1 (Fig. 6A). In the dorsal CA1, both AP5 and Mla treated-rats showed shorter latency than saline- injected rats after the training (Fig. 6B, F2,16 = 16.411, P < 0.0001). Conversely in the ventral CA1, neither AP5 nor Mla treatment affected the latency (Fig. 6C, F2,15 = 0.773, P = 0.48). Discussion Rat hippocampus is known to contain approximately 311 500 CA1 pyramidal neurons, receiving 13 059-28 697 CA3–CA1 syn- apses and up to 1 742 temporoammonic synapses from entorhi- nal cortex per single neuron (Bezaire and Soltesz 2013). Although both structural and functional heterogeneity are known in dorso/ventral or left /right CA1 neurons, the location of learning-induced synaptic plasticity has not been speciﬁed in the broad CA1 area. Here we found that the training increased AMPA receptor-mediated responses at dorsal CA3– CA1 synapses in both hemispheres, whereas ventral CA3–CA1 synapses did not show the plasticity in either hemisphere. The speciﬁed CA1 subﬁelds of learning-induced plasticity provide a synaptic evidence of dorso/ventral heterogeneity at the syn- apse level. Self-Entropy of the Frequency in Ventral CA1 Neurons (C) Ventral synapses exhibited greater single-channel current than dorsal synapses, although the training did not affect the current. (D) An example of nonstationary ﬂuctuation analysis for GABAA receptor current. (E) Mean number of open Cl−channels at dorsal and ventral CA1 synapses. IA training signiﬁcantly increased the number of open channels only at dorsal synapses. Ventral GABAergic synapses possessed more channels than dorsal synapses. (F) Neither training nor CA1 region affected the single-channel current. The number of cells in each group is shown at the bottom of each bar. Error bars indicate + SEM. P < 0.05 versus untrained. #P < 0.05 versus dorsal. analysis further visualized the density distribution (Fig. 4N). The average levels of self-entropy were 16.8 ± 2.7 bits (untrained) and 16.3 ± 1.7 bits (IA trained) per single CA1 neu- ron (Fig. 4P). nor the training affected the single-channel current (Fig. 5F). These results suggest that the training promotes postsynaptic plasticity by increasing the number of GABAA receptor- channels at dorsal but not ventral CA1 synapses. The Number of Postsynaptic GABAA Receptor Channels 5E) at ventral CA1 synapses may provide further evidence of dorso/ventral heterogeneity at the synapses. Nonstationary ﬂuctuation analysis further revealed evi- dence at a single channel level. We found the training signiﬁ- cantly increased the postsynaptic number of open AMPA receptors at dorsal CA1 synapses, whereas the training did not affect the ventral CA1 synapses (Fig. 5B). By combining in vivo gene delivery and in vitro patch-clamp recordings, we previ- ously demonstrated that contextual learning depends on syn- aptic delivery of GluA1-containing AMPA receptors at dorsal CA1 synapses at the molecular level (Mitsushima et al. 2011). The increase in the number of open channels without changes in the single-channel current further revealed learning-induced current changes at the AMPA receptor-mediated CA1 synapses. The dorsal and ventral hippocampus play different roles based on distinct input and output connections (Swanson and Cowan 1977). Spatial and contextual memory appears to depend only on the dorsal hippocampus (Moser and Moser 1998; Strange et al. 2014), whereas ventral hippocampal lesions alter stress responses and emotional behavior (Henke 1990; Kjelstrup et al. 2002). In untrained rats, ventral CA1 neurons tended to show greater mEPSC frequency (mEPSC, F1,98 = 3.241, P = 0.075) and exhibited greater mIPSC frequency than dorsal neurons (mIPSC, F1,98 = 40.120, P < 0.0001; Fig. 4). This heteroge- neity was already reported Millior et al. (2016), showing greater basal releases of both GABA and glutamate at ventral CA1 syn- apses. Moreover, as to the postsynaptic heterogeneity, ventral neurons showed greater mE(I)PSC amplitude than dorsal neu- rons (mEPSC, F1,98 = 11.324, P = 0.0011; mIPSC, F1,98 = 11.795, P = Nonstationary ﬂuctuation analysis further revealed evi- dence at a single channel level. We found the training signiﬁ- cantly increased the postsynaptic number of open AMPA receptors at dorsal CA1 synapses, whereas the training did not affect the ventral CA1 synapses (Fig. 5B). By combining in vivo gene delivery and in vitro patch-clamp recordings, we previ- ously demonstrated that contextual learning depends on syn- aptic delivery of GluA1-containing AMPA receptors at dorsal CA1 synapses at the molecular level (Mitsushima et al. 2011). The increase in the number of open channels without changes in the single-channel current further revealed learning-induced current changes at the AMPA receptor-mediated CA1 synapses. The Number of Postsynaptic GABAA Receptor Channels To examine whether IA alters the number of GABAA receptors, we used mIPSC responses to calculate the number of opening GABAA receptors at dorsal or ventral CA1 synapses (Fig. 5D). The number of open GABAA receptors was larger in IA-trained rats than untrained rats at dorsal CA1 synapses (t96 = 2.30, P = 0.024), but not at ventral CA1 synapses (t81 = 0.44, P = 0.66, Fig. 5E). Although ventral synapses possessed more Cl−chan- nels than dorsal synapses (F1,179 = 6.532, P = 0.011), neither area Cerebral Cortex, 2019, Vol. 29, No. 5 2192 Figure 6. Intra-CA1 injection of plasticity blockers impairs the IA learning. (A) Experimental design of bilateral intra-CA1 injection and IA training. (B) Microinjection of AP5 or methyllycaconitine (Mla) into the dorsal CA1 impaired learning. (C) The injection into the ventral CA1 did not affect the performance. The number of rats in each group is shown at the bottom of each bar. Vertical gray and black bars indicate guide cannula and injector. The number indicates posterior coordinate from bregma. Error bars indicate + SEM. P < 0.01 versus saline (Sal). ure 6. Intra-CA1 injection of plasticity blockers impairs the IA learning. (A) Experimental design of bilateral intra-CA1 injection and IA training. (B) Micr AP5 or methyllycaconitine (Mla) into the dorsal CA1 impaired learning. (C) The injection into the ventral CA1 did not affect the performance. The number h group is shown at the bottom of each bar. Vertical gray and black bars indicate guide cannula and injector. The number indicates posterior coordi gma. Error bars indicate + SEM. P < 0.01 versus saline (Sal). Figure 6. Intra-CA1 injection of plasticity blockers impairs the IA learning. (A) Experimental design of bilateral intra-CA1 injection and IA training. (B) Microinjection of AP5 or methyllycaconitine (Mla) into the dorsal CA1 impaired learning. (C) The injection into the ventral CA1 did not affect the performance. The number of rats in each group is shown at the bottom of each bar. Vertical gray and black bars indicate guide cannula and injector. The number indicates posterior coordinate from bregma. Error bars indicate + SEM. *P < 0.01 versus saline (Sal). 0.0009; Fig. 3), suggesting greater postsynaptic AMPA/GABAA current at ventral CA1 synapses in untrained rats. Finally, the greater single current of AMPA receptor (Fig. 5C) and more post- synaptic GABAA channels (Fig. The Number of Postsynaptic GABAA Receptor Channels Although laterality was not clear in our laboratory conditions, right side of CA1 exhibited more power of gamma oscillation and spine density than left side in rats reared in the spatially enriched conditions (Shinohara et al. 2013). In humans, patients with unilateral damage to the right hippocampus exhibit spatial memory deﬁcits (Abrahams et al. 1996), whereas damage to the left hippocampus impairs verbal semantic representation (Richardson et al. 2004). In the present study, the right sides of synapses tended to have greater AMPA/NMDA ratios, mEPSC amplitudes, and self-entropy than the left side after training, though the laterality was not signiﬁcant. Fibers through the ven- tral hippocampal commissure are known to connect bilateral CA1 (Amaral and Witter 1995; Kawakami et al. 2003), inducing high coherence of CA1 theta waves during running or REM sleep in the freely moving state (Patel et al. 2012). As unilateral CA1 blockade of AMPA receptor delivery (Mitsushima et al. 2011, 2013) fail to impair the IA learning, bilateral CA1 neurons may work together to compensate for impairment of the other in rats in normal laboratory conditions. The dorsal and ventral hippocampus play different roles based on distinct input and output connections (Swanson and Cowan 1977). Spatial and contextual memory appears to depend only on the dorsal hippocampus (Moser and Moser 1998; Strange et al. 2014), whereas ventral hippocampal lesions alter stress responses and emotional behavior (Henke 1990; Kjelstrup et al. 2002). In untrained rats, ventral CA1 neurons tended to show greater mEPSC frequency (mEPSC, F1,98 = 3.241, P = 0.075) and exhibited greater mIPSC frequency than dorsal neurons (mIPSC, F1,98 = 40.120, P < 0.0001; Fig. 4). This heteroge- neity was already reported Millior et al. (2016), showing greater basal releases of both GABA and glutamate at ventral CA1 syn- apses. Moreover, as to the postsynaptic heterogeneity, ventral neurons showed greater mE(I)PSC amplitude than dorsal neu- rons (mEPSC, F1,98 = 11.324, P = 0.0011; mIPSC, F1,98 = 11.795, P = The question arises as to whether the synaptic strength contributes to memory. In regards to the excitatory synapses, Learning Promotes Synaptic Diversity in CA1 Sakimoto et al. \| 2193 2193 Synapses regulate cell ﬁring according to the all-or-none principle (Kandel et al. 2013). If a neuron is considered an all- or-none device (Cannon 1922; Wiener 1961), one neuron can handle 1-bit of memory per clock cycle (log2 2 = 1 bit; Hartley 1928). The Number of Postsynaptic GABAA Receptor Channels Since the Ser408–409 phosphorylation is known to prevent clathrin adaptor protein 2-mediated GABAA receptor internalization (Jovanovic et al. 2004; Lu et al. 2010; Petrini and Barberis 2014), the Ser408–409 phosphorylation may contribute to increase the number of GABAA receptor channels (Fig. 5E) without changing Cl−current per channel (Fig. 5F). Synaptic dysfunction is well correlated with cognitive decline in Alzheimer’s disease (Terry et al. 1991). Amyloid beta (Aβ42) is well known as a major causative agent (Shankar et al. 2008; Querfurth and LaFerla 2010; Penzes et al. 2011; Sevigny et al. 2016), and long-term exposure to Aβ42 impairs the AMPA receptor trafﬁcking by reducing synaptic distribution of CaMKII in cultured pyramidal neurons (Gu et al. 2009). As regard target molecule, hippocampal neurons that lack GluA3 were resistant against Aβ-mediated synaptic depression and spine loss, sug- gesting that Aβ initiates synaptic and memory deﬁcits by removing GluA3-containing AMPA receptors from synapses (Reinders et al. 2016). On the other hand, as to the inhibitory synapses, Aβ42 speciﬁcally binds to nicotinic α7 receptors (Wang et al. 2000) promoting the learning-induced plasticity at the GABAA receptor-mediated inhibitory synapses (Mitsushima et al. 2013; Townsend et al. 2016). Application of Aβ42, being known to block the nicotinic α7 receptor-mediated cholinergic response (Liu et al. 2001), quickly weakens GABAA receptor- mediated synaptic currents via downregulation of GABAA receptors (Ulrich 2015). Understanding the learning-induced plasticity in speciﬁc CA1 subﬁelds as well as the quantiﬁcation of synaptic diversity is necessary to diagnose the functional impairment in cognitive disorders, which may help to identify potential targets for therapeutic intervention. Sequential recording of mEPSCs and mIPSCs enables analy- sis of the strength of excitatory and inhibitory inputs in each neuron one-by-one. The increase in amplitude may indicate additional receptor delivery into the synapses, whereas the increase in frequency could result in an increase in the number of functional synapses or the probability of vesicle release from the presynaptic terminal (Kerchner and Nicoll 2008; Mitsushima et al. 2013). IA training clearly increased both mE(I)PSC ampli- tudes and frequencies in both hemispheres of dorsal CA1 neu- rons, whereas the training increased neither mE(I)PSC amplitudes nor frequencies in ventral CA1 neurons, regardless of the hemi- sphere. Moreover, the performance clearly impaired by the bilat- eral blockade of the plasticity in dorsal, but not ventral CA1 subﬁelds, suggesting a speciﬁc role of the dorsal CA1 synapses for contextual learning (Fig. 6). The Number of Postsynaptic GABAA Receptor Channels Based on the principle, computational theory proposed a role of the hippocampus as a kind of memory device (Marr 1977). We previously found a logarithmic relationship between the number of cells blocking plasticity and learning perfor- mance (Mitsushima et al. 2011), providing an evidence of binary processing of contextual information, such as what, where, or when. We hypothesized that the plasticity at excit- atory/inhibitory synapses may change the binary processing of ﬁring to encode memory. In support of this, real-time recordings of multiple unit activity of dorsal CA1 neurons fur- ther showed task-dependent diversiﬁed feature of ripple-like on/off ﬁrings (Ishikawa and Mitsushima 2016; Tomokage et al. 2018). Since selective elimination of the ripple-like events dur- ing post-training consolidation periods impairs the perfor- mance in dorsal hippocampus-dependent task (Girardeau et al. 2009), the events may contribute to code experienced information. contextual learning requires AMPA receptor delivery, as bilat- eral CA1 blockade of AMPA receptor delivery impairs learning (Mitsushima et al. 2011). Recently, Takemoto et al. further developed a technique to inactivate synaptic GluA1 AMPA receptors in vivo using chromophore-assisted light inactivation (CALI). Since optical inactivation of synaptic AMPA receptors successfully erased acquired-fear memory (Takemoto et al. 2017), newly delivered GluA1-containing AMPA receptors into the CA1 synapses seems to be required for contextual memory formation. contextual learning requires AMPA receptor delivery, as bilat- eral CA1 blockade of AMPA receptor delivery impairs learning (Mitsushima et al. 2011). Recently, Takemoto et al. further developed a technique to inactivate synaptic GluA1 AMPA receptors in vivo using chromophore-assisted light inactivation (CALI). Since optical inactivation of synaptic AMPA receptors successfully erased acquired-fear memory (Takemoto et al. 2017), newly delivered GluA1-containing AMPA receptors into the CA1 synapses seems to be required for contextual memory formation. Learning also affects the GABAA receptor-mediated inhibitory synapses, but the plasticity seems to be task-dependent at CA1 synapses (Cui et al. 2008; Mitsushima et al. 2013). Spatial learning presynaptically increases GABA release probability (Cui et al. 2008), whereas contextual learning postsynaptically strengthened GABAA receptor-mediated inhibitory synapses (Mitsushima et al. 2013). Since optogenetic inactivation of somatostain-expressing interneurons in bilateral dorsal CA1 impaired the contextual fear learning, the learning seems to require the GABAergic inputs from somatostain-expressing interneurons at basal dendrites of dorsal CA1 neurons (Lovett-Barron et al. 2014). We further found a rapid phosphorylation of Ser408–409 GABAA receptor β3 subunit within 5 min after the training (Sakimoto et al. 2017). The Number of Postsynaptic GABAA Receptor Channels Although the IA training failed to affect the synaptic plasticity in either hemisphere of ventral CA1 neurons, optogenetic cell body inhibition study revealed essential role of ventral CA1 neurons for social discrimination task (Okuyama et al. 2016). Authors’ Contributions Here we propose a new approach to quantify the learning- induced synaptic diversity in 4 CA1 subﬁelds. The subﬁeld- speciﬁc increase in self-entropy at dorsal, but not ventral, CA1 synapses in trained rats further provides an evidence of learn- ing. Since bilateral blockade of the synaptic diversity clearly impaired the learning performance (Mitsushima et al. 2013, Ono and Mitsushima 2017), we hypothesized that the increased self-entropy may code a piece of experienced information after training. Present results not only conﬁrmed previous ﬁndings, but also speciﬁed the subregion, suggesting a crucial role for IA learning (Fig. 6). Considering the total number of dorsal CA1 neurons (Andersen et al. 2006; Bezaire and Soltesz 2013), a pos- sible increase in total self-entropy after IA training is estimated to be 12 550 000 bits. In any case, the quantiﬁcation of the syn- aptic diversity in speciﬁc CA1 subﬁelds would be a useful approach for diagnostic evaluation of cognitive disorders. D.M., Y.S., and J.M. wrote the article. Y.S., J.M., and D.M. per- formed the experiments. 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https://openalex.org/W4312601438	https://agrocienciauruguay.uy/index.php/agrociencia/article/download/255/215	English	null	Water Interception by Crop Mulch Avena strigosa in Irrigated No-Tillage System	Agrociencia	2,015	cc-by	425	Water Interception by Crop Mulch Avena strigosa in Irrigated No-Tillage System Rodrigues da Rocha M*¹, Carlesso R², Petry M T², Basso L J³, Schons de Ávila V³ ¹Doutoranda do Programa de Pós-Graduação em Engenharia Agrícola (PPGEA) da Universidade Federal de Santa Maria (UFSM). ²Professor (a) do Departamento de Engenharia Rural - UFSM. ³Mestrando (a) do PPGEA - UFSM. E-mail: marta.da.rocha@gmail.com ²Professor (a) do Departamento de Engenharia Rural - UFSM. ³Mestrando (a) do PPGEA - UFSM. E-mail: marta.da.rocha@gmail.com Agrociencia Uruguay, Special Issue Agrociencia Uruguay, Special Issue 26 Abstract One of the difficulties of the irrigation management is determining the effective rainfall, that is, the water that is indeed available to the crops, due to the influence of the climatic factors and the soil characteristics, as the surface roughness, the composition and the amount of mulch. Objectifying the quantification of the interception and the water storage through mulching in an irrigated no- tillage crop system this work was conducted on a Ultisol in Santa Maria, RS, Brazil; in the agricultural year of 2013/2014 with Avena strigosa crop waste. The period of the evaluations was from 60 days, numbering nine events; in which three levels of mulch, (bare soil, 2 and 4 t ha-1 of dry matter - DM) under three irrigation levels (4, 8 e 16 mm) in a micro sprinkler system, with a bi-factorial distribution scheme in three repetitions. The water content in the mulching were quantified before, 3; 6 and 24 h after the irrigations, collecting the mulch, randomly in the experimental units, with the assistance of a sampling board of 0.09 m² and the content of water in the soil, with sensors FDR installed in depths 0.00-0.10; 0.10 -0.25; 0.25-0.55 e 0.55-0.85 m. The content of water in soil from different levels of mulch, which received 4mm of irrigation, didn’t diverge, suggesting a bigger specification of the soil profile when small doses of irrigation are evaluated. In relation to the water in the mulch, it is observed that three hours after the irrigation, the mulch has the same content of water it had before the irrigation, not having difference between the two mulches levels in the whole period evaluated. The water interception through the mulch drifted from 0.5 to 1 mm, in the treatments 2 and 4 t ha-1 of DM, tending to reduce with time, considering the collections done thereupon the irrigations. The water interception through the mulch must be offset in an irrigation crop system in irrigated no-tillage, particularly as it is worked with low irrigation volume. Keywords: irrigation management, no-tillage, mulch
https://openalex.org/W2796620788	https://www.frontiersin.org/articles/10.3389/fmicb.2018.00693/pdf	English	null	A Carbohydrate Moiety of Secreted Stage-Specific Glycoprotein 4 Participates in Host Cell Invasion by Trypanosoma cruzi Extracellular Amastigotes	Frontiers in microbiology	2,018	cc-by	11,985	Citation: Keywords: Trypanosoma cruzi, extracellular amastigotes, Ssp-4 glycoprotein, galectin-3 Florentino PTV, Real F, Orikaza CM, da Cunha JPC, Vitorino FNL, Cordero EM, Sobreira TJP and Mortara RA (2018) A Carbohydrate Moiety of Secreted Stage-Speciﬁc Glycoprotein 4 Participates in Host Cell Invasion by Trypanosoma cruzi Extracellular Amastigotes. Front. Microbiol. 9:693. doi: 10.3389/fmicb.2018.00693 Florentino PTV, Real F, Orikaza CM, da Cunha JPC, Vitorino FNL, Cordero EM, Sobreira TJP and Mortara RA (2018) A Carbohydrate Moiety of Secreted Stage-Speciﬁc Glycoprotein 4 Participates in Host Cell Invasion by Trypanosoma cruzi Extracellular Amastigotes. Front. Microbiol. 9:693. doi: 10.3389/fmicb.2018.00693 A Carbohydrate Moiety of Secreted Stage-Speciﬁc Glycoprotein 4 Participates in Host Cell Invasion by Trypanosoma cruzi Extracellular Amastigotes Pilar T. V. Florentino1, Fernando Real2, Cristina M. Orikaza3, Julia P. C. da Cunha4, Francisca N. L. Vitorino4, Esteban M. Cordero1,5, Tiago J. P. Sobreira6 and Renato A. Mortara3* 1 Department of Microbiology, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, Brazil, 2 Institut Cochin, INSERM U1016, Paris, France, 3 Department of Microbiology, Immunology and Parasitology, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, Brazil, 4 Special Laboratory of Cell Cycle, Center of Toxins, Immune-Response and Cell Signaling (CeTICS), Butantan Institute, São Paulo, Brazil, 5 Facultad de Ciencias, Centro de Genómica y Bioinformática, Universidad Mayor, Santiago, Chile, 6 Bindley Bioscience Center, Purdue University, West Lafayette, IN, United States ORIGINAL RESEARCH published: 10 April 2018 doi: 10.3389/fmicb.2018.00693 Keywords: Trypanosoma cruzi, extracellular amastigotes, Ssp-4 glycoprotein, galectin-3 Reviewed by: Reviewed by: Carlos Robello, Institut Pasteur de Montevideo, Uruguay Reviewed by: Carlos Robello, Institut Pasteur de Montevideo, Uruguay Mariane B. Melo, Massachusetts Institute of Technology, United States Robert Braidwood Sim, University of Oxford, United Kingdom Mariane B. Melo, Massachusetts Institute of Technology, United States Robert Braidwood Sim, University of Oxford, United Kingdom Correspondence: Renato A. Mortara ramortara@unifesp.br Correspondence: Renato A. Mortara ramortara@unifesp.br Specialty section: This article was submitted to Infectious Diseases, a section of the journal Frontiers in Microbiology Specialty section: This article was submitted to Infectious Diseases, a section of the journal Frontiers in Microbiology Received: 16 February 2018 Accepted: 23 March 2018 Published: 10 April 2018 Received: 16 February 2018 Accepted: 23 March 2018 Published: 10 April 2018 Edited by: Celio Geraldo Freire-de-Lima, Universidade Federal do Rio de Janeiro, Brazil Furthermore, Ssp-4 is secreted by EAs, either free or associated with parasite vesicles, and can participate in host-cell interactions. The results presented here describe the possible role of a carbohydrate moiety of T. cruzi surface glycoproteins in host cell invasion by EA forms, highlighting the potential of these moieties as therapeutic and vaccine targets for the treatment of Chagas’ disease. Edited by: Celio Geraldo Freire-de-Lima, Universidade Federal do Rio de Janeiro, Brazil Edited by: Celio Geraldo Freire-de-Lima, Universidade Federal do Rio de Janeiro, Brazil Edited by: Celio Geraldo Freire-de-Lima, Universidade Federal do Rio de Janeiro, Brazil Trypanosoma cruzi is the etiologic agent of Chagas’ disease. It is known that amastigotes derived from trypomastigotes in the extracellular milieu are infective in vitro and in vivo. Extracellular amastigotes (EAs) have a stage-speciﬁc surface antigen called Ssp-4, a GPI-anchored glycoprotein that is secreted by the parasites. By immunoprecipitation with the Ssp-4-speciﬁc monoclonal antibodies (mAb) 2C2 and 1D9, we isolated the glycoprotein from EAs. By mass spectrometry, we identiﬁed the core protein of Ssp-4 and evaluated mRNA expression and the presence of Ssp-4 carbohydrate epitopes recognized by mAb1D9. We demonstrated that the carbohydrate epitope recognized by mAb1D9 could promote host cell invasion by EAs. Although infectious EAs express lower amounts of Ssp-4 compared with less-infectious EAs (at the mRNA and protein levels), it is the glycosylation of Ssp-4 (identiﬁed by mAb1D9 staining only in infectious strains and recognized by galectin-3 on host cells) that is the determinant of EA invasion of host cells. Furthermore, Ssp-4 is secreted by EAs, either free or associated with parasite vesicles, and can participate in host-cell interactions. The results presented here describe the possible role of a carbohydrate moiety of T. cruzi surface glycoproteins in host cell invasion by EA forms, highlighting the potential of these moieties as therapeutic and vaccine targets for the treatment of Chagas’ disease. Trypanosoma cruzi is the etiologic agent of Chagas’ disease. It is known that amastigotes derived from trypomastigotes in the extracellular milieu are infective in vitro and in vivo. Extracellular amastigotes (EAs) have a stage-speciﬁc surface antigen called Ssp-4, a GPI-anchored glycoprotein that is secreted by the parasites. By immunoprecipitation with the Ssp-4-speciﬁc monoclonal antibodies (mAb) 2C2 and 1D9, we isolated the glycoprotein from EAs. By mass spectrometry, we identiﬁed the core protein of Ssp-4 and evaluated mRNA expression and the presence of Ssp-4 carbohydrate epitopes recognized by mAb1D9. We demonstrated that the carbohydrate epitope recognized by mAb1D9 could promote host cell invasion by EAs. Although infectious EAs express lower amounts of Ssp-4 compared with less-infectious EAs (at the mRNA and protein levels), it is the glycosylation of Ssp-4 (identiﬁed by mAb1D9 staining only in infectious strains and recognized by galectin-3 on host cells) that is the determinant of EA invasion of host cells. Ssp-4 Glycosylation Is Associated With Host Cell Invasion by T. cruzi EAs Host cell invasion is a property exhibited by EAs of some by T. cruzi strains maintained in vitro. First, we conﬁrmed that the parasite strains G and CL can be categorized as infectious and less-infectious EA strains, respectively, based on HeLa cell invasion assays. Figure 1A shows that the number of internalized parasites from the G strain within HeLa cells is two logs higher (p < 0.001) than the number from the CL strain, allowing us to classify these strains as infectious and less infectious, respectively. Kahn et al. (1996) have observed that amastigotes, but not trypomastigotes or epimastigotes, interact with host macrophages via mannose surface receptors (MRIs). The cell surface protein galectin-3 (Gal-3), which belongs to the galectin family and recognizes β-galactosides, has been previously implicated in the interaction of T. cruzi with host cell membranes (Moody et al., 2000; Kleshchenko et al., 2004; Vray et al., 2004; Pineda et al., 2015). In addition, Machado et al. (2014) observed the recruitment of galectin-3 at invasion sites of EAs in macrophage cells. Next, the expression of Ssp-4 on the surfaces of infectious and less infectious EAs was assessed by immunoﬂuorescence using two monoclonal antibodies speciﬁc for the Ssp-4 antigen, namely, mAb2C2 and mAb1D9, the latter being employed to identify carbohydrate epitopes associated with Ssp-4 (Barros et al., 1997). The carbohydrate epitope recognized by mAb1D9 is highly expressed in infectious EAs and is absent in less infectious EAs; conversely, mAb2C2 only recognizes Ssp-4 on the surfaces of less infectious strains (Figure 1B). This result was reproduced in EA lysates immunostained with mAb1D9 and mAb2C2 by Western blotting (Figure 1C): while the carbohydrate epitope recognized by mAb1D9 is completely absent in the less-infectious CL strain, the epitope recognized by mAb2C2 is detected in both strains, although this recognition is weak in EAs of the infectious G strain (Figure 1C, arrow). The EAs from group I strains (such as the G strain) were found to enter mammalian cells much more eﬃciently than parasites from groups II (Y strain) or VI (CL strain) (Fernandes and Mortara, 2004; Mortara et al., 2005; da Silva et al., 2006; Fernandes et al., 2007). Diﬀerent studies have shown that the expression of protein and carbohydrate epitopes varies between T. cruzi strains and that these variations are correlated with parasite infectivity (Mortara et al., 1988, 1992; Verbisck et al., 1998; da Silva et al., 2006; Yoshida, 2006). INTRODUCTION The ﬂagellated protozoan Trypanosoma cruzi is the etiologic agent of Chagas’ disease and is responsible for an estimated 6–7 million individuals infected worldwide, mostly in Latin America (World Health Organization [WHO], 2017). This parasite has four deﬁned morphological stages: two infective forms called metacyclic and bloodstream trypomastigotes and two replicative forms April 2018 \| Volume 9 \| Article 693 Frontiers in Microbiology \| www.frontiersin.org 1 Glycosylated Ssp-4 Participates in T. cruzi Infection Florentino et al. known as amastigotes and epimastigotes (Clayton, 2010). Although T. cruzi amastigotes are usually found in the cytoplasm of infected cells of the mammalian host, these forms can also be found in the extracellular milieu due to trypomastigote diﬀerentiation or early lysis of infected cells (Andrews et al., 1987; Ley et al., 1988) or due to cytolysis at inﬂamed sites of infection during the chronic stage of Chagas’ disease (Scharfstein and Morrot, 1999). Although limited in their ability to mimic intracellular amastigotes, EAs present the same set of stage-speciﬁc surface antigens as Ssp-4 (Andrews et al., 1987), the structural and functional features of which are not yet fully understood. In the present report, we have revealed the Ssp-4 protein sequence and observed a correlation between the expression of protein and carbohydrate epitopes and the strain infectivity proﬁle. Our ﬁndings suggest that parasite infectivity correlates with posttranslational modiﬁcations in Ssp-4, resulting in the expression of distinct carbohydrate epitopes on EA surfaces. We also demonstrated that the highly infective G strain secretes an Ssp-4 epitope (recognized by mAb1D9) into vesicle trails adhered to HeLa cells, which may contribute to galectin-3 recruitment during host cell invasion by EAs. These extracellular amastigotes (EAs) are proxies for their intracellular counterparts as they share morphological and immunochemical markers and are capable of invading and sustaining infection cycles in mammalian cells (Nogueira and Cohn, 1976; Ley et al., 1988). However, unlike the infective trypomastigote forms, EAs invade HeLa cells in an actin- dependent mechanism, forming a phagocytic cup that surrounds these parasites (Mortara, 1991; Procópio et al., 1999), suggesting that EAs display functionally distinct membrane proteins that interact with a diﬀerent set of host cell receptors. The membrane proteins on the surfaces of EAs are recognized by host cell receptors, and the roles of these proteins in actin-dependent invasion remain elusive. Ssp-4 Glycosylation Is Associated With Host Cell Invasion by T. cruzi EAs We have previously observed two secreted proteins from EAs, p21 and mevalonate kinase, that mediate host cell signaling during invasion (da Silva et al., 2009; Ferreira et al., 2016). EAs also express on their surfaces a major glycoprotein, stage-speciﬁc protein 4 (Ssp-4), initially described by Andrews et al. (1987). This protein is gradually released into extracellular milieu, which is mediated by an endogenous phosphatidylinositol-speciﬁc phospholipase C (PI-PLC) (Andrews et al., 1988). We have demonstrated that Ssp-4 was also released in membrane trails when EAs were attached to poly-L-lysine-coated glass (Barros et al., 1996). Two monoclonal antibodies detect diﬀerent epitopes of Ssp- 4, mAb2C2 (Andrews et al., 1987) and mAb1D9, this last one recognizes a carbohydrate epitope (Barros et al., 1997). Moreover, highly infective EAs from the G strain showed reduced parasite invasion when incubated with mAb1D9 (da Silva et al., 2006), suggesting the involvement of Ssp-4 in host cell invasion. The inverse correlation between the detection of the Ssp- 4 carbohydrate epitope by mAb1D9 and the infectivity of EAs was investigated using other T. cruzi strains. Figures 2A,B show the membrane distribution and expression of Ssp-4 epitopes (detected by mAb2C2 and mAb1D9 by immunoﬂuorescence and Western blotting, respectively) in T cruzi strains Tc863 (Tc III), Tc1522 (Tc I), and Tc1994 (TcBat) compared with the infectious G strain (Tc I). Highly infective EAs from the G and Tc1522 strains presented higher reactivity to mAb1D9 and lower reactivity to mAb2C2 (Figures 2B,C). Conversely, Tc863 exhibited low infectivity and lacked the carbohydrate epitope recognized by mAb1D9; however, this strain exhibited strong reactivity to mAb2C2. Tc1994 (TcBat) showed intermediate April 2018 \| Volume 9 \| Article 693 Frontiers in Microbiology \| www.frontiersin.org 2 Glycosylated Ssp-4 Participates in T. cruzi Infection Florentino et al. FIGURE 1 \| Ssp-4 epitope expression correlates with infectivity of extracellular amastigotes from the G and CL strains. (A) Number of internalized parasites in 300 cells per coverslip. Extracellular amastigotes (EAs) from the G and CL strains were incubated with HeLa cells (MOI 10:1) for 2 h. Mean and SD are represented in the graphic. Three independent experiments were performed in duplicate. ∗∗∗p < 0.001. (B) EAs of the G or CL strains immunostained with mAb1D9 and mAb2C2 (green); nuclei and kinetoplasts were labeled with DAPI (blue). White arrows indicate the distribution of Ssp-4 epitopes on parasite cell surfaces. Scale bar: 5 µm. Ssp-4 Glycosylation Is Associated With Host Cell Invasion by T. cruzi EAs (C) Western blotting of EAs from the G and CL lysates was incubated with mAb2C2 and mAb1D9. Red arrows indicate the Ssp-4 band. As a control, EA lysates were incubated with anti-α-tubulin antibody. FIGURE 1 \| Ssp-4 epitope expression correlates with infectivity of extracellular amastigotes from the G and CL strains. (A) Number of internalized parasites in 300 cells per coverslip. Extracellular amastigotes (EAs) from the G and CL strains were incubated with HeLa cells (MOI 10:1) for 2 h. Mean and SD are represented in the graphic. Three independent experiments were performed in duplicate. ∗∗∗p < 0.001. (B) EAs of the G or CL strains immunostained with mAb1D9 and mAb2C2 (green); nuclei and kinetoplasts were labeled with DAPI (blue). White arrows indicate the distribution of Ssp-4 epitopes on parasite cell surfaces. Scale bar: 5 µm. (C) Western blotting of EAs from the G and CL lysates was incubated with mAb2C2 and mAb1D9. Red arrows indicate the Ssp-4 band. As a control, EA lysates were incubated with anti-α-tubulin antibody. mAb1D9. Consistent with this result, IPs from the control serum were not recognized by mAb2C2 or mAb1D9 (Figure 3B). infectivity compared with the other isolates and expressed epitopes recognized with similar strength by both mAb1D9 and mAb2C2. g y g The 93 kDa bands were subjected to mass spectrometric analysis followed by a database search using the T. cruzi CL Brener (hybrid clone) genome database (Supplementary Table S1). The mAb2C2 immunoprecipitates from the CL strain showed high identity with sequences from two CL Brener haplotypes, namely, TcCLB.507089.170 (Esmeraldo- like haplotype) and TcCLB.506725.20 (non-Esmeraldo-like haplotype), with 16% coverage. Both sequences code for the same 67 kDa hypothetical protein. Interestingly, mass spectrometric analysis of the 93 kDa band from the immunoprecipitation of the G strain with mAb2C2 and mAb1D9 also identiﬁed the same protein (TcCLB.506725.20) with 19.6 and 17.2% coverage, respectively (Supplementary Table S1). Proteins identiﬁed by mass spectrometry in the IP of the G strain are listed in Supplementary Table S2; TcCLB.506725.20 had the highest score among the proteins. Frontiers in Microbiology \| www.frontiersin.org Ssp-4 Expression Does Not Correlate With EA Infectivity y As we have established that the presence of the Ssp-4 carbohydrate epitope recognized by mAb1D9 correlates with EA infectivity, we sought to identify the Ssp-4 sequence and evaluate whether the mRNA and protein expression of Ssp- 4 is also correlated with host cell invasion. We employed an immunoprecipitation assay on lysates of less-infectious EAs by using either mAb2C2 or control serum and speciﬁcally detected a band of 93 kDa associated with Ssp-4 (Figure 3A). Western blotting with mAb2C2 conﬁrmed the presence of a band corresponding to a 93-kDa protein in the mAb2C2 immunoprecipitates that is absent when immunoprecipitation is performed using control serum (red arrows in Figure 3A). Immunoprecipitation of G strain lysates using mAb1D9 and mAb2C2 also detected a band of 93 kDa, which was detected with In silico analysis predicted an N-terminal signal peptide with high probability, indicating that this protein is secreted (Figure 3B and Supplementary Figure S1). An enzymatic Frontiers in Microbiology \| www.frontiersin.org April 2018 \| Volume 9 \| Article 693 Frontiers in Microbiology \| www.frontiersin.org 3 Glycosylated Ssp-4 Participates in T. cruzi Infection Florentino et al. FIGURE 2 \| Ssp-4 epitope expression correlates with infectivity of T. cruzi isolates. (A) Immunoﬂuorescence of extracellular amastigotes (EAs) from the G strain and the Tc863 (863), Tc1994 (1994), and Tc1522 (1522) isolates with mAb1D9 or mAb2C2 (green); nuclei and kinetoplasts were DAPI labeled (blue). White arrows indicate the distribution of carbohydrate epitopes on parasite cell surfaces. Scale bars: 3 µm. (B) Western blotting of T. cruzi EAs from the G strain and the Tc863, Tc1994, and Tc1522 isolates (863, 1994, and 1522, respectively) incubated with mAb2C2 and mAb1D9; anti-α-tubulin antibody was used for normalization. (C) Number of internalized parasites (EAs) in 300 cells per coverslip of T. cruzi isolates. Three independent experiments were performed in triplicate or duplicate. ∗p < 0.05; ∗∗∗p < 0.001. FIGURE 2 \| Ssp-4 epitope expression correlates with infectivity of T. cruzi isolates. (A) Immunoﬂuorescence of extracellular amastigotes (EAs) from the G strain and the Tc863 (863), Tc1994 (1994), and Tc1522 (1522) isolates with mAb1D9 or mAb2C2 (green); nuclei and kinetoplasts were DAPI labeled (blue). White arrows indicate the distribution of carbohydrate epitopes on parasite cell surfaces. Scale bars: 3 µm. (B) Western blotting of T. Frontiers in Microbiology \| www.frontiersin.org Ssp-4 Expression Does Not Correlate With EA Infectivity cruzi EAs from the G strain and the Tc863, Tc1994, and Tc1522 isolates (863, 1994, and 1522, respectively) incubated with mAb2C2 and mAb1D9; anti-α-tubulin antibody was used for normalization. (C) Number of internalized parasites (EAs) in 300 cells per coverslip of T. cruzi isolates. Three independent experiments were performed in triplicate or duplicate. ∗p < 0.05; ∗∗∗p < 0.001. (shown in blue) and VGENFDDSWASDLRR (shown in red) (Supplementary Table S3). Only MAP2 was selected for polyclonal antibody production in mice. The polyclonal antibody against the MAP2 peptide reacted with a band of 93 kDa from EA lysates immunoprecipitated with mAb2C2 (Figure 3E), conﬁrming that the MAP2 peptide is part of the protein core of Ssp-4. domain with alpha/beta hydrolase activity, hydrophobic domains in the N- and C-terminal regions, six potential N-glycosylation sites, and eight potential O-glycosylation sites were also predicted by bioinformatics. Although this protein does not have a classic glycosylphosphatidylinositol (GPI)- anchor domain, a highly hydrophobic C-terminal domain of approximately 20 amino acids is predicted, and without this terminal domain, Ssp-4 has 100% probability of being anchored to GPI. These data conﬁrm that the hypothetical protein identiﬁed by mass spectrometry is Ssp-4, as previously identiﬁed by mAb2C2 (Andrews et al., 1988). We sequenced nucleotides from the T. cruzi CL Brener, G and CL strains, and the protein alignment revealed that the Ssp-4 sequence is well conserved across all strains and among CL Brener haplotypes, clone Dm28c, and the Sylvio strain from the genome database (Supplementary Figure S2A). Protein identity between all strains was analyzed and was found to be at least 95.7% (Supplementary Figure S2B). The identiﬁed Ssp-4 sequence allowed us to compare the mRNA expression of Ssp-4 in infectious and less-infectious EA strains; we observed that Ssp-4 mRNA expression, measured The immunogenic peptides of the predicted protein were assessed by prediction of the tertiary structure of the protein using homology modeling and threading by two diﬀerent algorithms (I-Tasser and Yasara) (Figure 3D). In these models, the peptides with higher immunogenic potential from the protein are displayed in diﬀerent colors (blue, green, red, and yellow). From the structural analysis, peptides LREFVRSTERNR (named MAP2, shown in yellow) and EARDQQALTQLR (shown in green) predicted to be more exposed on the protein surface compared to peptides LNWGADAKEMYTEYR April 2018 \| Volume 9 \| Article 693 4 o et al. Glycosylated Ssp-4 Participates in T. Ssp-4 Expression Does Not Correlate With EA Infectivity cruzi Infection E 3 \| Ssp-4 identiﬁcation by mass spectrometry revealed a hypothetical protein from the CL Brener database that was more highly expressed in the CL strain. munoprecipitation (IP) with mAb2C2 or control serum (CS) in protein extracts of extracellular amastigotes (EAs) from the CL strain was performed in the ce of protein A-Sepharose. Colloidal Coomassie-stained polyacrylamide gel of the IP with mAb2C2 or CS and Western blotting with mAb2C2 of the whole cell WC) from extracellular amastigotes (EAs) of the CL and unbound (UB) fraction and IP of 2C2 and CS. Red arrows indicate the band of 93 kDa. (B) IP med with mAb1D9, mAb2C2, and CS in protein extracts of EAs from the G strain. Western blotting of the WC from the G strain and IPs with mAb1D9. Red ndicates the band of 93 kDa. (C) Scheme of the hypothetical protein bearing the Ssp-4 epitope identiﬁed by mass spectrometry, revealing domains ntered by in silico analysis. (D) Structural models of the Ssp-4 putative protein predicted by I-Tasser and Yasara. MAP2 peptides are in yellow. In green, blue, d are the other peptides selected by I-Tasser and Yasara model (MAP1, MAP3, and MAP4, respectively). (E) IP of protein extracts of EAs of CL with mAb2C2, uently probed with MAP2 in Western blotting. CR i l f ld hi h i l i f i i MAP2 ib d (Fi 4B C) Th f h i i f Glycosylated Ssp-4 Participates in T. cruzi Infection Florentino et al. FIGURE 3 \| Ssp-4 identiﬁcation by mass spectrometry revealed a hypothetical protein from the CL Brener database that was more highly expressed in the CL strain. (A) Immunoprecipitation (IP) with mAb2C2 or control serum (CS) in protein extracts of extracellular amastigotes (EAs) from the CL strain was performed in the presence of protein A-Sepharose. Colloidal Coomassie-stained polyacrylamide gel of the IP with mAb2C2 or CS and Western blotting with mAb2C2 of the whole cell lysate (WC) from extracellular amastigotes (EAs) of the CL and unbound (UB) fraction and IP of 2C2 and CS. Red arrows indicate the band of 93 kDa. (B) IP performed with mAb1D9, mAb2C2, and CS in protein extracts of EAs from the G strain. Western blotting of the WC from the G strain and IPs with mAb1D9. Red arrow indicates the band of 93 kDa. Ssp-4 Expression Does Not Correlate With EA Infectivity (C) Immunoﬂuorescence (IF) of EAs of the G and CL strains incubated with MAP2 (green); nuclei and kinetoplasts were labeled with DAPI (blue). Scale bar: 5 µm. by phagocytic cups formed by host cell actin (Figure 5C and Supplementary Figure S3). carbohydrate epitopes recognized by mAb1D9 could be related to EAs infectivity. These phagocytic cups associated with the interactions of infectious EAs are also rich in galectin-3 (Gal-3); Figure 6A shows that the actin of the phagocytic cup and Gal-3 colocalize in regions of interaction between host cells and infectious EAs tagged with mAb1D9. The host cell origin of Gal-3 was conﬁrmed by Western blotting on extracts of host cells or EAs alone and of host cells interacting with EAs (Figure 6B). Additionally, host cells infected by EAs present 20% more Gal-3 than non-infected host cells (Figure 6B). The direct interaction between the Ssp- 4 carbohydrate epitope recognized by mAb1D9 and host cell Gal-3 was assessed by immunoprecipitation using anti-Gal-3; Figure 6C shows that only the lysates of infected host cells presented reactivity with mAb1D9 (red arrow), indicating that the carbohydrate epitope recognized by mAb1D9 and displayed on Ssp-4 is a β-galactoside, with which Gal-3 is known to speciﬁcally interact (Hughes, 1999). Ssp-4 Expression Does Not Correlate With EA Infectivity (C) Scheme of the hypothetical protein bearing the Ssp-4 epitope identiﬁed by mass spectrometry, revealing domains encountered by in silico analysis. (D) Structural models of the Ssp-4 putative protein predicted by I-Tasser and Yasara. MAP2 peptides are in yellow. In green, blue, and red are the other peptides selected by I-Tasser and Yasara model (MAP1, MAP3, and MAP4, respectively). (E) IP of protein extracts of EAs of CL with mAb2C2, subsequently probed with MAP2 in Western blotting. MAP2 antibody (Figures 4B,C). Therefore, the invasiveness of infectious strains is not associated with Ssp-4 mRNA or protein expression. These data suggest that Ssp-4 glycosylation and by qPCR, is at least twofold higher in less-infectious strains compared to infectious EAs (Figure 4A). This diﬀerence was conﬁrmed by Western blotting and immunoﬂuorescence with a April 2018 \| Volume 9 \| Article 693 Frontiers in Microbiology \| www.frontiersin.org 5 Glycosylated Ssp-4 Participates in T. cruzi Infection Florentino et al. FIGURE 4 \| Ssp-4 protein expression is increased in the less infective strain. (A) Relative mRNA expression of Ssp-4 from extracellular amastigotes (EAs) of the CL strain compared to EAs from the G strain in three independent experiments performed in triplicate. ∗∗p < 0.01. (B) Whole cell lysates (WC) of EAs of the G and CL strains were incubated with MAP2 in Western blotting; anti-α-tubulin antibody was used for normalization. Red arrows indicate Ssp-4. (C) Immunoﬂuorescence (IF) of EAs of the G and CL strains incubated with MAP2 (green); nuclei and kinetoplasts were labeled with DAPI (blue). Scale bar: 5 µm. RE 4 \| Ssp-4 protein expression is increased in the less infective strain. (A) Relative mRNA expression of Ssp-4 from extracellular amastigotes (EAs) of th compared to EAs from the G strain in three independent experiments performed in triplicate ∗∗p < 0 01 (B) Whole cell lysates (WC) of EAs of the G and FIGURE 4 \| Ssp-4 protein expression is increased in the less infective strain. (A) Relative mRNA expression of Ssp-4 from extracellular amastigotes (EAs) of the CL strain compared to EAs from the G strain in three independent experiments performed in triplicate. ∗∗p < 0.01. (B) Whole cell lysates (WC) of EAs of the G and CL strains were incubated with MAP2 in Western blotting; anti-α-tubulin antibody was used for normalization. Red arrows indicate Ssp-4. Frontiers in Microbiology \| www.frontiersin.org EAs Secrete Glycosylated Ssp-4 That May or May Not Be Associated With Vesicles Because in silico analysis identiﬁed a signal peptide on the Ssp-4 protein core (Figure 3C and Supplementary Figures S1, S2), we assessed the secretion of the glycosylated form of Ssp-4 (detected by mAb1D9) using confocal and electron microscopy and SDS– PAGE/silver staining on EA supernatant fractions. First, morphological analysis of the microscopy images revealed that infectious EAs release vesicles to the extracellular medium, and these vesicles are either attached to poly-L-lysine- coated substrates or to host cell surfaces (Figures 5A,B). Vesicles of regular shape (100–200 nm in diameter) are secreted in trails (Figures 5A,B, arrows). These vesicle trails display the carbohydrate epitope recognized by mAb1D9 and are shed locally at sites of close contact between EAs and host cells, characterized After demonstrating that the carbohydrate moiety of Ssp-4 is a β-galactoside that can be recognized by host cell surface April 2018 \| Volume 9 \| Article 693 Frontiers in Microbiology \| www.frontiersin.org 6 Glycosylated Ssp-4 Participates in T. cruzi Infection Florentino et al. Florentino et al. Glycosylated Ssp-4 Participates in T. cruzi Infection FIGURE 5 \| Extracellular amastigotes release Ssp-4 associated with vesicles interacting with host cell. (A) Scanning electron microscopy of extracellular amastigotes (EAs) adhered to coverslips coated with poly-L-lysine or Vero cells. Scale bars: 2 and 5 µm. (B) Transmission electron microscopy of HeLa cells incubated for 30 min with EAs from the G strain (colored in red). Scale bar: 1 µm. Black arrows indicate EA secreted vesicles. (C) Immunoﬂuorescence of HeLa cells incubated for 30 min with EAs from G (MOI 10:1) and mAb1D9 (green), DAPI (blue), and phalloidin-TRITC (red). Upper panels: images of a focal plane showing vesicles secreted by EAs of the G strain (green) associated with actin (red); bottom panels: three-dimensional reconstruction from a Z-series acquired by a confocal microscope. Scale bar: 2 µm. Arrows indicate vesicular structures secreted by EAs. (D) Extracellular amastigotes (EAs) from the G and Y strains were incubated for 6 h in RPMI medium without fetal bovine serum. Supernatant was collected and fractionated in three different populations: V2 (pelleted after 2 h of ultracentrifugation), V16 (pelleted after 16 h ultracentrifugation), and VF (supernatant from pellet V16). Western blotting with mAb1D9 and silver-stained SDS–PAGE of the different fractions. Whole cell (WC) lysate of EAs of the G strain was used as control. FIGURE 5 \| Extracellular amastigotes release Ssp-4 associated with vesicles interacting with host cell. Frontiers in Microbiology \| www.frontiersin.org EAs Secrete Glycosylated Ssp-4 That May or May Not Be Associated With Vesicles (A) Scanning electron microscopy of extracellular amastigotes (EAs) adhered to coverslips coated with poly-L-lysine or Vero cells. Scale bars: 2 and 5 µm. (B) Transmission electron microscopy of HeLa cells incubated for 30 min with EAs from the G strain (colored in red). Scale bar: 1 µm. Black arrows indicate EA secreted vesicles. (C) Immunoﬂuorescence of HeLa cells incubated for 30 min with EAs from G (MOI 10:1) and mAb1D9 (green), DAPI (blue), and phalloidin-TRITC (red). Upper panels: images of a focal plane showing vesicles secreted by EAs of the G strain (green) associated with actin (red); bottom panels: three-dimensional reconstruction from a Z-series acquired by a confocal microscope. Scale bar: 2 µm. Arrows indicate vesicular structures secreted by EAs. (D) Extracellular amastigotes (EAs) from the G and Y strains were incubated for 6 h in RPMI medium without fetal bovine serum. Supernatant was collected and fractionated in three different populations: V2 (pelleted after 2 h of ultracentrifugation), V16 (pelleted after 16 h ultracentrifugation), and VF (supernatant from pellet V16). Western blotting with mAb1D9 and silver-stained SDS–PAGE of the different fractions. Whole cell (WC) lysate of EAs of the G strain was used as control. in all three fractions, demonstrating that infectious EAs release either free glycosylated Ssp-4 or glycosylated Ssp-4 in association with diﬀerent secreted vesicles. No diﬀerences in electrophoretic mobility were observed between vesicle- free and vesicle-associated Ssp-4 protein. This ﬁnding is consistent with previous observations about the T. cruzi secretome from metacyclic typomastigotes, where the stage- speciﬁc GPI-anchored protein GP82 detected in vesicle-free fractions had a mobility proﬁle that matched that of the protein present in vesicle-enriched fractions (Bayer-Santos et al., 2013). components and is secreted in vesicles during interactions with host cells at sites of EA invasion, we evaluated whether glycosylated Ssp-4 could also be found in vesicle-free fractions of infectious EA supernatants (Figure 5D). Fractions were obtained by ultracentrifugation of the EA supernatant after 2 or 16 h, resulting in precipitates containing ectosomes (V2 fraction) and exosomes (V16 fraction), respectively. The remaining supernatant is considered free of vesicles (VF fraction). Figure 5D shows that although SDS–PAGE/silver staining demonstrated diﬀerent protein proﬁles between supernatant populations, glycosylated Ssp-4 was detected by mAb1D9 April 2018 \| Volume 9 \| Article 693 Frontiers in Microbiology \| www.frontiersin.org 7 Glycosylated Ssp-4 Participates in T. cruzi Infection Florentino et al. EAs Secrete Glycosylated Ssp-4 That May or May Not Be Associated With Vesicles FIGURE 6 \| Galectin-3 is recruited to extracellular amastigotes from the G strain at sites of invasion and interacts with the Ssp-4 carbohydrate epitope recognized by mAb1D9. (A) Immunoﬂuorescence with mAb1D9 (blue) and anti-Gal-3 (green). HeLa cells were incubated with extracellular amastigotes (EAs) from the G strain (MOI 10:1) for 30 min. The actin cytoskeleton was labeled with 647 phalloidin (red), and DAPI (white) was used to label nuclei and kinetoplasts. White arrows indicate EAs surrounded by actin and Gal-3. Scale bar: 5 µm. (B) Western blotting of EAs interacting with HeLa cells performed with anti-Gal-3 and mAb1D9. Samples were normalized with anti-α-actin. HeLa cells were incubated with EAs of the G strain (MOI 40:1) for 40 min. Whole cell (WC) lysate from infected cells (H+G), and controls only from HeLa (H) and EAs of the G strain (G) were obtained. (C) Immunoprecipitation (IP) was performed for H+G and controls of H and G lysates with anti-Gal-3 coupled to protein G-Sepharose. Red arrow indicates the band that reacted with mAb1D9 on the IP H+G. FIGURE 6 \| Galectin-3 is recruited to extracellular amastigotes from the G strain at sites of invasion and interacts with the Ssp-4 carbohydrate epitope recognized by mAb1D9. (A) Immunoﬂuorescence with mAb1D9 (blue) and anti-Gal-3 (green). HeLa cells were incubated with extracellular amastigotes (EAs) from the G strain (MOI 10:1) for 30 min. The actin cytoskeleton was labeled with 647 phalloidin (red), and DAPI (white) was used to label nuclei and kinetoplasts. White arrows indicate EAs surrounded by actin and Gal-3. Scale bar: 5 µm. (B) Western blotting of EAs interacting with HeLa cells performed with anti-Gal-3 and mAb1D9. Samples were normalized with anti-α-actin. HeLa cells were incubated with EAs of the G strain (MOI 40:1) for 40 min. Whole cell (WC) lysate from infected cells (H+G), and controls only from HeLa (H) and EAs of the G strain (G) were obtained. (C) Immunoprecipitation (IP) was performed for H+G and controls of H and G lysates with anti-Gal-3 coupled to protein G-Sepharose. Red arrow indicates the band that reacted with mAb1D9 on the IP H+G. DISCUSSION carbohydrate epitope recognized by mAb1D9 and EA infectivity, adding these observations to those from the sequencing of the Ssp-4 protein core, the measurement of the expression of this protein in infectious versus less-infectious EAs and the analysis of the nature and function of the carbohydrate moieties in parasite infectivity. Mortara et al. (1999) ﬁrst established correlation between Ssp- 4 recognition by mAb1D9 and EA infectivity by demonstrating that EAs of more infective strains present higher reactivity with mAb1D9. However, da Silva et al. (2006) did not observe the same correlation between mAb1D9 reactivity and EA infectivity in isolates from chagasic patients. Although correlation was not demonstrated, the authors veriﬁed that blockage of the carbohydrate epitope with mAb1D9 could inhibit EA invasion, suggesting a role for carbohydrate moieties of Ssp-4 in host cell invasion. We conﬁrmed the correlation between the Ssp-4 Mortara et al. (1999) ﬁrst established correlation between Ssp- 4 recognition by mAb1D9 and EA infectivity by demonstrating that EAs of more infective strains present higher reactivity with mAb1D9. However, da Silva et al. (2006) did not observe the same correlation between mAb1D9 reactivity and EA infectivity in isolates from chagasic patients. Although correlation was not demonstrated, the authors veriﬁed that blockage of the carbohydrate epitope with mAb1D9 could inhibit EA invasion, suggesting a role for carbohydrate moieties of Ssp-4 in host cell invasion. We conﬁrmed the correlation between the Ssp-4 y The EAs from the most infective strains, G and clone Tc1552, which belong to the same phylogenetic group (Tc I), exhibited high reactivity with mAb1D9 and low reactivity with mAb2C2 (Figures 1, 2). Conversely, less-infectious EAs, such as those of the CL strain (Tc VI) and the Tc863 isolate (Tc III), showed high reactivity with mAb2C2 and low reactivity with mAb1D9. These April 2018 \| Volume 9 \| Article 693 Frontiers in Microbiology \| www.frontiersin.org 8 Glycosylated Ssp-4 Participates in T. cruzi Infection Florentino et al. results suggest that the post-translational modiﬁcations of Ssp-4 diﬀer among T. cruzi strains, and this phenotype directly reﬂects parasite infectivity. our group in microarray assays. In the previous experiments, a 21-kDa protein (P21) and a mevalonate kinase (MVK) were also detected, being more highly expressed in the infectious EAs of the G strain than in the less-infectious CL strain; these two factors were shown to contribute to host cell invasion (da Silva et al., 2009; Ferreira et al., 2016). DISCUSSION Here, we conﬁrmed by quantitative PCR that Ssp-4 is more highly expressed in less- infectious strains (Figure 4A), although infectious EAs exhibit a highly glycosylated Ssp-4 (as revealed by mAb1D9). Thus, we concluded that even though Ssp-4 provides the substrate for other posttranslational modiﬁcations, host cell invasion is not determined by Ssp-4 protein expression alone but by glycosylation of Ssp-4 (Figure 4). The ﬁrst description of Ssp-4 revealed that mAb2C2 recognizes a surface GPI-anchored glycoprotein, which is cleaved and released into extracellular milieu in the presence of PI-PLC (Andrews et al., 1988). Subsequently, Olivas- Rubio et al. (2009) identiﬁed a partial sequence from the DGF-1 multigenic family, but identiﬁcation of potential domains on the putative protein was challenging. These challenges led us to identify Ssp-4 using approaches other than immunostaining, such as mass spectrometry (MS) of mAb2C2- and mAb1D9-immunoprecipitated proteins, protein sequencing with bioinformatic analysis and quantitative PCR. Considering that glycosylated Ssp-4 is secreted and that infectious EAs secrete large amounts of vesicle trails when adhered to poly-L-lysine surfaces covered by glycosylated Ssp-4 or mammalian host cell surfaces (Figures 5A,B) (Barros et al., 1996), we investigated whether or not Ssp-4 interacted with these vesicles to modulate EA infection by either inhibiting or promoting host cell invasion. A recent study observed a similar pattern of secreted vesicles from ﬂagellar membranes of Trypanosoma brucei that act as carriers of virulence factors, contributing to host immune evasion and erythrocyte remodeling (Szempruch et al., 2016). First, vesicles displaying glycosylated Ssp-4 are secreted locally at sites of EA-host-cell interaction, namely, the phagocytic cup formed during the process of EA internalization, suggesting that these vesicles could be recognized by host cell receptors and could ultimately promote host cell invasion. Therefore, we sought to identify a possible receptor that could bind to carbohydrates displayed by Ssp-4. Several groups have demonstrated that Gal-3 participates in host cell infection by T. cruzi; Machado et al. (2014) observed the recruitment of Gal-3 during invasion and after parasitic escape from vacuoles in peritoneal macrophages infected with EAs of the G strain. DISCUSSION Considering that EAs are internalized by non-professional phagocytes via an actin-dependent process similar to phagocytosis (Mortara, 1991; Procópio et al., 1998; Fernandes et al., 2013) and that Gal-3 has been shown to play a crucial role during phagocytosis in macrophages (Sano et al., 2003), we investigated the association of Gal-3 with the Ssp-4 carbohydrate epitope recognized by mAb1D9 during host cell invasion by EAs. We have demonstrated that Gal- 3 is recruited to the EA entry site (Figure 6A), is more abundant in infected cells than in uninfected cells (Figure 6B) and co-immunoprecipitates with Ssp-4 recognized by mAb1D9 (Figure 6C). As Gal-3 speciﬁcally recognizes β-galactosides, we concluded that a β-galactose antigen displayed by Ssp-4 is the carbohydrate moiety recognized by host cells. Exogenous Gal- 3 is able to activate Rac-1 in corneal epithelial cells (Saravanan et al., 2009). Interestingly, our group has previously revealed the participation of proteins associated with the Rho-GTPase family, such as Rac-1, in EA internalization (Fernandes and Mortara, 2004; Bonﬁm-Melo et al., 2018). Members of these families are responsible for mediating the recruitment of the actin cytoskeleton with cell surface receptors. Thus, increased Gal-3 availability could mediate Rac-1 activation and formation of phagocytic cups involved in host cell invasion of EAs. y q Based on the CL Brener hybrid clone genomic database, haplotype sequences encode a hypothetical protein that is highly expressed (mRNA and protein) in amastigote forms (Atwood et al., 2005; Minning et al., 2009). Additionally, Ssp-4 sequences exhibit homology with a hypothetical protein from Trypanosoma rangeli and Leishmania ssp., but not Trypanosoma brucei, which indicates that these proteins could be associated with the presence of an intracellular cycle deﬁned by the amastigote form. In silico analysis of the amino acid sequences identiﬁed by MS led to the identiﬁcation of Ssp-4 domains previously described by Andrews et al. (1988) as potential sites to be targeted to the plasma membrane and secreted, i.e., the presence of N- and O-glycosylation sites and a possible GPI-anchor. Potential glycosylation sites observed explain the fact that hypothetical protein encountered by MS present 67 kDa, and by Western blotting molecular weight detect a band between 93 kDa. Finally, an α/β-hydrolase motif was identiﬁed. Frontiers in Microbiology \| www.frontiersin.org Immunoprecipitation Ssp-4 was immunoprecipitated (IP) using protein A-Sepharose (Sigma-Aldrich) with mAb2C2 (IgG2a). Lysate was prepared from 109 EAs of the CL or G strains with lysis buﬀer (50 mM Tris–HCl, 150 mM NaCl, 1% Triton X-100, pH 7.4). The lysate was then incubated with protein A-Sepharose for 1 h at 4◦C for pre-clearing. Next, the supernatant from the pre- clearing was incubated with protein A-Sepharose and mAb2C2 (1:5, ascitic ﬂuid) overnight at 4◦C. After incubation, the pellet containing protein A-Sepharose attached to mAb2C2 and Ssp- 4 was washed with washing buﬀer (20 mM Tris–HCl, pH 7.5) with diﬀerent NaCl concentrations (0.5, 0.3, 0.1 M). The pellet was then resuspended with 4 × SDS–PAGE sample buﬀer (0.1 M Tris–HCl, 10% SDS, 0.5 mM bromophenol blue, 10% glycerol, 12% β-mercaptoethanol, pH 6.8). As a control, we used a non-immune mouse serum for immunoprecipitation instead of mAb2C2. Alternatively, immunoprecipitation was performed using EAs from the G strain lysate with mAb1D9 (IgG3) and protein G-Sepharose (Sigma-Aldrich). Host Cells HeLa cells and Vero cells (Instituto Adolfo Lutz, São Paulo, Brazil) were cultivated by successive passaging in RPMI 1640 medium (Atena Biotecnologia) supplemented with 10% heat- inactivated fetal bovine serum (FBS; Vitrocell), 100 U/mL penicillin and 100 µg/mL streptomycin (Sigma-Aldrich). Mass Spectrometric Analysis p y The IPs from the EAs of the CL strain with mAb2C2 and the control serum (CS) and IPs from the EAs of the G strain with mAb2C2, mAb1D9, and CS were subjected to SDS–PAGE and stained with colloidal Coomassie. The band of 93 kDa from all mAb2C2 and CS IPs were excised from the gel and destained with 50 mM ammonium bicarbonate in 50% acetonitrile (ACN) solution. Then, the bands were dehydrated with 100% ACN. Proteins were reduced with 10 mM dithiothreitol and alkylated with 50 mM iodoacetamide. Gel bands were washed with 50 mM NH4HCO3/50% ACN and then with 100% ACN. The bands were then incubated with porcine trypsin (Sigma-Aldrich) solution (400 ng) for 16 h at 37◦C for protein digestion. The resulting peptides were resuspended in 0.1% formic acid, and an aliquot (4.5 µL) was injected into a Q-TOF Ultima mass spectrometer (Waters) coupled to a nano-chromatography system (nanoACQUITY, Waters). Samples were desalted and concentrated using a pre-column (Symmetry C18, 180 µm × 20 mm, Waters) and eluted onto a capillary reversed-phase C18 column (10 cm × 75 µm, Waters) at a ﬂow rate of 600 nL/min. Peptides were fractionated with a linear gradient from 5 to 40% ACN in 0.1% formic acid for 15 min. The eluted peptides were directly injected into the mass spectrometer (MS) via an electrospray. The MS spectrum of phosphoric acid was acquired simultaneously to acquisition of the sample spectra. The MS/MS spectra of the three most intense peaks with two to four charges in the MS function were automatically acquired in the data-dependent acquisition (DDA) mode. The capillary voltage and reference cone were set at 3.2 kV and 100 V, respectively. All analyses were performed in positive ion mode. The spectra were acquired from 50 to 2000 m/z. DISCUSSION Previous study found that the trypomastigote surface prolyl oligopeptidase (POP) enzyme (Tc80), which contains an α/β-hydrolase domain, is capable of hydrolyzing substrates such as ﬁbronectin and collagen and was shown to be involved in non-phagocytic cell invasion (Bastos et al., 2005). However, the enzymatic functions of this feature of Ssp-4 were not investigated, and the contribution of this enzyme to host cell invasion remains unclear. To conﬁrm our MS data, a polyclonal antibody (MAP2) was produced based on the tertiary structure of Ssp-4 (Figure 3C). In this set of results, it was observed that MAP2 reacts with mAb2C2 immunoprecipitates (mAb2C2-IPs) from less infectious EAs (Figure 3E). These data strongly suggest that the protein detected by mass spectrometry is the Ssp-4 that was previously described by Andrews et al. (1988). Immunostaining using the MAP2 antibody detected higher expression of Ssp-4 on non-infectious EAs, suggesting again that recognition of the carbohydrate epitope on Ssp-4 by mAb1D9 can hinder the interactions of the protein core epitopes (Figure 4B). The production of the MAP2 antibody, obtained after chemical synthesis of peptides generated by bioinformatics and protein modeling, conﬁrms the antigenic potential of Ssp-4 and renders feasible the production of an eﬃcient and safe vaccine against Chagas’ disease using a combination of the Ssp-4 protein core and carbohydrate moieties (Quijano-Hernandez and Dumonteil, 2011; Ma et al., 2015). The hypothetical protein (TcCLB.506725.20) identiﬁed by MS and considered here to be Ssp-4 was previously detected by April 2018 \| Volume 9 \| Article 693 9 Glycosylated Ssp-4 Participates in T. cruzi Infection Florentino et al. We have shown that post-translational modiﬁcations of surface proteins of T. cruzi EAs are more closely associated with parasite infectivity than the expression of the protein core. Additionally, glycosylated Ssp-4 can form a bridge between the parasite and host cell surface factors such as Gal-3, triggering EA internalization. In our model, we have shown that protein glycosylation plays a central role in the control of EA host cell invasion, highlighting the importance of post-translational modiﬁcations. The results of this study may contribute to the development of therapeutic agents and vaccines for the treatment of Chagas’ disease. 5 min to obtain serum containing polyclonal antibodies (MAP2). Control serum was obtained as a negative control by caudal vein puncture before the ﬁrst immunization. All animal procedures were approved by the local ethics committee (No. 1465020616). Parasites In this study, parasites from the G (Tc I) (Yoshida, 1983) and CL (Tc VI) strains (Brener and Chiari, 1963) were used. New isolates of T. cruzi were also used: the Tc863 (Tc III) isolate and clones Tc1522 (José strain; Tc I) and Tc1994 (Tcbat). These previously classiﬁed/typed isolates were a gift from Dr. Marta M. Geraldes Teixeira (ICB-USP, Trypanosomatid Bank). T. cruzi trypomastigote forms were collected from the supernatants of Vero cells cultivated with RPMI medium supplemented with 2.5% heat-inactivated FBS. The trypomastigotes were then incubated with liver infusion tryptose (Camargo, 1964) medium (pH 5.8) supplemented with 10% FBS for 16 h to obtain EAs (Tomlinson et al., 1995). Frontiers in Microbiology \| www.frontiersin.org Ssp-4 Antibodies Monoclonal antibody (mAb) 2C2, which recognizes an epitope of Ssp-4, was a gift from Norma Andrews (University of Maryland) (Andrews et al., 1987). The antibody mAb1D9 was obtained by immunization of BALB/c mice with amastigotes from clone D11 (derived from G strain). Peptides (Bio-Synthesis Inc.) were synthesized based on the Ssp-4 tertiary structure modeled by homology modeling and threading (described in section “Molecular Modeling and Structural Analyses”). We selected non-conjugated multiple antigenic peptide sequence (LREFVRSTERNR) for animal immunization. Brieﬂy, solubilized peptides (1 mg/mL) in phosphate-buﬀered saline (PBS; 137 mM NaCl, 10 mM phosphate, 2.7 mM KCl, pH 7.4) were inoculated into BALB/c mice in the presence of Freund’s complete or incomplete adjuvant (Sigma-Aldrich). Four immunizations (100 µL peptide solution/mouse) were performed intraperitoneally with 7-day interval. These mice were euthanized with lethal injections of ketamine/xylazine (250/50 mg/kg per mouse) 1 week after the last immunization. Then, cardiac puncture was performed on these mice, and the collected blood was centrifuged at 1,200 × g for April 2018 \| Volume 9 \| Article 693 Frontiers in Microbiology \| www.frontiersin.org 10 Glycosylated Ssp-4 Participates in T. cruzi Infection Florentino et al. Peptides from the IPs of the G strain were injected onto an LTQ-Orbitrap Velos (Thermo). Masses from the raw Q-TOF ﬁles were corrected using a lock mass of m/z 784.823, and the raw data were converted to the .pkl format using the ProteinLynx Global Server (Waters). Orbitrap raw ﬁles were converted into .mgf ﬁles using MSConverter. Searches were performed in MASCOT (2.4.1 version) against a Trypanosoma cruzi CL Brener database (downloaded in 2017/05, 21,169 sequences) by considering a peptide and fragment mass tolerance of 0.5 Da (for Q-TOF ﬁles) and peptide and fragment mass tolerance of 10 ppm and 0.6 Da, respectively. Carbamidomethylation of cysteine and oxidation of methionine residues were speciﬁed as ﬁxed and variable modiﬁcations, respectively. To obtain a ﬁnal list of identiﬁed proteins, masses smaller than 60 kDa and higher than 110 kDa were excluded as were proteins identiﬁed as control samples. Finally, proteins identiﬁed with scores lower than 80 were also excluded. and Ssp-4 Forward (5′-cctctgacattgacccgttatt-3′) and Reverse (5′- gtaagttggattggtgtggta-3′). RNA extraction from the EAs of the G and CL strains was performed with the RNeasy Protect Mini Kit (Qiagen). Next, cDNA was generated using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Transmission and Scanning Electron HeLa cells incubated with EAs for 30 min were ﬁxed with 2.5% glutaraldehyde and 2% formaldehyde in 0.1 M sodium cacodylate buﬀer at room temperature for 1 h and then processed for transmission electron microscopy (TEM) according to procedures described elsewhere (Arruda et al., 2012). Cells were observed in a JEOL 1200 EXII electron microscope (JEOL Ltd., Peabody, MA, United States). For scanning electron microscopy (SEM), EAs adhered to Vero cells or to coverslips covered with 0.1% poly-L-lysine (Sigma-Aldrich) were ﬁxed with 4% paraformaldehyde, washed with 0.1 M cacodylate solution, post- ﬁxed with osmium tetroxide, treated with tannic acid, dehydrated Ssp-4 Antibodies Relative quantiﬁcation was monitored by intercalation of SYBRTM Green Master Mix (Life Technologies) in the double-stranded DNA in qPCR reactions; normalized values calculated with a 11cycle threshold (Ct) and relative expression (2−11Ct) with the G strain as a reference are shown. GAPDH is the housekeeping gene; this gene is constitutively expressed in T. cruzi. 1www.expasy.org 2http://gpcr.biocomp.unibo.it/predgpi/pred.htm In Silico Analysis Bioinformatics was performed using the ExPASy1 platform. The signal peptide was predicted by SignalP v. 4.1. The TargetP v. 1.1 platform was used to predict subcellular location. Hydrophobic domains were predicted by the TMHMM 2.0 server. Potential sites of N- and O-glycosylation were identiﬁed by NetNGlyc 1.0 and NetOGlyc 4.0, respectively. Interproscan v. 4.8 software was used to identify diﬀerent protein domains. Prediction of GPI-anchoring sites was performed with the PredGPI2 program. Molecular Modeling and Structural Analyses The three-dimensional structure of the putative Ssp-4 sequenced by mass spectrometry was modeled using I-Tasser (Yang et al., 2015) and Yasara3. The model with the highest C-score from I-Tasser and the hybrid model from Yasara were selected for structural analysis. The solvent-accessible surface area (SASA) of each amino acid was calculated using DSSP 2.2.1 (Touw et al., 2015). The most exposed peptides in both models were selected as the most immunogenic peptides to be synthesized (Bio-Synthesis Inc.) for the production of the antibody against the Ssp-4 protein region (section “Ssp-4 Antibodies”). 3www.yasara.org Cloning of the Ssp-4 Coding Sequence The open reading frame encoding the putative Ssp-4 protein identiﬁed by mass spectrometry was ampliﬁed by PCR using T. cruzi genomic DNA as a template. The ampliﬁcation was performed using the Ssp4F (5′-atggctctaaagagtatgcggaaa-3′) and Ssp4R (5′-ttaatggttcaaattgctgtaaaa-3′) oligonucleotides and Pfu DNA polymerase (Fermentas). Amplicons from the G and CL strains and the CL Brener clone were resolved on agarose gels, and the bands at the expected size were excised and puriﬁed. Puriﬁed amplicons were A-tailed prior to ligation into the pGEM R⃝-T Easy vector (Promega). Plasmid DNA isolated from positive clones was sequenced on an ABI3500 genetic analyzer (Applied Biosystems) using the BigDye R⃝Terminator v3.1 Cycle Sequencing Kit (Life Technologies). Sequences were aligned by BioEdit v. 7.2.5 using the ClustalW algorithm. The assembled nucleotide sequences encoding the putative Ssp-4 protein from the G and CL strains were deposited in GenBank under accession numbers KX581696 and KX581697, respectively. Western Blotting The EA lysates (20 µg) were incubated with 4 × sample buﬀer and subjected to SDS–PAGE (Laemmli, 1970). Proteins from the gel were transferred to nitrocellulose membranes to perform the immunoblotting (Towbin et al., 1979). Membranes were blocked for 1 h with ECL blocking agent (GE Healthcare), TBS (50 mM Tris–HCl, 150 mM NaCl, pH 7.4), and TBS-T (TBS with 0.1% Tween 20). Then, nitrocellulose membranes were incubated with mAb1D9 (1:500, ascites), mAb2C2 (1:500 ascites), MAP2 (1:500, mouse antiserum), or anti-α-tubulin (1:1000, Sigma-Aldrich) overnight with 5% bovine serum albumin (Sigma-Aldrich) in TBS-T. Next, the membranes were incubated for 1 h with a secondary antibody, anti-mouse IgG-peroxidase (Sigma-Aldrich), and visualized by a chemiluminescence detector (UVITEC) in the presence of luminol with the ECL Prime Western blotting detection reagent (GE Healthcare). 1www.expasy.org 2http://gpcr.biocomp.unibo.it/predgpi/pred.htm Relative Quantiﬁcation of mRNA by Real-Time PCR (qPCR) For real-time quantitative PCR (qPCR), we synthesized the following primers: GAPDH Forward (5′-agcgcgcgtctaagacttaca- 3′) and Reverse (5′-tggagctgcggttgtcatt-3) (Cordero et al., 2008) April 2018 \| Volume 9 \| Article 693 Frontiers in Microbiology \| www.frontiersin.org 11 Glycosylated Ssp-4 Participates in T. cruzi Infection Florentino et al. Then, the coverslips were incubated with the primary antibodies mAb1D9 (mouse IgG) and anti-Galectin-3 (rat IgG; a gift from Dr. Roger Chammas, Universidade de São Paulo) for 1 h at room temperature. Next, the samples were incubated with the secondary antibodies Alexa Fluor IgG mouse 488 and Alexa Fluor IgG rat 568 in the presence of 1 µm DAPI (Invitrogen) and 200 units/mL phalloidin 647 (Invitrogen) for 1 h at room temperature. with ethanol, dried in a critical point dryer (Bal-Tec AG, Balzers, Liechtenstein), and sputtered with gold. Samples were observed in an FEI Quanta FEG 250 SEM (Hillsboro, OR, United States). Statistics All results are representative of three independent experiments with at least two biological replicates. Excel and GraphPad Prism 4.0 were employed for data plotting and statistical analysis. Statistical tests included Student’s t-test and ANOVA, and a statistical threshold of p < 0.05 was used. EA Supernatant Fractionation The EAs supernatant fractionation was adapted from Bayer- Santos et al. (2013). Brieﬂy, parasites from the G strain were incubated in RPMI medium without FBS for 6 h at 37◦C (108 EAs/mL). Then, the parasites were removed by centrifugation at 3,000 × g for 10 min, and supernatant was collected. The supernatant was ﬁltered by using a 0.45- µm-pore membrane and subjected to ultracentrifugation at 100,000 × g for 2 h to obtain the ﬁrst pellet, which was enriched with larger vesicles (V2). The supernatant resulting from this ultracentrifugation was again subjected to ultracentrifugation at 100,000 × g for 16 h to generate the second pellet, which was enriched with smaller vesicles (V16), and the supernatant contained vesicle-free soluble (VF) proteins. Infection of Host Cells Funding was provided by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) through grants and fellowships number (2011/51475-3 and 2012/23150-5), Conselho Nacional de Desenvolvimento Cientíﬁco e Tecnológico (CNPq) fellowship number 302068/2016-3 to RM, and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). Suspension containing 105 HeLa cells in RPMI supplemented with 10% FBS per well (24-well plate) was plated on coverslips and incubated overnight at 37◦C in CO2 (5%). Next, EAs (Multiplicity of Infection; MOI: 10:1) were added for 2 h at 37◦C. After incubation, the cells were washed 5–7 times with PBS. Then, the cells were ﬁxed with Bouin’s solution (Sigma- Aldrich) for 5 min and stained with Giemsa for 1 h (da Silva et al., 2006). Internalized parasites were quantiﬁed by light microscopy (Bonﬁm-Melo et al., 2015). AUTHOR CONTRIBUTIONS PF, FR, CO, and RM designed and coordinated the study and wrote the paper. JdC, PF, and FV performed the proteomic experiments and analyses. EC, PF, and CO designed and performed DNA sequencing experiments. TS designed the three- dimensional structure analysis and peptide selection for antibody production. All authors analyzed the results and approved the ﬁnal version of the manuscript. Parasites Immunoﬂuorescence and Confocal Imaging Samples were examined by a Leica TCS SP5 confocal system (Leica Microsystems, Wetzlar, Germany) using a 100 × NA 1:44 oil immersion objective. Images were processed by Imaris software (Bitplane AG, Andor Technology, Belfast, United Kingdom). Parasites Immunoﬂuorescence and Confocal Imaging The EAs were attached to coverslips covered with 0.1% poly-L-lysine (Sigma-Aldrich) for 50 min or HeLa cells for 30 min at 37◦C and ﬁxed with 4% paraformaldehyde. After ﬁxation, coverslips were incubated with blocking solution, PGN (0.2% gelatin, 0.1% NaN3, diluted in PBS), for 1 h. Next, samples were incubated with mAb1D9, mAb2C2 (1:100), or MAP2 (1:100) diluted in PGN supplemented with 0.25% saponin (PGN-S; Sigma-Aldrich) for 1 h at room temperature. Next, coverslips were incubated with a secondary antibody, Alexa Fluor 488 IgG-mouse (Invitrogen), diluted in PGN-S (1:100) for 1 h at room temperature. Coverslips were then incubated with 1 µM 4′,6-diamidino-2-phenylindole (DAPI) for 15 min. After three washes with PBS, coverslips were mounted in glycerol buﬀered with 0.1 M Tris (pH 8.6) and 0.1% p-phenylenediamine. Samples were examined by a Leica TCS SP5 confocal system (Leica Microsystems, Wetzlar, Germany) using a 100 × NA 1:44 oil immersion objective. Images were processed by Imaris software (Bitplane AG, Andor Technology, Belfast, United Kingdom). The EAs were attached to coverslips covered with 0.1% poly-L-lysine (Sigma-Aldrich) for 50 min or HeLa cells for 30 min at 37◦C and ﬁxed with 4% paraformaldehyde. After ﬁxation, coverslips were incubated with blocking solution, PGN (0.2% gelatin, 0.1% NaN3, diluted in PBS), for 1 h. Next, samples were incubated with mAb1D9, mAb2C2 (1:100), or MAP2 (1:100) diluted in PGN supplemented with 0.25% saponin (PGN-S; Sigma-Aldrich) for 1 h at room temperature. For Western blotting, EAs (MOI 40:1) were incubated with HeLa cells for 40 min at 37◦C in CO2 (5%). Infected HeLa cells, control non-infected HeLa cells, and EA suspensions were lysed with lysis buﬀer (50 mM Tris–HCl, 150 mM NaCl, 1% Triton X-100, pH 7.4) for 30 min at 4◦C. Samples were subjected to Western blotting with primary antibodies anti-Gal-3 (1:250), mAb1D9 (1:500), and anti-α-actin (1:1000; Cell Signaling), and then, immunoprecipitation with anti-Gal-3 and protein G-Sepharose (Sigma-Aldrich) was performed as described above. Band densitometry was analyzed by UVIBAND image quantiﬁcation software. Next, coverslips were incubated with a secondary antibody, Alexa Fluor 488 IgG-mouse (Invitrogen), diluted in PGN-S (1:100) for 1 h at room temperature. Coverslips were then incubated with 1 µM 4′,6-diamidino-2-phenylindole (DAPI) for 15 min. After three washes with PBS, coverslips were mounted in glycerol buﬀered with 0.1 M Tris (pH 8.6) and 0.1% p-phenylenediamine. Frontiers in Microbiology \| www.frontiersin.org REFERENCES Bonﬁm-Melo, A., Zanetti, B. F., Ferreira, E. R., Vandoninck, S., Han, S. W., Van Lint, J., et al. (2015). Trypanosoma cruzi extracellular amastigotes trigger the protein kinase D1-cortactin-actin pathway during cell invasion. Cell. Microbiol. 17, 1797–1810. doi: 10.1111/cmi.12472 Andrews, N. W., Hong, K. S., Robbins, E. S., and Nussenzweig, V. (1987). Stage- speciﬁc surface antigens expressed during the morphogenesis of vertebrate forms of Trypanosoma cruzi. Exp. Parasitol. 64, 474–484. doi: 10.1016/0014- 4894(87)90062-2 Brener, Z., and Chiari, E. (1963). [Morphological Variations Observed in Diﬀerent Strains of Trypanosoma Cruzi. Rev. Inst. Med. Trop. Sao Paulo 5, 220–224. Andrews, N. W., Robbins, E., Ley, V., and Nussenzweig, V. (1988). Stage- speciﬁc surface antigens during the morphogenesis of Trypanosoma cruzi: developmentally regulated expression of a glycosyl-phosphatidylinositol anchored glycoprotein of amastigotes. Mem. Inst. Oswaldo Cruz 83(Suppl. 1), 561–562. doi: 10.1590/S0074-02761988000500067 Camargo, E. P. (1964). Growth and Diﬀerentiation in Trypanosoma Cruzi. I. Origin of Metacyclic Trypanosomes in liquid media. Rev. Inst. Med. Trop. Sao Paulo 6, 93–100. Clayton, J. (2010). Chagas disease 101. Nature 465, S4–S5. doi: 10.1038/ nature09220 Arruda, D. C., Santos, L. C., Melo, F. M., Pereira, F. V., Figueiredo, C. R., Matsuo, A. L., et al. (2012). Beta-Actin-binding complementarity-determining region 2 of variable heavy chain from monoclonal antibody C7 induces apoptosis in several human tumor cells and is protective against metastatic melanoma. J. Biol. Chem. 287, 14912–14922. doi: 10.1074/jbc.M111.322362 Cordero, E. M., Gentil, L. G., Crisante, G., Ramirez, J. L., Yoshida, N., Anez, N., et al. (2008). Expression of GP82 and GP90 surface glycoprotein genes of Trypanosoma cruzi during in vivo metacyclogenesis in the insect vector Rhodnius prolixus. Acta Trop. 105, 87–91. doi: 10.1016/j.actatropica.2007. 08.004 da Silva, C. V., Kawashita, S. Y., Probst, C. M., Dallagiovanna, B., Cruz, M. C., da Silva, E. A., et al. (2009). Characterization of a 21kDa protein from Trypanosoma cruzi associated with mammalian cell invasion. Microbes Infect. 11, 563–570. doi: 10.1016/j.micinf.2009.03.007 Atwood, J. A. III, Weatherly, D. B., Minning, T. A., Bundy, B., Cavola, C., Opperdoes, F. R., et al. (2005). The Trypanosoma cruzi proteome. Science 309, 473–476. doi: 10.1126/science.1110289 Barros, H. C., Da Silva, S., Verbisck, N. V., Araguth, M. F., Tedesco, R. C., Procópio, D. O., et al. (1996). Release of membrane-bound trails by Trypanosoma cruzi amastigotes onto modiﬁed surfaces and mammalian cells. J. Eukaryot. Microbiol. 43, 275–285. doi: 10.1111/j.1550-7408.1996.tb03990.x da Silva, C. V., Luquetti, A. O., Rassi, A., and Mortara, R. A. (2006). ACKNOWLEDGMENTS Alternatively, EAs were added to HeLa cells adhered to 6- well plates and incubated for 40 min at 37◦C in CO2 (5%). An immunoﬂuorescence assay was performed on the coverslips; after EA (MOI 10:1) incubation, the cells were washed with PBS and ﬁxed with 4% paraformaldehyde (PFA) for 15 min. We thank Martha Teixeira (Universidade de São Paulo) for T. cruzi isolates; João Bosco Pesquero (Universidade Federal de São Paulo) for the DNA sequencing service; Rita Sinigaglia April 2018 \| Volume 9 \| Article 693 Frontiers in Microbiology \| www.frontiersin.org 12 Glycosylated Ssp-4 Participates in T. cruzi Infection Florentino et al. Coimbra, André Aguillera, Márcia Tanakae, and Patricia Milanez from Centro de Microscopia Eletrônica (UNIFESP) for sample processing and TEM and SEM imaging; and Roger Chammas (Universidade de São Paulo) for the Gal-3 antibody. the ClustalW algorithm. Regions with 100% identity between the sequences are shown in black. White and gray regions represent amino acid divergence in at least one of the aligned sequences. In the legend, 1 and 2 represent the Esmeraldo-like haplotype and non-Esmeraldo-like haplotypes from the CL Brener database, respectively; 3 and 4 represent Dm28c and Sylvio database sequences, respectively; 5, 6 and 7 represent Ssp-4 coding sequences from the CL Brener clone and the G and CL strains isolated in this study, respectively. (B) Protein identity and divergence scores between all strains aligned. SUPPLEMENTARY MATERIAL TABLE S3 \| Solvent accessible surface area (SASA). The solvent accessible surface area (SASA) for each amino acid predicted by DSSP 2.2.1. SUPPLEMENTARY MATERIAL FIGURE S3 \| Vesicle trails covered with Ssp-4 carbohydrate epitopes on EAs of the G strain adhered to poly-L-lysine. Extracellular amastigotes (EAs) were attached onto coverslips coated with poly-L-lysine for 50 min at 37◦C. Then, the parasites were ﬁxed with 4% paraformaldehyde and incubated with blocking solution for 1 h. Samples were incubated with mAb1D9 (green) and DAPI (blue). Left panels: immunoﬂuorescence images obtained from one plane. Arrows indicate released vesicle trails from parasites. Right panels: Differential interference contrast (DIC). Scale bar: 2 µm. The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2018.00693/full#supplementary-material The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2018.00693/full#supplementary-material FIGURE S1 \| In silico analysis of the Ssp-4 sequence. Analysis of the Ssp-4 protein sequence identiﬁed by mass spectrometry using the ExPASy platform. The results obtained from the server are as follows: (A) prediction of signal peptide (SignalP v. 4.1); (B) prediction of transmembrane helices (TMHMM v. 2.0); (C) prediction of GPI-anchor (PredGPI); (D) prediction of subcellular localization (TargetP v. 1.1); (E) Interproscan v. 4.8; (F) prediction of N-glycosylation (NetNGlyc v. 1.0); and (G) prediction of O-glycosylation (NetOGlyc v. 4.0). FIGURE S1 \| In silico analysis of the Ssp-4 sequence. Analysis of the Ssp-4 protein sequence identiﬁed by mass spectrometry using the ExPASy platform. The FIGURE S1 \| In silico analysis of the Ssp-4 sequence. Analysis of the Ssp-4 protein sequence identiﬁed by mass spectrometry using the ExPASy platform. The results obtained from the server are as follows: (A) prediction of signal peptide (SignalP v. 4.1); (B) prediction of transmembrane helices (TMHMM v. 2.0); (C) prediction of GPI-anchor (PredGPI); (D) prediction of subcellular localization (TargetP v. 1.1); (E) Interproscan v. 4.8; (F) prediction of N-glycosylation (NetNGlyc v. 1.0); and (G) prediction of O-glycosylation (NetOGlyc v. 4.0). TABLE S1 \| Proteins and peptides identiﬁed by mass spectrometry. TABLE S2 \| List of identiﬁed proteins from EAs of the G strain immunoprecipitated with mAb2C2 and mAb1D9. (TargetP v. 1.1); (E) Interproscan v. 4.8; (F) prediction of N-glycosylation (NetNGlyc v. 1.0); and (G) prediction of O-glycosylation (NetOGlyc v. 4.0). TABLE S3 \| Solvent-accessible surface area (SASA). The solvent-accessible FIGURE S2 \| Ssp-4 protein alignment revealed a conserved backbone between different T. cruzi strains. 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Rep. 6:24610. doi: 10.1038/srep24610 April 2018 \| Volume 9 \| Article 693 Frontiers in Microbiology \| www.frontiersin.org 13 Glycosylated Ssp-4 Participates in T. cruzi Infection Florentino et al. Hughes, R. C. (1999). Secretion of the galectin family of mammalian carbohydrate- binding proteins. Biochim. Biophys. Acta 1473, 172–185. doi: 10.1016/S0304- 4165(99)00177-4 Procópio, D. O., da Silva, S., Cunningham, C. C., and Mortara, R. A. (1998). REFERENCES V., Andreoli, W. K., Silva, R. B., et al. (1999). Features of host cell invasion by diﬀerent infective forms of Trypanosoma cruzi. Mem. Inst. Oswaldo Cruz 94(Suppl. 1), 135–137. doi: 10.1590/S0074-02761999000700014 Yoshida, N. (2006). Molecular basis of mammalian cell invasion by Trypanosoma cruzi. An. Acad. Bras. Cienc. 78, 87–111. doi: 10.1590/S0001- 37652006000100010 Nogueira, N., and Cohn, Z. (1976). Trypanosoma cruzi: mechanism of entry and intracellular fate in mammalian cells. J. Exp. Med. 143, 1402–1420. doi: 10.1084/ jem.143.6.1402 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. Olivas-Rubio, M., Hernandez-Martinez, S., Talamas-Rohana, P., Tsutsumi, V., Reyes-Lopez, P. A., and Rosales-Encina, J. L. (2009). cDNA cloning and partial characterization of amastigote speciﬁc surface protein from Trypanosoma cruzi. Infect. Genet. Evol. 9, 1083–1091. doi: 10.1016/j.meegid.2009.05.016 Copyright © 2018 Florentino, Real, Orikaza, da Cunha, Vitorino, Cordero, Sobreira and Mortara. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Pineda, M. A., Corvo, L., Soto, M., Fresno, M., and Bonay, P. (2015). Interactions of human galectins with Trypanosoma cruzi: binding proﬁle correlate with genetic clustering of lineages. Glycobiology 25, 197–210. doi: 10.1093/glycob/cwu103 Procópio, D. O., Barros, H. C., and Mortara, R. A. (1999). Actin-rich structures formed during the invasion of cultured cells by infective forms of Trypanosoma cruzi. Eur. J. Cell Biol. 78, 911–924. doi: 10.1016/S0171-9335(99)80093-4 April 2018 \| Volume 9 \| Article 693 Frontiers in Microbiology \| www.frontiersin.org 14
https://openalex.org/W2984478896	http://www.scielo.br/pdf/ram/v20n6/1678-6971-ram-20-06-eRAMD190061.pdf	English	null	MANAGERS’ INFLUENCE ON COMPANY CAPABILITIES	RAM. Revista de Administração Mackenzie	2,019	cc-by	10,641	Submission: Apr. 8, 2019. Acceptance: July 11, 2019. ISSN 1678-6971 (electronic version) • RAM, São Paulo, 20(6), eRAMD190061, 2019 Dossier, doi:10.1590/1678-6971/eRAMD190061 ISSN 1678-6971 (electronic version) • RAM, São Paulo, 20(6), eRAMD190061, 2019 Dossier, doi:10.1590/1678-6971/eRAMD190061 MANAGERS’ INFLUENCE ON COMPANY CAPABILITIES To cite this paper: Costa, J. C. N., Camargo, S. M., Toaldo, A. M. M., & Didonet, S. R. (2019). Managers’ influence on company capabilities. Revista de Administração Mackenzie, 20(6). doi:10.1590/1678-6971/ eRAMD190061 1 Universidade Federal do Paraná (UFPR), Curitiba, PR, Brazil. 1 Universidade Federal do Paraná (UFPR), Curitiba, PR, Brazil. 2 Centro Universitário Internacional (Uninter), Curitiba, PR, Brazil. 2 Centro Universitário Internacional (Uninter), Curitiba, PR, Brazil. This is an open-access article distributed under the terms of the Creative Commons Attribution License. This paper may be copied, distributed, displayed, transmitted or adapted if provided, in a clear and explicit way, the name of the journal, the edition, the year and the pages on which the paper was originally published, but not suggesting that RAM endorses paper reuse. This licensing term should be made explicit in cases of reuse or distribution to third parties. It is not allowed the use for commercial purposes. Este artigo pode ser copiado, distribuído, exibido, transmitido ou adaptado desde que citados, de forma clara e explícita, o nome da revista, a edição, o ano e as páginas nas quais o artigo foi publicado originalmente, mas sem sugerir que a RAM endosse a reutilização do artigo. Esse termo de licenciamento deve ser explicitado para os casos de reutilização ou distribuição para terceiros. Não é permitido o uso para fins comerciais. Juliana C. N. Costa, Shirlei M. Camargo, Ana M. M. Toaldo, Simone R. Didonet Absorptive capacity. Marketing capability. Managers’ characteristics. Tenure. Age. ABSTRACT Purpose: This study aims to verify the moderating role of managers’ characteristics, age, and tenure (time in the sector, position, company), in the relation between the realized absorptive capacity (RACAP) and the architectural marketing capabilities (CAM). Purpose: This study aims to verify the moderating role of managers’ characteristics, age, and tenure (time in the sector, position, company), in the relation between the realized absorptive capacity (RACAP) and the architectural marketing capabilities (CAM). Originality/value: The present study considers the human element as a factor that affects the relations between the capacities of the company. It contributes theoretically to help understand what can impact the formulation and implementation of marketing strategies and theoretically strengthen the role of the human element. As a practical contribution, it has been shown that it is not enough to seek external knowledge, it is necessary that it is transformed and then used effectively in the design and implementation of marketing strategies. Design/method/approach: Quantitative research, with transverse tem poral data collection. This study empirically tested the hypotheses based on a sample of 343 marketing managers from Brazilian manufacturing industries. Data were collected through a survey. Data were processed by means of modeling of structural equations in AMOS software. Findings: The characteristics of managers (age and tenure) moderate the relationship between a part of RACAP (knowledge transformation) and CAM (architectural marketing capability). More experienced managers should be valued because it has been proven that in this sector, they make a difference when it comes to transforming knowledge and using it in their strategies. Design/method/approach: Quantitative research, with transverse tem poral data collection. This study empirically tested the hypotheses based on a sample of 343 marketing managers from Brazilian manufacturing industries. Data were collected through a survey. Data were processed by means of modeling of structural equations in AMOS software. Findings: The characteristics of managers (age and tenure) moderate the relationship between a part of RACAP (knowledge transformation) and CAM (architectural marketing capability). More experienced managers should be valued because it has been proven that in this sector, they make a difference when it comes to transforming knowledge and using it in their strategies. ISSN 1678-6971 (electronic version) • RAM, São Paulo, 20(6), eRAMD190061, 2019 doi:10.1590/1678-6971/eRAMD190061 KEYWORDS Absorptive capacity. Marketing capability. Managers’ characteristics. Tenure. Age. ISSN 1678-6971 (electronic version) • RAM, São Paulo, 20(6), eRAMD190061, 2019 doi:10.1590/1678-6971/eRAMD190061 Managers’ influence on company capabilities ISSN 1678-6971 (electronic version) • RAM, São Paulo, 20(6), eRAMD190061, 2019 doi:10.1590/1678-6971/eRAMD190061 1. INTRODUCTION Companies are continually looking for ways to improve their processes, products, and services in order to achieve their goals. For this, they need to differentiate themselves from their competitors by achieving sustainable competitive advantages. One way to achieve such advantages is through organizational capabilities. g p Capabilities are a company’s abilities to combine, develop, and exploit resources to create competitive advantage (Murray & Chao, 2005; Ruiz- -Ortega & García-Villaverde, 2008; Kaufmann & Roesch, 2012). There are several types of capabilities in a company. One of the most relevant is the absorptive capacity (ACAP), which is the ability to take in external knowledge and use it for commercial purposes (Zahra & George, 2002). As Morgan, Zou, Vorhies, and Katsikeas (2003) argue, knowledge creates the most strategically significant resources, so it is essential to understand the logic of its attainment. ACAP is concerned with understanding the processes of acquisition, assimilation, transformation, and exploitation of knowledge (Zahra & George, 2002). However, we can divide ACAP into two phases: potential absorptive capacity (PACAP), formed by the acquisition and assimilation of knowledge; and realized absorptive capacity (RACAP), containing the stages of transformation and exploitation of external knowledge (Zahra & George, 2002). Another important existing capability in organizations is the architec tural marketing capability (AMC), which involves the skills for planning and implementing marketing strategies within a company. This capability is also relevant for leveraging other types of capabilities, such as specialized and cross-functional capabilities (Morgan, 2012). In other words, architectural marketing skills are essential because they help formulate and implement what was planned, transforming it into a perceived value offer for the public (Morgan et al., 2003). Therefore, one can perceive that there is a relation ship between ACAP and AMC, because knowledge is needed to formulate and implement the right marketing strategies. Companies can obtain and transform knowledge through ACAP. However, both capacities – ACAP and AMC – require the human element in order to exist. As Nieves and Haller (2014) point out, human capital is considered a vital resource to ensure the realization of several key capabilities that allow for sustainable competitive advantage. Despite its relevance, many theories do not consider it. 1. INTRODUCTION However, these theories have reached the limit of their explanatory power, leading researchers to become interested in 3 3 ISSN 1678-6971 (electronic version) • RAM, São Paulo, 20(6), eRAMD190061, 2019 doi:10.1590/1678-6971/eRAMD190061 ISSN 1678-6971 (electronic version) • RAM, São Paulo, 20(6), eRAMD190061, 2019 doi:10.1590/1678-6971/eRAMD190061 Juliana C. N. Costa, Shirlei M. Camargo, Ana M. M. Toaldo, Simone R. Didonet Juliana C. N. Costa, Shirlei M. Camargo, Ana M. M. Toaldo, Simone R. Didonet Juliana C. N. Costa, Shirlei M. Camargo, Ana M. M. Toaldo, Simone R. Didonet how human factors affect corporate outcomes (Finkelstein, Hambrick, & Cannella, 2009). Bromiley and Rau (2016, p. 174) argue that “strategy academics have an ongoing concern with the strategy process – the mechanisms by which organizations formulate and implement the strategy”. According to the authors, there is a significant and growing flow of research in this area that focuses on the role of the top management (CEOs/COOs, other senior managers, and top management teams) during the development and imple mentation of a strategy. As Hiller, Beauchesne, and Whitman (2013) note, in order to understand company strategies and actions, it is necessary to understand better the individual characteristics of top executives who make decisions on behalf of organizations. Furthermore, Bendig, Strese, Flatten, Costa, and Brettel (2018) empiri cally validated a model that related the micro-fundamentals of dynamic capabilities to the personality of the chief executive officer (CEO). The authors understood how company leaders indirectly influence the dynamic capabilities of the company by shaping individual learning conditions. Bach & Lee (2018) also investigated the relationship between corporate perfor mance and executive characteristics from the perspective of upper echelons theory (Hambrick & Mason, 1984). To exercise realized absorptive capacity (RACAP), it is necessary to merge new and existing knowledge in order to use them later (Flatten, Engelen, Zahra, & Brettel, 2011). That is, the manager’s experience can make a difference when it comes to transforming the knowledge acquired and using it in the formulation and implementation of a company’s marketing strategies. Finally, there are indications that the characteristics of managers, represented in this study by age and tenure (time the executive has been in the sector, company, and position), play a moderating role in the RACAP- AMC relationship. Therefore, the objective of this article was to verify the moderating role of the managers’ characteristics (age and tenure) in the relation between RACAP and AMC. ISSN 1678-6971 (electronic version) • RAM, São Paulo, 20(6), eRAMD190061, 2019 doi:10.1590/1678-6971/eRAMD190061 1. INTRODUCTION The article is intended to cover the literature gap by explaining how the characteristics of managers (human element) intensify the influence of RACAP on AMC and seeks to deepen understanding of the specific elements of RACAP in relation to AMC. Specifically, it remains to be understood how transformation and exploitation influence architectural marketing capabilities when moderated by the human element. As mentioned earlier, ACAP and AMC are essential capabilities for companies and are interrelated, since it is ACAP that provides the knowledge that AMC requires. Despite Managers’ influence on company capabilities the relevance of their relationship, we found no evidence of studies that investigated them together. We enhanced this study by introducing the managers’ characteristics as a moderating factor in this relationship, since they have been shown to influence strategic decisions (Eisenhardt & Schoonhoven 1990; Boeker, 1997; Hambrick, 2007; Chen, Kang, & Butler, 2019). In a 2016 study, Bromiley and Rau claim that while the CEO’s experience, tenure, and age influence the company’s results, the “how” of this occurrence is a sophisticated element. Therefore, this research contributes to the theory by demonstrating that the better a company manages its ability to learn from external knowledge, the more significant the differentiation between it and the competition will be, since it will be more costly to copy products and processes that are always being improved. Besides, firms increase this complexity due to causal ambiguity (Lippman & Rumelt, 1982) generated through the integration of different capacities (e.g., RACAP and AMC). g g p g Briefly, causal ambiguity refers to the uncertainty regarding what ele ments cause differences in efficiency between firms. It prevents competitors who seek to imitate a company from knowing what to imitate or what to do, helping to preserve the condition of heterogeneity (Reed & DeFillippi, 1990) and leading to competitive advantage. This article is structured as follows: first, we present a brief theoretical overview of the variables involved in the study (absorptive capacity, archi tectural marketing capabilities, and the managers’ characteristics). Next, we discuss our methodological choices and provide our results. Finally, we discuss our findings and the considerations they raise. ISSN 1678-6971 (electronic version) • RAM, São Paulo, 20(6), eRAMD190061, 2019 doi:10.1590/1678-6971/eRAMD190061 2.1 Realized absorptive capacity (RACAP) Individual learning is the basis for organizational knowledge absorption. However, according to Cohen and Levinthal (1990), such knowledge will only be useful if it is translated into organizational capacities to assist in the development of specific resources. In this paper, we present the results obtained by Zahra and George (2002) as a dynamic capability, which assists in the reconfiguration of resources in dynamic environments (Eisenhardt & Martin, 2000; Teece, Pisano, & Shuen, 1997; Burcharth, Lettl, & Ulhøi, 2015). Dynamic capabilities are heterogeneous among companies, which leads to the distinction of strategies because they are based on path dependence 5 5 ISSN 1678-6971 (electronic version) • RAM, São Paulo, 20(6), eRAMD190061, 2019 doi:10.1590/1678-6971/eRAMD190061 Juliana C. N. Costa, Shirlei M. Camargo, Ana M. M. Toaldo, Simone R. Didonet Juliana C. N. Costa, Shirlei M. Camargo, Ana M. M. Toaldo, Simone R. Didonet and use unique assets and idiosyncratic processes. This factor leads to the creation of sustainable competitive advantage as a result of the dynamic capacities in the companies (Teece et al., 1997). Path dependence is the pathway that has already been followed by the company and demonstrates that the company’s history is indeed relevant to the decisions to be made (Teece et al., 1997). When a company faces a decision, the path it chooses to pursue will be a function of its current position and future paths. However, the path it has already traveled shapes its current position (Teece et al., 1997). Therefore, all the trajectory already traced by the company, which involves its experiences, errors, achievements, successes, and failures, will influence it at the time of decision making. This temporal logic links with the observable characteristics of the managers were used in this study. Absorptive capacity (ACAP) is the company’s ability to acquire, assimilate, transform, and exploit knowledge from outside the organization (Zahra & George, 2002). The present study used the ACAP division proposed by Zahra and George (2002) and followed by Flatten et al. (2011), Jiménez- -Barrionuevo, García-Morales, and Molina (2011), and Chauvet (2014). This framework separates absorptive capacity into stages: acquisition, assimilation, transformation, and exploitation. These four phases are then organized into two: PACAP and RACAP. Potential absorptive capacity covers the acquisition and assimilation stages, while realized absorptive capacity encompasses the transformation and exploitation phases. ISSN 1678-6971 (electronic version) • RAM, São Paulo, 20(6), eRAMD190061, 2019 doi:10.1590/1678-6971/eRAMD190061 2.1 Realized absorptive capacity (RACAP) The transformation stage of realized absorptive capacity deals with “the company’s ability to develop and refine routines that aim to facilitate the combination of existing knowledge and new knowledge acquired and assimilated” (Zahra & George, 2002, p. 190). In turn, there is exploitation, which for Zahra and George (2002), as for Cohen and Levinthal (1990), is the company’s ability to incorporate new knowledge into its operations. Through the systems of appropriation of this knowledge that is externally absorbed and finally exploited, the company tends to generate competitive advantage (Zahra & George, 2002). The company can face different situations related to its new external knowledge. It may, for example, generate several marketing strategies from a small amount of such knowledge. Alternately, it may generate a broad knowledge base, but not have the capacity to exploit it (Lane, Koka, & Pathak, 2006). One way to work on this knowledge base is through the capabilities that the organization already has, such as its architectural capability. The latter has a critical role in the company’s ability to formulate and implement marketing strategies, and will, therefore, be addressed below. 6 Managers’ influence on company capabilities 2.2 Architectural marketing capability (AMC) According to Santos-Vijande, Sanzo-Pérez, Trespalacios Gutiérrez, and Rodríguez (2012), interest in studying marketing skills has recently increased. Morgan (2012) conceptualizes marketing capabilities as the acquisition, combination, and transformation of marketing resources into offers that the market values. This author further classifies marketing capabilities into four types: specialized (the marketing mix); interoperability (e.g., customer relationship management – CRM – and new product development); dynamics (e.g., market learning); and planning and implementation of strategies (Morgan, 2012). Due to their importance in the planning and implementation process of a company’s marketing strategies, an analysis of AMCs was undertaken as part of this study. These capabilities can be defined as the processes by which companies plan appropriate combinations of knowledge and other resources available to implement and execute in their markets what has been planned, transforming it into a perceived value offer for their publics (Morgan et al., 2003). Planning refers to a company’s ability to develop marketing strategies to leverage specialized and multifunctional capabilities and resources in the pursuit of competitive advantages (Morgan, 2012). Implementation refers to the ability to acquire, combine, and distribute the resources necessary to reach the strategies previously defined (Morgan, 2012). That is, the company needs to be able to plan and implement its marketing strategies and thus achieve a better result. However, Hiller et al. (2013) argue that, in order to understand company strategies and actions, it is necessary to deepen the knowledge of the individual characteristics of senior executives who make decisions on behalf of organizations. Thus, the human element and its characteristics, in addition to being relevant to realized absorptive capacity, also have an essential relation to AMCs, and are relevant in the strategic planning and implementation. Therefore, in this research, we investigated managers’ characteristics. We will discuss those further in the next section. ISSN 1678-6971 (electronic version) • RAM, São Paulo, 20(6), eRAMD190061, 2019 doi:10.1590/1678-6971/eRAMD190061 2.3 Managers’ characteristics Human capital plays a crucial role in developing and maintaining the capabilities of a company. Nieves and Haller (2014), therefore, consider it a fundamental resource for a series of essential capabilities to be realized, and thus, for competitive advantage to be sustained. Hambrick and Mason ISSN 1678-6971 (electronic version) • RAM, São Paulo, 20(6), eRAMD190061, 2019 doi:10.1590/1678-6971/eRAMD190061 Juliana C. N. Costa, Shirlei M. Camargo, Ana M. M. Toaldo, Simone R. Didonet Juliana C. N. Costa, Shirlei M. Camargo, Ana M. M. Toaldo, Simone R. Didonet (1984), creators of the “upper echelons theory”, have already seen the necessity of including the human element in organizational studies. Their theory states that managers’ characteristics predict a part of company results. This is because, as managers are different from each other, they will have different knowledge bases, which may lead them to make different decisions (Adner & Helfat, 2003; Marimuthu & Kolandaisamy, 2009). Penrose (1959) made the argument that top managers are resources that influence the organization’s performance. This can happen through well-formulated and implemented strategies. Therefore, while an organiza tion’s employees are essential for the efficient and effective operation of the business, not all groups have equal weight. Specifically, strategic managers are a group whose importance in generating and maintaining business success has been demonstrated by several authors (Penrose, 1959; Hambrick & Mason, 1984; Castanias & Helfat, 1991; Lado & Wilson, 1994; Guedes & Conceição Gonçalves, 2019). Some researchers have found evidence that executive demographic profiles, at both the individual and team levels, correlate with company strategies and results (Eisenhardt & Schoonhoven, 1990; Boeker, 1997; Hambrick, 2007). According to Hambrick and Mason (1984), one can divide personal characteristics into two types: psychological properties, which are not the focus of this study; and observable experiences. These observable experiences can be several, but age and tenure (time in position, organization, or industry) are the two most-used demographic variables in managerial studies (Hiller et al., 2013). One can state that the three types of tenure are linked, since the time a manager has been in their position is linked with the time for which they have been in their sector, as well as in their industry. Although they are related, the theory indicates that they can be considered separately (Finkelstein et al., 2009). There is considerable evidence of the relevance of the tenure phenomenon. ISSN 1678-6971 (electronic version) • RAM, São Paulo, 20(6), eRAMD190061, 2019 doi:10.1590/1678-6971/eRAMD190061 2.3 Managers’ characteristics However, there is still a demand for the elaboration of how this process happens and of the concept itself (Finkelstein et al., 2009). The same occurs with the age characteristic, since it can be related to tenure to the extent that the manager accumulates experience and time in the company. The individual’s aging process may play a role in determining how individual changes over time can affect the performance of the work itself (Waldman & Avolio, 1993; Sturman, 2003). These influences, however, are still considered controversial. For instance, some authors (Miller, 1991; Herrmann & Datta, 2006; Helfat et al., 2009) argue that the more experience in the company, the 8 Managers’ influence on company capabilities Managers’ influence on company capabilities higher the tendency to maintain the status quo, the lower the likelihood of taking risks, and the less capacity to absorb and use external knowledge. However, more significant experience may result in greater awareness of complex managerial environments (Herrmann & Datta, 2006). 2.4 Characterization of the sample and respondents Firms in the southeast region of Brazil, followed by the south region, made up the bulk of our sample, which is in line with the characteristics of the Brazilian gross domestic products (GDP) generation data. The other companies, which made up less than 10% of the sample, were located in the northeast or midwest, or were multinational. According to the Serviço Brasileiro de Apoio às Micro e Pequenas Empresas (Sebrae)classification (2016) by number of employees and industry, the sample is characterized, for the most part (around 86%), by micro, small, and medium-sized companies. These data indicate that the profile of the manufacturing industry in Brazil is in line with our study sample, since data from the Annual Social Information Report (Relação Anual de Informações Sociais – Rais) (2015) point to a similar profile. At the individual level, the desired respondents were managers who were involved in the organization’s marketing strategies. In order to respect this premise, there was a filter question in the questionnaire that eliminated some respondents, leaving only those with the appropriate profile for the research. Regarding the age of the respondents, 44.76% of them were at or below 35 years of age. The remainder (55.24%) were older than 35. The oldest respondent was 67 years old. Regarding tenure, 53.49% of respondents had up to four years of expe rience in their roles, while 46.51% had more than four years of experience. Meanwhile, 48.84% of respondents had up to seven years of experience, and 51.16% had over seven years in the sector. Finally, 46.8% of respondents had up to five years of tenure at their companies, while 53.2% had more than five years. 2.5 Relationship between variables A more extensive tenure is often an essential indicator of managerial experience and accumulated knowledge that could potentially influence the degree of exploitation and exploration (Finkelstein & Hambrick, 1996; Abebe & Angriawan, 2014). Both exploitation and exploration refer to the develop ment of new knowledge, but the former uses pre-existing knowledge in the company, while the second seeks it externally (Vorhies, Orr, & Bush, 2011). 9 9 ISSN 1678-6971 (electronic version) • RAM, São Paulo, 20(6), eRAMD190061, 2019 doi:10.1590/1678-6971/eRAMD190061 Juliana C. N. Costa, Shirlei M. Camargo, Ana M. M. Toaldo, Simone R. Didonet Juliana C. N. Costa, Shirlei M. Camargo, Ana M. M. Toaldo, Simone R. Didonet That is, longer tenure will influence the process of obtaining and using knowledge. Therefore, the human element is essential for ACAP, which, as well as exploitation and exploration, is related to organizational learning. The human element is relevant in understanding the dynamics among organizational capacities. In this sense, it is necessary to have synergy with the company’s internal resources, such as intellectual capital, to ensure that ACAP offers a sustainable competitive advantage based on the development of strategies (Engelman, Fracasso, Schmidt, & Zen, 2017). Following the concepts of Cohen and Levinthal (1990), the character and role of ACAP in the assimilation and exploitation of knowledge suggest that at both, the individual and organizational levels, prior knowledge makes it possible to assimilate and explore new knowledge. However, it was not only Cohen and Levinthal (1990) who highlighted human capital. Zahra and George (2002), when they expanded the conception of ACAP, maintained human capital as an essential factor in acquiring and exploiting acquired knowledge. Other authors who have taken the human element into account are Liao, Welsch, and Stoica (2003), Daghfous (2004), Jansen, Van Den Bosch, and Volberda (2005), Todorova and Durisin (2007), Camisón and Fóres (2011), and Hotho, Becker-Ritterspach, and Saka-Helmhout (2012). p Human capital can be considered a fundamental resource to ensure the realization of a series of essential capabilities that allow the sustainability of advantage over rivals (Nieves & Haller, 2014). Thus, the knowledge, skills, and collective characteristics of an organization’s employees and managers create capabilities to gain competitive advantage (Lengnick-Hall & Lengnick Hall, 2003). Rodenbach and Brettel (2012) and Von den Driesch, Costa, Flatten, and Brettel (2015) state that managerial experience, as well as age, influences marketing (dynamic) capabilities, depending on environmental conditions. ISSN 1678-6971 (electronic version) • RAM, São Paulo, 20(6), eRAMD190061, 2019 doi:10.1590/1678-6971/eRAMD190061 2.5 Relationship between variables That is, we infer that more experienced managers can help in the development of the marketing capabilities of the companies, which are represented by AMC in this research. Morgan et al. (2003), in another study, indicated that personal knowledge is vital for the development and use of AMC. Thus, there are indications that tenure (and, we infer, age as well) can influence both, RACAP and AMC, playing a moderating role in this relationship. According to Baron and Kenny (1986), a moderating variable is one that affects the direction and the strength of the relationship between the independent variable (RACAP) and the dependent variable (AMC). That is, the experience and age of the manager can influence how they use the knowledge acquired within a company when planning and implementing 10 10 Managers’ influence on company capabilities Managers’ influence on company capabilities Managers’ influence on company capabilities marketing strategies. However, since RACAP is composed of two phases (transformation and exploitation), we decided to analyze it in a disaggregated way. Thus: marketing strategies. However, since RACAP is composed of two phases (transformation and exploitation), we decided to analyze it in a disaggregated way. Thus: • H1: We hypothesize that (a) the age of the manager; (b) their tenure in the sector; (c) their tenure in the office; and (d) their tenure in the com pany moderate the relation between knowledge transformation (one phase of the realized absorptive capacity) and architectural marketing capabilities. • H2: We hypothesize that (a) the age of the manager; (b) their tenure in the sector; (c) their tenure in the office; and (d) their tenure in the company moderates the relation between knowledge exploitation (one phase of the realized absorptive capacity) and architectural marketing capabilities. • H2: We hypothesize that (a) the age of the manager; (b) their tenure in the sector; (c) their tenure in the office; and (d) their tenure in the company moderates the relation between knowledge exploitation (one phase of the realized absorptive capacity) and architectural marketing capabilities. Figure 2.5.1 THEORETICAL MODEL Architectural marketing capabilities RACAP – transformation RACAP – exploration H1 a) Age b) Industry tenure c) Job pos. tenure d) Firm tenure H2 Source: Elaborated by the authors. RACAP – transformation Source: Elaborated by the authors. Source: Elaborated by the authors. 3. METHODOLOGY The study used a quantitative approach, with transverse data collection and non-probabilistic sampling by adhesion (Creswell, 2010). We performed data collection through a survey, conducted between November 2016 11 11 Juliana C. N. Costa, Shirlei M. Camargo, Ana M. M. Toaldo, Simone R. Didonet Juliana C. N. Costa, Shirlei M. Camargo, Ana M. M. Toaldo, Simone R. Didonet and January 2017, with marketing managers or employees involved with the marketing strategy of their companies. Six researchers conducted the interviews with a sample characterized by companies in the transformation industry throughout the nation, classified according to the National Classification of Economic Activities (Classificação Nacional de Atividades Econômicas – Cnae). Regarding the measurement instrument, we used scales that were already tested and fully published in international journals, and the absorptive capacity scale was drawn from Flatten et al.’s (2011) study. The architectural marketing capabilities scale used in the study is one developed by Vorhies and Morgan (2005). Even so, considering the use of scales applied outside the Brazilian environment, some procedures were adopted to guarantee the validity and reliability of the scale. The translation/back-translation method, face validity, and pre-test with six specialists were then used to verify the comprehension of the question naire. We also performed an exploratory factorial analysis (EFA) to verify the distinct stages of RACAP, as well as convergent and discriminant validity analysis (Hair, Babin, Money, & Samouel, 2005). Furthermore, by using Harman’s test, we verified the common method bias. After data collection, we performed data cleaning procedures, such as missing data, out of range values, non-engaged responses, asymmetry and kurtosis (with a multivariate analysis of normality), and identification of outliers. After this step, the final sample consisted of 343 respondents. Then, the data analysis and the hypothesis test were carried out employing structural equation modeling using SPSS and AMOS statistical software. ISSN 1678-6971 (electronic version) • RAM, São Paulo, 20(6), eRAMD190061, 2019 doi:10.1590/1678-6971/eRAMD190061 3.1 Data treatment and results Regarding the multivariate data normality, the sum of the critical ratio (C.R.) resulted in a value of 19.45. Therefore, we state that the data are not normal. The referenced value represents the normalized Mardia (1970, 1974) estimate of multivariate kurtosis, although it is not explicitly stated as such (Byrne, 2010). Bentler and Wu (2005), based on Mardia’s estimate, suggests that in practice, values greater than 5 indicate non-normal data. In the case of the present study, the value of 19.45 represents this number. According to Pallant (2005), the non-normality of data is recurrent in the applied social sciences. Still, Hair, Black, Babin, Anderson, and Tatham (2009) state that, in samples with more than 200 cases, the harmful effects 12 12 Managers’ influence on company capabilities Managers’ influence on company capabilities of non-normality are reduced, which allowed us to be less concerned with non-normal variables. The results of the scale reliability test are presented in Figure 3.1.1. It can be observed through Cronbach’s alphas that present values are above 0.07 (Hair et al., 2005). Figure 3.1.1 SCALE RELIABILITY TEST Construct Alpha Knowledge transformation 0.90 Knowledge exploitation 0.88 Architectural marketing capabilities 0.95 Source: Elaborated by the authors. Figure 3.1.1 Studies in which two or more constructs are measured using the same method may have skewed effects. Another factor that can bias the effects is having only one respondent per company. When this occurs, one must worry about the covariance observed between the constructs, since it can be due to the use of the same method of measurement (Lowry & Gaskin, 2014; Podsakoff, MacKenzie, & Podsakoff, 2012). Therefore, to verify the possible interference of common method bias, the Harman (1976) test was performed, employing exploratory factor analysis and setting a factor, without rotation. The only forced factor was 47.6% (< 50%) (Podsakoff, MacKenzie, & Podsakoff, 2003). When the Harman test is employed, the results indicate that common method bias does not interfere with the model, since its value is less than 50%. That is, the explanatory power of a variable is not mostly in a single factor. Subsequently, we tested for the discriminant and convergent validities of the model. Figure 3.1.2 shows the results. For the construct to have discriminant validity, the square root of the average variance extracted (AVEs) (values highlighted in bold diagonal) should be higher than any corresponding correlation (Fornell & Larcker, 1981). ISSN 1678-6971 (electronic version) • RAM, São Paulo, 20(6), eRAMD190061, 2019 doi:10.1590/1678-6971/eRAMD190061 3.1 Data treatment and results The square root was verified; therefore, that data reached the discriminant validity of all the constructs, according to the criteria stipulated in the literature. g p We ensured convergent validity of the model through mean extracted variance (AVE) and composite reliability (CR), following the recommendations of Hair et al. (2009) (AVE > .50; CR > .70). Therefore, the values in Figure 3.1.2 indicate that both AVE and CR were achieved. 13 13 13 ISSN 1678-6971 (electronic version) • RAM, São Paulo, 20(6), eRAMD190061, 2019 doi:10.1590/1678-6971/eRAMD190061 ta, Shirlei M. Camargo, Ana M. M. Toaldo, Simone R. Didon Juliana C. N. Costa, Shirlei M. Camargo, Ana M. M. Toaldo, Simone R. Didonet Figure 3.1.2 CONSTRUCTS’ AVERAGE VARIANCE EXTRACTED (AVE) AND COMPOSITE RELIABILITY (CR) CR AVE Exp Tra Arq Exp 0.885 0.720 0.849 Tra 0.900 0.692 0.434 0.832 Arq 0.960 0.923 0.344 0.432 0.961 AMOS bootstraping (343 cases, 2000 runs); model fit: (CMIN/DF = 2.506; NFI = 0.938; CFI = 0.962; RMSEA = 0.066; SRMR = 0.0378; HOELTER 0.05 = 0.168; HOELTER 0.01 = 0.182). Source: Elaborated by the authors. Figure 3.1.2 CONSTRUCTS’ AVERAGE VARIANCE EXTRACTED (AVE) AND COMPOSITE RELIABILITY (CR) Once the adjustments of the structural model were verified (results shown in the footer of Figure 3.1.2) and were satisfied with the values stipulated in the literature (Byrne, 2010), we tested the hypotheses. As a result, the manager’s age, tenure in the industry, and tenure in the office moderated the relationship between transformation (the first phase of the RACAP) and AMCs. However, the data did not support the hypotheses regarding the moderation between exploitation (the second phase of the RACAP) and AMCs. We present the results in Figure 3.1.3. Specifically, it was found that the higher the manager’s age, the stronger the relationship between knowledge transformation (independent variable – IV) and architectural capacities (dependent variable – DV) (H1a), with β = 0.47 (more experienced managers) versus β = 0.17 (younger managers). There was no statistically significant moderation related to the age of managers in the relationship between knowledge exploitation and AMCs (H2a). Tenure in the industry also moderates the relation between transfor mation (of knowledge) and AMCs (H1b), as represented by β = 0.52 for managers with longer sector tenure and β = 0.11 for managers with shorter tenures. As for H2a, time in the sector did not have a moderating effect on the relationship between IV and DV in H2b. In H1c, there was a moderation of the manager’s post-tenure in the relationship between transformation (of knowledge) and AMCs. The more time in the same position, the higher the intensity of this relation, with β = 0.58 (higher post-tenure) versus β = 0.06 (lower post-tenure). Data did not support H2c, so there was no moderation of the post-tenure in the relation ship between exploitation and AMCs. The variable tenure in the company moderates the relation proposed in H1d between transformation of knowledge and AMC. The longer the tenure 14 14 14 Managers’ influence on company capabilities Managers’ influence on company capabilities Managers’ influence on company capabilities in the company, the stronger this relation. β = 0.57 represents this finding (longer tenure in the company) versus β = 0.04 (shorter tenure in the company). Following the logic of the other findings, data did not support H2d, so there is also no moderation in the relationship between the IV and the DV proposed in this study. Figure 3.1.2 CONSTRUCTS’ AVERAGE VARIANCE EXTRACTED (AVE) AND COMPOSITE RELIABILITY (CR) Figure 3.1.3 HYPOTHESES TESTING RESULTS Hypotheses Proposed relation Moderation Status β (standardized coefficient) More years Fewer years H1 TRA - > AMC a) Age Supported 0.47 0.17 b) Industry tenure Supported 0.52 0.11 c) Job pos. tenure Supported 0.58 0.06 d) Firm tenure Supported 0.57 0.04 H2 EXP - > AMC a) Age Rejected – – b) Industry tenure Rejected – – c) Job pos. tenure Rejected – – d) Firm tenure Rejected – – Source: Elaborated by the authors. Figure 3.1.3 In the section, we presented the study’s final considerations, its theoretical and managerial contributions, its limitations, and suggestions for future research. ISSN 1678-6971 (electronic version) • RAM, São Paulo, 20(6), eRAMD190061, 2019 doi:10.1590/1678-6971/eRAMD190061 4. DISCUSSION OF RESULTS AND FINAL CONSIDERATIONS This study aimed to verify the moderating role of managers’ charac teristics (age and tenure) in the relationship between RACAP and AMCs. The findings contribute to the literature on management strategy by demon strating that a manager’s age and tenure have a moderating effect on the relation between knowledge transformation and AMCs. This result means that the higher the manager’s age and the more time they have spent in 15 15 ISSN 1678-6971 (electronic version) • RAM, São Paulo, 20(6), eRAMD190061, 2019 doi:10.1590/1678-6971/eRAMD190061 Juliana C. N. Costa, Shirlei M. Camargo, Ana M. M. Toaldo, Simone R. Didonet Juliana C. N. Costa, Shirlei M. Camargo, Ana M. M. Toaldo, Simone R. Didonet Juliana C. N. Costa, Shirlei M. Camargo, Ana M. M. Toaldo, Simone R. Didonet their industry, position, and company, the stronger the relationship between knowledge transformation and the ability to formulate and implement marketing strategies. In the context of RACAP, transforming knowledge means developing and refining routines to facilitate the combination of existing knowledge with new knowledge (Zahra & George, 2002). In turn, a part of architectural marketing capabilities focuses on processes through which firms plan appropriate combinations, among other things, of knowledge (Morgan et al., 2003). Finally, there is a need for RACAP in order for AMCs to be used. That is, it is necessary to merge the new and existing knowledge when planning marketing strategies. However, according to some authors (Cohen & Levinthal, 1990; Ziek & George, 2002; Nieves & Haller, 2014), the human element influences the realization of a series of capabilities – in the case of this research, of RACAP and AMC. Since managers are distinct from each other (in this case, in terms of age and tenure), they may have different knowledge bases, which will lead them to make different decisions (Adner & Helfat, 2003; Marimuthu & Kolandaisamy, 2009). That is, as demonstrated in this research, when combining existing knowledge with the new (transforming it) and applying it to the strategic planning of a company, more experienced managers have the advantage. Therefore, a major theoretical contribution of this study is its proof of the moderating effect of the characteristics of the managers on the rela tionship between realized absorptive capacity and architectural marketing capabilities (RACAP-AMC). Identifying this effect helps to increase the understanding of which factors may impact the formulation and implemen tation of marketing strategies. ISSN 1678-6971 (electronic version) • RAM, São Paulo, 20(6), eRAMD190061, 2019 doi:10.1590/1678-6971/eRAMD190061 Managers’ influence on company capabilities Managers’ influence on company capabilities 2019). The present findings are in agreement with these authors, strength ening the role of the human element in the researched context. The characteristics of the individuals studied (age and tenure) moderate the relation between the proposed capabilities. This, in turn, sheds light on the human element, which has been little explored in the marketing capacities literature. As it is understood that managers’ attributes impact the company (Boeker, 1997; Hambrick, 2007; Guedes & Conceição Gonçalves, 2019; Dhir & Shukla, 2018) and, as found in this research, specifically influence the relationship between external knowledge and AMC, a study window is opened to explore the role of new individual characteristics in the strategic relationships of organizations. Another finding was the moderation of the managers’ characteristics only in the external knowledge transformation phase in AMCs (H1a, H1b, H1c, H1d). Managers’ characteristics did not moderate the exploitation element. Through the two-step scale questions, the transformation of knowledge was revealed to be more strategic, whereas the exploitation of this knowledge reflected more operational factors (Flatten et al., 2011). According to Flatten et al. (2011), the exploitation phase refers to encouraging the development of new products, revising technologies and adapting them with new knowl edge, and working more efficiently when adopting new technologies. In turn, the transformation phase refers to structuring, combining, applying, and otherwise using the acquired knowledge. Although strategic managers are essential in both phases of transformation, data have shown that those with more experience are more effective in strengthening the relationship between RACAP and AMC than younger and less experienced ones. There fore, longer tenure and a manager’s age intensifies the relationship between knowledge transformation and the formulation and implementation of mar keting strategies, but not the relationship between knowledge exploitation and AMC. Organization employees are essential for effective and efficient business conduct. However, according to Penrose (1959), managers have a more significant influence on processes. Therefore, not all groups have the same weight of influence in organizational activities. Specifically, strategic managers are a group whose importance in the generation and maintenance of business success has been demonstrated by several authors (Penrose, 1959; Hambrick & Mason, 1984; Castanias & Helfat 1991; Lado & Wilson, 1994). Still, Hambrick and Mason (1984) were the developers of the “upper echelons theory”, which suggests companies’ results are predicted in part by the characteristics of top managers. 4. DISCUSSION OF RESULTS AND FINAL CONSIDERATIONS Another theoretical contribution made by the study was to fill the literature gap by explaining how the managers’ characteristics (the human element) intensify the influence of RACAP on AMC. The study results deepened the understanding of the specific elements of RACAP in its relationship with AMC. Precisely, it was revealed to what degree transfor mation and exploitation influence architectural marketing capabilities when moderated by the human element. Since the foundational text of Cohen and Levinthal (1990), absorptive capacities studies have addressed the human element. Other authors also consider individuals as transforming agents when it comes to the absorption of external knowledge (Zahra & George, 2002; Jansen et al., 2005; Todorova & Durisin, 2007; Camisón & Fóres, 2011; Hotho et al., 2012; Chen et al., 16 16 ISSN 1678-6971 (electronic version) • RAM, São Paulo, 20(6), eRAMD190061, 2019 doi:10.1590/1678-6971/eRAMD190061 Managers’ influence on company capabilities The results of the present research agree with those of the authors mentioned above. 17 17 ISSN 1678-6971 (electronic version) • RAM, São Paulo, 20(6), eRAMD190061, 2019 doi:10.1590/1678-6971/eRAMD190061 Juliana C. N. Costa, Shirlei M. Camargo, Ana M. M. Toaldo, Simone R. Didonet Juliana C. N. Costa, Shirlei M. Camargo, Ana M. M. Toaldo, Simone R. Didonet Regarding the practical implications of this study, since the conclusions point to the moderating influence of managers’ characteristics on the rela tionship between the capabilities studied, it is suggested that companies consider such factors at the time of project team formation, in order to plan and implement strategies for a new product. In this case, as there are elements of knowledge absorbed externally, managers with more experience and time in their position, company, and sector tend to influence the rela tion between the transformation of knowledge and AMC positively. Another contribution would be to clarify for companies that it is not enough to worry only about seeking external knowledge. Such knowledge needs to be transformed and then used effectively in the formulation and implementation of marketing strategies. In addition, it was pointed out that, unlike some theoreticians and researchers who see longer tenure/age to be related to a lack of flexibility and daring (Miller, 1991; Herrmann & Datta, 2006; Helfat et al., 2009), experienced managers need be valued, because they, as established in the present study, play a fundamental role in effectively transforming acquired knowledge into strategies. We also admit that with the human element considered in the relation ship between the transformation of external knowledge and the capability to formulate and implement marketing strategies, the causal ambiguity (Lippman & Rumelt, 1982) of the company tends to be more complicated. In this way, uncertainties regarding the causes of differences in efficiency among firms are increased, preserving conditions of heterogeneity (Reed & DeFillippi, 1990) concerning competition and leading to the attainment of competitive advantage. Accordingly, companies focusing on more experi enced managers can make their processes more complex, making it difficult for competitors to understand. As a limitation of this study, one can cite the fact that it is a “portrait” of a specific period in time. Therefore, we suggest longitudinal research, since ACAP is a dynamic capability (Zahra & George, 2002) and architectural marketing capabilities involve the implementation of strategies. Thus, time can influence both factors. ISSN 1678-6971 (electronic version) • RAM, São Paulo, 20(6), eRAMD190061, 2019 doi:10.1590/1678-6971/eRAMD190061 Managers’ influence on company capabilities In this way, we could have reinforced knowledge about the topics discussed here. Besides that, the study sample was non-probabilistic, and we collected data in a single sector (manufacturing), which may limit its generalization. Thus, we suggest replicating the study in other sectors in order to ascertain if the variables analyzed behave in the same way. Another suggestion for a future study would be to test the moderating effect of specific training of the 18 18 Managers’ influence on company capabilities Managers’ influence on company capabilities Managers’ influence on company capabilities managers on the company’s capabilities or to investigate other characteris tics, especially the psychological ones (following the same logic of the study by Bendig et al., 2018), interpretations and information filters (Finkelstein et al., 2009). managers on the company’s capabilities or to investigate other characteris tics, especially the psychological ones (following the same logic of the study by Bendig et al., 2018), interpretations and information filters (Finkelstein et al., 2009). ISSN 1678-6971 (electronic version) • RAM, São Paulo, 20(6), eRAMD190061, 2019 doi:10.1590/1678-6971/eRAMD190061 RESUMO Objetivo: Verificar o papel moderador das características dos gestores, como idade e tenure (tempo no setor, cargo, empresa), na relação entre a capacidade absortiva realizada (RACAP) e a capacidade arquitetural de marketing (CAM). Objetivo: Verificar o papel moderador das características dos gestores, como idade e tenure (tempo no setor, cargo, empresa), na relação entre a capacidade absortiva realizada (RACAP) e a capacidade arquitetural de marketing (CAM). Originalidade/valor: O presente estudo considera o elemento humano como fator que afeta as relações entre as capacidades da empresa. Con tribui teoricamente ao auxiliar o entendimento do que pode impactar a formulação e implementação de estratégias de marketing, além de forta lecer teoricamente o papel do elemento humano. Como contribuição prática, demonstrou-se que não basta apenas buscar o conhecimento externo, mas também é necessário que ele seja transformado para então ser utilizado de maneira eficaz na elaboração e aplicação das estratégias de marketing. Design/metodologia/abordagem: Trata-se de pesquisa de caráter quanti tativo, com corte temporal transversal. Este estudo testou empiricamen te as hipóteses com base em uma amostra de 343 gestores de marketing de indústrias brasileiras de manufatura. Os dados foram coletados por meio de survey e tratados por meio de modelagem de equações estrutu rais no software AMOS. Resultados: As características dos gestores (idade e tenure) moderam a relação entre uma parte da RACAP (transformação do conhecimento) e a CAM (capacidade arquitetural de marketing). Gestores mais experien tes devem ser valorizados, pois comprovou-se que, nesse setor, eles fazem a diferença na hora de transformar o conhecimento e utilizá-lo em suas estratégias. 19 19 19 ISSN 1678-6971 (electronic version) • RAM, São Paulo, 20(6), eRAMD190061, 2019 doi:10.1590/1678-6971/eRAMD190061 SSN 1678-6971 (electronic version) • RAM, São Paulo, 20(6), eRAMD190061, 2019 doi:10.1590/1678-6971/eRAMD190061 Juliana C. N. Costa, Shirlei M. Camargo, Ana M. M. Toaldo, Simone R. Didonet PALAVRAS-CHAVE Capacidade absortiva. Capacidade de marketing. Características dos ges tores. Tenure. Idade. Capacidade absortiva. Capacidade de marketing. Características dos ges tores. Tenure. Idade. REFERENCES Abebe, M. A., & Angriawan, A. (2014). Organizational and competitive influences of exploration and exploitation activities in small firms. Journal of Business Research, 67(3), 339–345. doi:10.1016/j.jbusres.2013.01.015 Adner, R., & Helfat, C. E. (2003). 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Searching for the inverted U-shaped relationship between time and performance: Meta-analyses of the experience/perfor mance, tenure/performance, and age/performance relationships. Journal of Management, 29(5), 609–640. doi:10.1016/S0149-2063_03_00028-X Teece, D. Managers’ influence on company capabilities ISSN 1678-6971 (electronic version) • RAM, São Paulo, 20(6), eRAMD190061, 2019 doi:10.1590/1678-6971/eRAMD190061 SSN 1678-6971 (electronic version) • RAM, São Paulo, 20(6), eRAMD190061, 2019 doi:10.1590/1678-6971/eRAMD190061 Transformation Please rate your business unit in terms of external knowledge transformation. Seven-point scale running 1 = strongly disagree to 7 = strongly agree. TRA_1) Our employees have the ability to structure and to use collected knowledge. TRA_1) Our employees have the ability to structure and to use collected knowledge. knowledge. TRA_2) Our employees are used to absorbing new knowledge as well as to preparing it for further purposes and making it available. TRA_3) Our employees successfully link existing knowledge with new i i h TRA_2) Our employees are used to absorbing new knowledge as well as to preparing it for further purposes and making it available. TRA_2) Our employees are used to absorbing new knowledge as well as to preparing it for further purposes and making it available. TRA_3) Our employees successfully link existing knowledge with new insights. TRA_4) Our employees are able to apply new knowledge in their practical work. TRA_4) Our employees are able to apply new knowledge in their practical work. Scale Realized absorptive capacity (RACAP – transformation and exploration) (Hooley et al., 2005) Realized absorptive capacity (RACAP – transformation and exploration) (Hooley et al., 2005) Managers’ influence on company capabilities J., Pisano, G., & Shuen, A. (1997). Dynamic capabilities and strategic management. Strategic Management Journal, 18(7), 509–533. doi:10.1002/(SICI)1097-0266(199708)18:7<509::AID-SMJ882>3.0. CO;2-Z Todorova, G., & Durisin, B. (2007). Absorptive capacity: Valuing a reconcep tualization. Academy of Management Review, 32(3), 774–786. doi:10.5465/ AMR.2007.25275513 Von den Driesch, T., Costa, M. E. S., Flatten, T. C., & Brettel, M. (2015). How CEO experience, personality, and network affect firms’ dynamic capabilities. European Management Journal, 33(4), 245–256. doi:10.1016/ j.emj.2015.01.003 Vorhies, D. W., & Morgan, N. A. (2005). Benchmarking marketing capabilities for sustainable competitive advantage. Journal of Marketing, 69(1), 80–94. doi:10.1509/jmkg.69.1.80.55505 Vorhies, D. W., Orr, L. M., & Bush, V. D. (2011). Improving customer- focused marketing capabilities and firm financial performance via marketing exploration and exploitation. Journal of the Academy of Marketing Science, 39(5), 736–756. doi:10.1007/s11747-010-0228-z 25 25 25 ISSN 1678-6971 (electronic version) • RAM, São Paulo, 20(6), eRAMD190061, 2019 doi:10.1590/1678-6971/eRAMD190061 ISSN 1678-6971 (electronic version) • RAM, São Paulo, 20(6), eRAMD190061, 2019 doi:10.1590/1678-6971/eRAMD190061 Juliana C. N. Costa, Shirlei M. Camargo, Ana M. M. Toaldo, Simone R. Didonet Juliana C. N. Costa, Shirlei M. Camargo, Ana M. M. Toaldo, Simone R. Didonet Waldman, D. A., & Avolio, B. J. (1993) Aging and work performance in perspective: Contextual and developmental considerations. Research in Personnel and Human Resources Management, 11, 133–162. Zahra, S. A., & George, G. (2002). Absorptive capacity: A review, reconcep tualization, and extension. Academy of Management Review, 27(2), 185–203. doi:10.5465/amr.2002.6587995 26 26 ISSN 1678-6971 (electronic version) • RAM, São Paulo, 20(6), eRAMD190061, 2019 doi:10.1590/1678-6971/eRAMD190061 Managers’ influence on company capabilities Exploration Please rate your business unit in terms of new external knowledge explora tion (please, consider all your business units, such as R&D, production, marketing, and accounting) g g -point scale running 1 = strongly disagree to 7 = strongly agree). g g even-point scale running 1 = strongly disagree to 7 = strongly agree). XP_1) Our management supports the development of prototypes. EXP_2) Our company regularly reconsiders technologies and adapts them in accordance with new knowledge. EXP_2) Our company regularly reconsiders technologies and adapts them in accordance with new knowledge. EXP_3) Our company has the ability to work more effectively by adopting new technologies. EXP_3) Our company has the ability to work more effectively by adopting new technologies. Architectural marketing capabilities (Vorhies & Morgan, 2005) Please rate your business unit relative to your major competitors in terms of its marketing capabilities in the following areas. Please rate your business unit relative to your major competitors in terms of its marketing capabilities in the following areas. 27 27 ISSN 1678-6971 (electronic version) • RAM, São Paulo, 20(6), eRAMD190061, 2019 doi:10.1590/1678-6971/eRAMD190061 ISSN 1678-6971 (electronic version) • RAM, São Paulo, 20(6), eRAMD190061, 2019 Juliana C. N. Costa, Shirlei M. Camargo, Ana M. M. Toaldo, Simone R. Didonet Juliana C. N. Costa, Shirlei M. Camargo, Ana M. M. Toaldo, Simone R. Didonet Juliana C. N. Costa, Shirlei M. Camargo, Ana M. M. Toaldo, Simone R. Didonet Seven-point scale running 1 = much worse than competitors to = much better than competitors. Marketing planning PC 1.1) Marketing planning skills. PC 1.2) Ability to effectively segment and target market. PC 1.3) Marketing management skills and processes. PC 1.4) Developing creative marketing strategies. PC 1.5) Thoroughness of marketing planning processes. PC 1.1) Marketing planning skills. PC 1.2) Ability to effectively segment and target market. PC 1.3) Marketing management skills and processes. PC 1.4) Developing creative marketing strategies. PC 1.5) Thoroughness of marketing planning processes. SSN 1678-6971 (electronic version) • RAM, São Paulo, 20(6), eRAMD190061, 2019 doi:10.1590/1678-6971/eRAMD190061 Marketing implementation MI 1.1) Allocating marketing resources effectively. MI 1.2) Organizing to deliver marketing programs effectively MI 1.3) Translating marketing strategies into action. MI 1.4) Executing marketing strategies quickly. MI 1.5) Monitoring marketing performance. MI 1.1) Allocating marketing resources effectively. MI 1.2) Organizing to deliver marketing programs effective MI 1.3) Translating marketing strategies into action. MI 1.4) Executing marketing strategies quickly. MI 1.5) Monitoring marketing performance. Manager’s profile MC_1) How old are you? _______ years old. MC_2) How long have you been working in this firm? MC_2) R: _______________ years. MC_3) How long have you been working in this sector (economic sector)? MC_2) R: _______________ years. MC_4) How long have you been working in this job position? MC_2) R: _______________ years. Manager’s profile MC_1) How old are you? _______ years old. MC_2) How long have you been working in this firm? MC_2) R: _______________ years. MC_3) How long have you been working in this sector (economic sector)? MC_2) R: _______________ years. MC_4) How long have you been working in this job position? MC_2) R: _______________ years. 28 28 28 ISSN 1678-6971 (electronic version) • RAM, São Paulo, 20(6), eRAMD190061, 2019 doi:10.1590/1678-6971/eRAMD190061 Managers’ influence on company capabilities ISSN 1678-6971 (electronic version) • RAM, São Paulo, 20(6), eRAMD190061, 2019 doi:10.1590/1678-6971/eRAMD190061 AUTHOR NOTES Juliana C. N. Costa, Programa de Pós-Graduação em Administração (PPGADM), Universidade Federal do Paraná (UFPR); Shirlei M. Camargo, Programa de Pós-Graduação em Administração (PPGADM), Universidade Federal do Paraná (UFPR); Ana M. M. Toaldo, Programa de Pós- Graduação em Administração (PPGA), Universidade Federal do Rio Grande do Sul (UFRGS); Simone R. Didonet, Faculdade de Ciências Econômicas (Face), Universidade Federal de Minas Gerais (UFMG). Juliana C. N. Costa is now Research Group Member at the Programa de Pós-Graduação em Administração (PPGADM) of Universidade Federal do Paraná (UFPR); Shirlei M. Camargo is now professor at the Escola de Gestão, Comunicação e Negócios of Centro Universitário Inter nacional (Uninter); Ana M. M. Toaldo is now professor at the Programa de Pós-Graduação em Administração (PPGADM) of Universidade Federal do Paraná (UFPR); and Simone R. Didonet is now professor at the Programa de Pós-Graduação em Administração (PPGADM) of Universi dade Federal do Paraná (UFPR). Correspondence concerning this article should be addressed to Juliana C. N. Costa, Avenida Prefeito Lothário Meissner, 632, Jardim Botânico, Curitiba, Paraná, Brasil, CEP 80210-170. E-mail: julianacncosta@gmail.com EDITORIAL PRODUCTION Publishing Coordination Jéssica Dametta Language Editor Daniel de Almeida Leão EDITORIAL BOARD Editors-in-chief Janette Brunstein Silvia Marcia Russi de Domênico Associated Editor Glória Charão Ferreira Technical Support Vitória Batista Santos Silva Layout Designer Emap Graphic Designer Libro Layout Designer Emap Graphic Designer Libro 29 29 ISSN 1678-6971 (electronic version) • RAM, São Paulo, 20(6), eRAMD190061, 2019 doi:10.1590/1678-6971/eRAMD190061
https://openalex.org/W3159463712	https://figshare.com/articles/journal_contribution/A_Comparative_Estimation_of_Alprazolam_in_Pharmaceutical_Formulations_by_Validated_HPLC_and_HPTLC_Techniques/14535412/1/files/27870460.pdf	English	null	A Comparative Estimation of Alprazolam in Pharmaceutical Formulations by Validated HPLC and HPTLC Techniques	Analytical chemistry letters	2,021	cc-by	233	i i A Comparative Estimation of Alprazolam in Pharmaceutical Formulations by Validated HPLC and HPTLC Techniques Aradhana Sharma 1,2, Kumar Gaurav 3 and Richa Srivastava 2* 1 Central Revenues Control Laboratory, Pusa Campus, New Delhi-110012, India 2 Department of Applied Chemistry, Delhi Technological University, New Delhi- 110042, India 3 Amity Institute of Biotechnology, Amity University, Gurugram 122 413, India Analytical Chemistry Letters https://www.tandfonline.com/loi/tacl ISSN: 2229-7928 (Print); ISSN: 2230-7532 (Online) Supplementary Data y = 17134x + 141397 R² = 0.9918 0.00 500000.00 1000000.00 1500000.00 2000000.00 2500000.00 3000000.00 0 20 40 60 80 100 120 140 160 Figure S1a. Calibration curve for HPLC of alprazolam Concentration (μμμμμg/ml) Area i Analytical Chemistry Letters ISSN: 2229-7928 (Print); ISSN: 2230-7532 (Online) Aradhana Sharma 1,2, Kumar Gaurav 3 and Richa Srivastava 2* 1 Central Revenues Control Laboratory, Pusa Campus, New Delhi-110012, India 2 Department of Applied Chemistry, Delhi Technological University, New Delhi- 110042, India p pp y, g y, 110042, India 3 Amity Institute of Biotechnology, Amity University, Gurugram 122 413, India y = 17134x + 141397 R² = 0.9918 0.00 500000.00 1000000.00 1500000.00 2000000.00 2500000.00 3000000.00 0 20 40 60 80 100 120 140 160 Figure S1a. Calibration curve for HPLC of alprazolam Concentration (μμμμμg/ml) Area Concentration (μμμμμg/ml) Figure S1a. Calibration curve for HPLC of alprazolam Concentration ng/band Peak Area Figure S1b Calibration curve for HPTLC of alprazolam Concentration ng/band Figure S1b Calibration curve for HPTLC of alprazolam
https://openalex.org/W4387419780	https://journal.spera.asn.au/index.php/AIJRE/article/download/646/793	English	null	Additional Professional Induction Strategy (APIS)	Australian & international journal of rural education	2,013	cc-by	8,123	ABSTRACT This paper describes a strategy, designed by a faculty of education in a regional Australian university, to induct pre-service educators into the education profession. It then focuses on one component of the strategy, an initiative called Education Commons. This initiative uses a model of critical reflection to engage pre-service educators in discussions about current and relevant educational topics. This aims to connect them into professional networks and to assist their induction into the education profession from the outset of their tertiary study. An analysis of a small data set – two small stories told by an early career teacher who had participated in Education Commons while at university – is investigated for evidence of the effect of the program. The analysis highlights the use of critical reflection and career development learning. Robyn Henderson Karen Noble Kathleen Cross University of Southern Queensland ADDITIONAL PROFESSIONAL INDUCTION STRATEGY (APIS): EDUCATION COMMONS, A STRATEGY TO SUPPORT TRANSITION TO THE WORLD OF WORK Robyn Henderson Karen Noble Kathleen Cross Australian and International Journal of Rural Education, Vol. 23 (1) 2013 INTRODUCTION Within the education sector, nationally and internationally, there has been a continued increase in the attrition rates of beginning teachers, with as many as 40% leaving or intending to leave the profession within the first five years of beginning their careers (Ewing & Manuel, 2005; Verstegen & Zhang, 2012). For beginning teachers in regional, rural and remote locations with limited social support, this percentage may be even higher. As a result, there has been a growing interest in the wellbeing of teachers across the entire education sector and various initiatives have been mounted in an attempt to redress these overwhelmingly negative statistics. Over a six year period in a regional Australian university, a small group of academic and professional support staff have worked collaboratively to design, implement, Australian and International Journal of Rural Education, Vol. 23 (1) 2013 43 evaluate and refine various additional programs to support education students and to value-add to their formal study. These initiatives, which form the Faculty of Education's Additional Professional Induction Strategy (APIS), provide pre-service educators, including undergraduate and postgraduate diploma students, with opportunities to explore the development of personal and professional identities beyond the coursework study that they undertake as part of their degree or postgraduate diploma. The programs are seen as complementary to the formal curriculum and indeed, as we move into an ever increasing accredited and competitive higher education context, they become an essential tool in providing a level of distinctiveness within the student learning journey and within the marketplace. This paper provides an overview of the Additional Professional Induction Strategy. It then introduces Education Commons, one of the programs that form part of the overall strategy. It locates that particular initiative within literature that refers to the construction of professional identities through professional learning, the enhancement of workforce capabilities, and career development learning. Through an analysis of two small stories from an early career teacher who had participated in the Education Commons program during her final years of Education study, the program is considered in terms of its effects in relation to career development learning and critical reflection. The paper concludes with a discussion of how the program seems to promote professional and personal learning that is potentially lifelong and lifewide. Australian and International Journal of Rural Education, Vol. 23 (1) 2013 INTRODUCING THE ADDITIONAL PROFESSIONAL INDUCTION STRATEGY (APIS) It focuses on “what to do when you don’t know what to do” and provides connections to support services within the university and specific advice about doing assignments, locating information and how to be a successful student. The PD initiative provides workshops that develop students’ overarching capabilities and actions (SOCA for short) and enables students to turn their developing academic knowledge into practical strategies for use in classrooms. The left-hand side of Figure 1 shows the additional support that is on offer for enhancing engagement and success in students’ university study. The FYI (For Your Information) program offers support in relation to academic and information literacies. It focuses on “what to do when you don’t know what to do” and provides connections to support services within the university and specific advice about doing assignments, locating information and how to be a successful student. The PD initiative provides workshops that develop students’ overarching capabilities and actions (SOCA for short) and enables students to turn their developing academic knowledge into practical strategies for use in classrooms. The right-hand side of Figure 1 shows the initiatives that aim to build professional identity and link to the workforce outside the university context. Education Commons helps pre-service educators build professional networks and to think critically about their educational practice and knowledges. The E2 initiative is a program that operates on one of the university’s campuses. It provides professional development activities that bring together educators from the community and pre- service educators and academics staff from the university. Sitting between the programs that work internally and those that link with the world outside university is the Transition to Teaching initiative, which plays a specific role in helping pre- service educators move from the university context into the workforce. Its focus includes the logistics of applying for jobs, preparing for interviews and becoming a registered teacher. Australian and International Journal of Rural Education, Vol. 23 (1) 2013 INTRODUCING THE ADDITIONAL PROFESSIONAL INDUCTION STRATEGY (APIS) Over the past six years, several initiatives have been established within the faculty of education at a regional Australian university to value-add to the formal study of pre- service educators. Each was developed by academic and professional support staff around what was perceived as a gap in students’ formal study. While each initiative catered for a specific aspect of students’ development as future educators, it became apparent that the approach was a disparate one and that there was a need to see how the initiatives fitted together and related to each other. Additionally, in these neoliberal times, there was a need to ensure that that the initiatives were being efficient in terms of the resources being used and that efforts were not being duplicated. Meetings were held between those involved, and the aims and target audiences of each initiative were mapped. This resulted in the establishment of an overarching framework that was called the Additional Professional Induction Strategy (APIS). As shown in Figure 1, APIS provides support for pre-service educators within the university context and aims to build success in their study to become future educators. It also offers professional learning and development that help to induct pre-service educators into the education profession. Australian and International Journal of Rural Education, Vol. 23 (1) 2013 Australian and International Journal of Rural Education, Vol. 23 (1) 2013 44 Figure 1. The components of the Additional Professional Induction Strategy (APIS) The left-hand side of Figure 1 shows the additional support that is on offer for enhancing engagement and success in students’ university study. The FYI (For Your Information) program offers support in relation to academic and information literacies. It focuses on “what to do when you don’t know what to do” and provides connections to support services within the university and specific advice about doing assignments, locating information and how to be a successful student. The PD initiative provides workshops that develop students’ overarching capabilities and actions (SOCA for short) and enables students to turn their developing academic knowledge into practical strategies for use in classrooms. Figure 1. The components of the Additional Professional Induction Strategy (APIS) g (APIS) (APIS) The left-hand side of Figure 1 shows the additional support that is on offer for enhancing engagement and success in students’ university study. The FYI (For Your Information) program offers support in relation to academic and information literacies. FOCUSING ON EDUCATION COMMONS One of the initiatives, Education Commons, was established as a way of inducting pre-service educators into the education profession from the outset of their university study. It was based on the premise that high attrition rates of “new” educators in the education workforce might be ameliorated if pre-service educators were able to build a professional identity from the beginning of their study. Traditionally, induction into the education workforce has been understood as Australian and International Journal of Rural Education, Vol. 23 (1) 2013 45 something that occurs once pre-service educators are at the end of their university study and are ready to make the transition into work (Beauchamp & Thomas, 2009; Sachs, 2005). Whilst transition programs remain an essential part of what universities should offer, Education Commons has attempted to provide a much broader approach that focuses on personal and professional identity building and critical reflection on and about educational practice. The capacity building approach works to develop the capabilities of pre-service educators to “be” teachers, to understand themselves as belonging to the education profession, and to become educators who are able to cope with the dynamic nature of today’s educational world. In building these capacities, Education Commons complements the Transition into Teaching component of APIS. The difference is that Education Commons focuses on building professional identity and other capabilities that help pre-service educators feel part of the profession (Ewing & Manuel, 2005). As described elsewhere (see Noble & Henderson, in press), the program is based on a set of principles that regard the following as important: In building these capacities, Education Commons complements the Transition into Teaching component of APIS. The difference is that Education Commons focuses on building professional identity and other capabilities that help pre-service educators feel part of the profession (Ewing & Manuel, 2005). As described elsewhere (see Noble & Henderson, in press), the program is based on a set of principles that regard the following as important:  the building of a community of practice where participants meet to discuss important educational topics and enhance critically reflective skills. FOCUSING ON EDUCATION COMMONS This engages them in dialogue (Wenger, 1998) and helps to build democratic professionalism (Sachs, 1999, 2005);  an understanding of the process of “becoming a teacher” or “becoming an educator” as occurring over an extended period of time and as a lifelong and lifewide enterprise (Alsup, 2006; Noble & Henderson, 2011); p ( p )  the importance of privileging space and opportunity to develop personal and professional identities (Alsup, 2006); p ( p )  the importance of privileging space and opportunity to develop personal and professional identities (Alsup, 2006); p ( p )  the situatedness of personal and professional identity development. In building on these understandings, Education Commons has used a cyclical two- step process. Each cycle begins with a panel of educators who are able to come to the university campus and who represent a range of educational sectors, including early childhood, primary, middle and secondary schooling, and vocational and further education. The discussion that the educators engage in is presented live to an audience of on-campus pre-service educators and academics, and it is video- recorded so that the artifacts can be made available in an online environment for all pre-service educators, regardless of their location or mode of study. Each panel has a focus educational topic that is current and relevant to pre-service educators and to educators in the field. However, there is no set agenda for the panel discussion. It begins with the panelists introducing themselves and identifying their interest/s in the topic, then the interactive discussion goes wherever the panelists and the audience take it. In building on these understandings, Education Commons has used a cyclical two- step process. Each cycle begins with a panel of educators who are able to come to the university campus and who represent a range of educational sectors, including early childhood, primary, middle and secondary schooling, and vocational and further education. The discussion that the educators engage in is presented live to an audience of on-campus pre-service educators and academics, and it is video- recorded so that the artifacts can be made available in an online environment for all pre-service educators, regardless of their location or mode of study. Each panel has a focus educational topic that is current and relevant to pre-service educators and to educators in the field. However, there is no set agenda for the panel discussion. Australian and International Journal of Rural Education, Vol. 23 (1) 2013 FOCUSING ON EDUCATION COMMONS It begins with the panelists introducing themselves and identifying their interest/s in the topic, then the interactive discussion goes wherever the panelists and the audience take it. The second step of the Education Commons process is a pedagogical conversation that provides opportunities for pre-service educators to unpack the points and ideas raised in the panel discussion and critically reflect on what was said, on their Australian and International Journal of Rural Education, Vol. 23 (1) 2013 46 learnings, and on links to past, present and future experiences in education. The video-recordings from the panel discussions provide stimulus materials where required. For on-campus pre-service educators, the second step has been offered as a face-to-face session about two weeks after the panel discussion. However, the online Education Commons site with its inbuilt tools to foster discussion, including discussion forums and Wimba classrooms, is available to all pre-service educators, panelists and academic staff to continue the conversations that began during the panel discussions. Twelve cycles of Education Commons usually occur during each academic year. All Education Commons events draw on a model of critical reflection (Macfarlane, Noble, Kilderry, & Nolan, 2005) that allows pre-service educators to think at a critical level. In particular, the model includes opportunities to think deeply about aspects of educational practice, to make links between theory and practice, and to think about other ways of “doing” educational practice. Macfarlane et al.’s four steps – confront, deconstruct, theorise, and think otherwise – have provided a useful framework for ensuring that pre-service educators move beyond the taken-for- granted and consider educational practice from multiple perspectives. When participants engage in collaborative critical reflection, they are able to customise and individualise their learning journeys (Noble & Henderson, in press) and achieve what Ryan (2011) refers to as “purposeful reflection” which enables “deep, active learning” (p. 101). According to Ryan (2012), the practice of critical reflection can be achieved at two levels: “making sense of experience” and “reimagining future experience” (p. 208). Like the theorise and think otherwise components of Macfarlane et al.’s (2005) model, Ryan’s (2012) second level aims to promote critical, more abstract, academic or professional reflection. Australian and International Journal of Rural Education, Vol. 23 (1) 2013 FOCUSING ON EDUCATION COMMONS Not only are participants “understanding the context of learning and the particular issues that might arise,” they understand “their own contribution to that context, including past experiences, values/philosophies and knowledge” and draw “on other evidence or explanation from the literature or relevant theories” (Ryan, 2012, p. 209). The use of critical reflection in Education Commons has been a useful strategy for ensuring that pre-service educators have space and opportunities to think about, reflect on, and make their own connections to educational topics. This approach to learning is a transformative one (Kalantzis & Cope, 2008). It is not about knowledge transmission; rather, it promotes active learning, whereby participants engage in discussions focused on “improving learning and professional practice” (Ryan, 2012, p. 209). Such discussions go beyond acceptance and maintenance of the status quo (Gur-Ze’ev, Masschelein, & Blake, 2001), encouraging participants to deconstruct and analyse issues (deconstruct and confront), to link theory and practice (theorise), and to think about multiple possibilities for practice (think otherwise) (Macfarlane et al., 2005). Additionally, Education Commons recognises that “becoming” an educator involves a learning journey that is complex and dynamic in nature (Alsup, 2006; Chong, Low, Australian and International Journal of Rural Education, Vol. 23 (1) 2013 47 & Goh, 2011; Temmerman, Noble, & Danaher, 2010). Within the community of practice (Wenger, 1998) of Education Commons, professional networks can develop as pre-service educators engage with educators from the field. Furthermore, there are opportunities to “engage in the complex integration of personal self, and the taking on of a culturally scripted, often narrowly defined professional role while maintaining individuality” (Alsup, 2006, p. 4). Critical reflection also allows participants to “become more in tune with their sense of self and with a deep understanding of how this self fits into a larger context which involves others” (Beauchamp & Thomas, 2009, p. 182). In taking up issues related to what it means to be an educator, Education Commons introduces key aspects of professional identity development. These include notions of being an educator, such as understanding the theory-practice nexus, considering rhetoric and reality, and building knowledge of perspectives and context; developing a sense of belonging to the profession, such as building networks, understanding a holistic perspective, and accepting the dynamic nature of education; and becoming an educator, with knowledge and experience of relevant and current issues and topics. Australian and International Journal of Rural Education, Vol. 23 (1) 2013 FOCUSING ON EDUCATION COMMONS A previous investigation into Education Commons and its development of professional identity identified collective agency as an important outcome of the program (Noble & Henderson, in press). It was found – through building relationships with other participants, exercising choice, networking, gaining a sense of belonging and connectedness, and connecting personally and professionally to educational practice and knowledge – that pre-service educators developed understandings about how to be, know and do (Gee, 1996) in the education profession. This suggested that the initiative was helping to build the capacity of pre-service educators and was preparing a workforce that was “flexible, sustainable, critically reflective and informed” (Noble & Henderson, in press). It also seemed that these qualities would be useful in times when “the future is fundamentally ‘unknowable’” (Jasman & McIlveen, 2011, p. 118). These findings suggest that there may be links with the field of career development learning, which “relates to learning about the content and process of career development or life/career management” (McMahon, Patton, & Tatham, 2003, p. 6). An examination of the principles of career development learning that were identified by Smith, Brooks, Lichtenberg, McIlveen, Torjul and Tyler (2009) indicated a similarity with the way Education Commons operates. For example, both rely on flexible partnerships, workplace experiences and a student-centred approach (Smith et al., 2009, p. 13). Additionally, the ability of Education Commons to draw on personal and professional dimensions of experience (Noble & Henderson, in press) reflects the efforts of career development learning to work “across all aspects of students’ lives” and for students to “systematically reflect … and articulate the acquired skills and experience” (Smith et al., 2009, p. 13). As highlighted in the Australian blueprint for career development (Ministerial Council for Education, Early Childhood Development and Youth Affairs [MCEECDYA], Australian and International Journal of Rural Education, Vol. 23 (1) 2013 48 2010), career development skills are essential for encouraging “learning by linking it to … hopes and dreams for the future” and enabling successful transition “between learning and work roles” (p. 9). According to McIlveen, Brooks, Lichtenberg, Smith, Torjul and Tyler (2011), the benefits of career development learning can include “reflecting upon past academic and workplace learning and assimilating new experiences into a burgeoning sense of professional identity” and facilitating “an individual’s accommodation of learning experiences that challenged previously held beliefs” (p. 151). FOCUSING ON EDUCATION COMMONS These descriptions suggest that Education Commons might foster career development learning and this warrants further investigation. Australian and International Journal of Rural Education, Vol. 23 (1) 2013 THE RESEARCH To investigate the idea that career development learning might indeed be a relevant way of conceptualising Education Commons and its effect on pre-service educators, we reviewed the data that have been collected as part of several research projects. Whilst we have previously looked broadly at the data (e.g., Noble & Henderson, in press), generally in relation to evaluating the program for learning and teaching purposes, we have decided to focus here on two small pieces of data. These were collected from an early career teacher who participated in Education Commons during the final years of her Education study as a pre-service educator. She returned to the university to participate in an Education Commons session after she had joined the teaching profession, and the data were generated as she talked with pre- service educators about her experiences as a beginning teacher and how Education Commons had given her ways of coping with difficult or challenging situations. From the data that were generated, we have extracted two small stories (Bamberg, 2004). Each tells a story of ongoing and past events (Bamberg & Georgakopoulou, 2008) that occurred during the early career teacher’s first weeks of being a teacher. It is widely recognised that narratives are used by people to represent their world, to explain the experiences they have had, and to position themselves in that world and in relation to others. In taking a sociocultural perspective, we recognise stories as evidence of a person’s discursive construction of “meaningful selves, identities, and realities” and as an attempt to make “sense of personal experiences” (Chase, 2011, p. 422). We analyse the two small stories using two analytical frames. The first is the four- stage learning taxonomy presented in the Australian blueprint for career development (MCEECDYA, 2010). This provides a conceptual framework for examining the developmental stages through which learners move as they engage in career development learning. The four stages involve: 1. acquiring and understanding knowledge; 2. applying this knowledge; 3. personalising learning, and 4. acting creatively on that learning (MCEECDYA, 2010, p. 25) 49 Australian and International Journal of Rural Education, Vol. 23 (1) 2013 The taxonomy includes information about what learners might be asked to do at each of the four levels, along with examples of performance indicators for each level. Some examples of these are provided in Table 1. THE RESEARCH 23 (1) 2013 50 Evaluate the impact of personal decisions on themselves or others Express their ideas/feelings Give their opinion Internalise experiences Question information and decisions Visualise options for themselves 4 Act Improve self-concept in order to contribute positively to life, learning and work Adapt products, concepts or scenarios Advise people Conceptualise ideas or projects Design new products or programs Edit a book or an article Elaborate new ideas or projects Facilitate transitions Guide or mentor others Innovate Invent new things Transfer skills, knowledges and attitudes to modify and/or create Transform behaviours and attitudes Table 2: Questions for locating examples of critical reflection Steps Questions Deconstruct Have these questions been considered?: What am I doing? How am I doing it? Has (classroom) practice been considered or analysed? Have aspects of (classroom) practice been identified as “normal” or “proper,” or as needing to be reconceptualised? Have aspects been identified as worthy of consideration to ensure more effective practice? Confront Have these questions been considered?: What is working? What is not working? What might I need to change? Has a specific focus for further consideration been identified? Have weaknesses or areas for change been considered? Has a problem or issue been identified? Has there been a decision that an aspect of practice needs to be modified or changed? Theorise Have these questions been considered?: How might I theorise this? How might I research this? What theories, research and evidence might I draw on? Has there been an attempt to locate further information? Have links been made between theory and practice? Have theories/research evidence been identified? Evaluate the impact of personal decisions on themselves or others Express their ideas/feelings Give their opinion Internalise experiences Question information and decisions Visualise options for themselves 4 Act Improve self-concept in order to contribute positively to life, learning and work Adapt products, concepts or scenarios Advise people Conceptualise ideas or projects Design new products or programs Edit a book or an article Elaborate new ideas or projects Facilitate transitions Guide or mentor others Innovate Invent new things Transfer skills, knowledges and attitudes to modify and/or create Transform behaviours and attitudes Table 2: Questions for locating examples of critical reflection Steps Questions Deconstruct Have these questions been considered?: What am I doing? How am I doing it? Has (classroom) practice been considered or analysed? THE RESEARCH With its framing as a developmental learning taxonomy, the Blueprint document highlights that “learners may not move through all four stages of the learning taxonomy. How far they progress will depend on their motivation and the context in which they use the skill, knowledge or attitude they have developed” (p. 26). The second analytical frame comes from Macfarlane, Noble, Kilderry and Nolan’s (2005) model of critical reflection. The four steps – deconstruct, confront, theorise, and think otherwise – provide a framework for examining the early career teacher’s stories. Because Education Commons had been conceptualised as drawing on the model of critical reflection, we were keen to investigate whether there was evidence in the two small stories of the model in use. In order to conduct the analysis, we adapted and designed a set of questions that could identify evidence of critical reflection (see Henderson, 2012, pp. 275-276; Macfarlane et al., 2005, p. 16). These questions are shown in Table 2. Table 1: The four-stage learning taxonomy from the Australian blueprint for career development (extracted from MCEECDYA, 2010, pp. 25-26) Stage Possible performance indicators Learner actions 1 Acquire Understand how individual characteristics contribute to achieving personal, social, educational and professional goals Classify information about people or things Codify new information Crosscheck information Explain new concepts Give examples to illustrate concepts Gather pertinent information Interview people Locate information Research a topic 2 Apply Adopt behaviours and attitudes conducive to reaching personal, social, educational and professional goals Apply acquired knowledge Develop a project Fix things Generalise acquired knowledge Learn about themselves Perform a task Plan using acquired knowledge Practise new skills Prepare a project Simulate a situation Solve a problem Try a new idea 3 Personalis e Assess personal characteristics and capitalise on those that contribute positively to the achievement of personal, educational, social and professional goals Analyse situations Be assertive Choose for themselves Comment on subjects and situations Decide for themselves Examine their decisions or reactions able 1: The four-stage learning taxonomy from the Australian blueprint for care velopment (extracted from MCEECDYA, 2010, pp. 25-26) Australian and International Journal of Rural Education, Vol. 23 (1) 2013 Australian and International Journal of Rural Education, Vol. TWO SMALL STORIES, CRITICAL REFLECTION AND CAREER DEVELOPMENT LEARNING In this section, we present the two small stories with our analysis and discussion of the evidence we found of the use of the model of critical reflection and characteristics of career development learning. We recognise that we begin with data that represent Education Commons positively. Indeed, the early career teacher prefaced her stories with the statement that “It’s only through … my notes and the discussions and skills that I’ve acquired through Education Commons that I’ve been able to deal with the many, many challenges I’ve had in my first term [of teaching].” In small story 1, the early career teacher described an experience on her second or third day of teaching. THE RESEARCH Have aspects of (classroom) practice been identified as “normal” or “proper,” or as needing to be reconceptualised? Have aspects been identified as worthy of consideration to ensure more effective practice? Confront Have these questions been considered?: What is working? What is not working? What might I need to change? Has a specific focus for further consideration been identified? Have weaknesses or areas for change been considered? Has a problem or issue been identified? Has there been a decision that an aspect of practice needs to be modified or changed? Theorise Have these questions been considered?: How might I theorise this? How might I research this? What theories, research and evidence might I draw on? Has there been an attempt to locate further information? Have links been made between theory and practice? Have theories/research evidence been identified? Has there been an attempt to draw in ideas from elsewhere to inform thinking about the problem/issue? Table 2: Questions for locating examples of critical reflection Australian and International Journal of Rural Education, Vol. 23 (1) 2013 51 Has there been an attempt to consider multiple perspectives or solutions to the identified problem or issue? Think otherwise Have these questions been considered?: What could I do differently? What aspects of my practice should I change? Has there been an attempt to re-think practice? Have multiple perspectives informed thinking? Has change been implemented? Has there been an attempt to re-think practice? Have multiple perspectives informed thinking? Has change been implemented? Australian and International Journal of Rural Education, Vol. 23 (1) 2013 Small story 1 Her initial actions meant that she had to confront the problem, which related to criticism from a parent that she was “unable to do [her] job properly.” Although we do not hear too many details about the deconstructing process that occurred, it was evident that the teacher made sense of the cause-effect demands made by the parent – “If it continued she’d be putting her child out of my class” – and she thought about how to manage the issue in ways other than crying and seeking support. The theorising step of critical reflection involved revisiting the processes of Education Commons and this resulted in the teacher reassuring herself that she could respond confidently as a teacher: “I’m the teacher” who has a “four year degree.” In revisiting her experiences of Education Commons and the confidence she had developed, she was able to find a way of managing the situation. In thinking otherwise, she resolved that she could say “I’m the teacher” and “this is how I intend to do things in my classroom.” However, she also left the way open for the parent to express an opinion and to be able to raise issues with her or with the principal. In terms of career development learning, the teacher’s story suggested that she was able to demonstrate many of the attributes that are identified in the four-stage learning taxonomy that is shown in Table 1 (MCEECDYA, 2010). She realised that four years in a teacher education program had allowed her to acquire knowledge that enabled her to do the job of a teacher (stage 1) and that she could apply that knowledge to the situation (stage 2). In particular, her application of that knowledge involved developing a plan, learning about herself, solving a problem, and trying a new idea. The teacher also demonstrated aspects of stage 3 of the four-stage model of career development learning, because she was able to personalise her learning. She analysed the situation and was assertive in her approach (“I’m the teacher”). She was able to comment on the situation, examine her reactions and her decision about how to resolve the situation, express her feelings and visualise options for herself. There was also some evidence that she was able to transfer skills and knowledge (stage 4) from her experiences of Education Commons to a problem that arose as part of her work as a teacher. Small story 1 Where shall I start? Okay for example, second or third day I had a parent come in and tell me how pathetic I was. I was a useless teacher, I was unable to do my job properly, I should be doing this, I should be doing that. I shouldn’t be doing this and if it continued she’d be pulling her child out of my class. This is my second or third day of school. It’s just as bad for me so I spent most of the morning in my principal’s office crying. It’s those sorts of things that just hit you and it’s not until you use things that we discuss in Education Commons that go, well no. It’s given me the confidence. I’ve done the four year degree. It’s like I’m going through my head, hello woman you know nothing. I’m the teacher. And that’s what you end up explaining to her in a term that I’m the teacher; if you’re not happy with the way I do things, come and tell me, come and tell my principal. But this is how I’ve started the term and this is how I intend to do things in my classroom, because you are the boss of your classroom. Your parents are part of your classroom 52 Australian and International Journal of Rural Education, Vol. 23 (1) 2013 but they aren’t in charge of it and it’s those sorts of things – those strategies – that helped me get through this first term. but they aren’t in charge of it and it’s those sorts of things – those strategies – that helped me get through this first term. The steps of the model of critical reflection are evident in small story 1. In telling about how she had to deal with a complaint from a parent, the early career teacher moved from being upset – “I spent most of the morning in my principal’s office crying” – to making a decision about how to manage the situation. Australian and International Journal of Rural Education, Vol. 23 (1) 2013 Small story 2 In small story 2, the early career teacher identified a particular issue in her class that she wanted to address. One of the children in the class had a pica, a compulsive eating of nonfood items, and the “mum’s not up for discussion yet.” The teacher’s description framed the issue in terms of the model of critical reflection. She began by deconstructing classroom observations: “putting particular textures into his mouth … rocking … he will repeat it over and over again.” She identified the child’s behaviours as “alarming signs of autism,” but realised that the child’s mother had not accepted that there might be something wrong with her child: “she’s just like no In small story 2, the early career teacher identified a particular issue in her class that she wanted to address. One of the children in the class had a pica, a compulsive eating of nonfood items, and the “mum’s not up for discussion yet.” The teacher’s description framed the issue in terms of the model of critical reflection. She began by deconstructing classroom observations: “putting particular textures into his mouth … rocking … he will repeat it over and over again.” She identified the child’s behaviours as “alarming signs of autism,” but realised that the child’s mother had not accepted that there might be something wrong with her child: “she’s just like, no there’s nothing wrong with my child.” In theorising an approach and coming up with an alternate plan (thinking otherwise), the teacher returned to her learnings about good teaching and research practices and found a way forward: “constant observations,” collecting and documenting evidence, while “not pushing the parents” until that evidence was collected. … rocking … he will repeat it over and over again.” She identified the child’s behaviours as “alarming signs of autism,” but realised that the child’s mother had not accepted that there might be something wrong with her child: “she’s just like, no there’s nothing wrong with my child.” In theorising an approach and coming up with an alternate plan (thinking otherwise), the teacher returned to her learnings about good teaching and research practices and found a way forward: “constant observations,” collecting and documenting evidence, while “not pushing the parents” until that evidence was collected. Aspects of the four-stage taxonomy of career development learning were also evident in small story 2. Small story 1 In other words, the small story indicated that the teacher was demonstrating elements of all four stages of the learning taxonomy, with particular strengths in stages 2 and 3 because she was applying her knowledge and personalising her learning to the situation at hand. Australian and International Journal of Rural Education, Vol. 23 (1) 2013 53 Small story 2 In small story 2, the early career teacher focused on a particular child in her class and her attempts to convince the child’s mother that the child was demonstrating behaviours that required attention. Like I have a little boy who’s showing alarming signs of autism; he has pica which is putting particular textures into his mouth, so paper, thumb tacks, I’ve had all sorts of things come out of his mouth, and rocking. He’ll sit on the floor when we’re having group time and he will just sit there and rock. I will be saying something and he will repeat it over and over again which is all alarming signs of autism. But mum’s not up for a discussion yet, she’s just like, no there’s nothing wrong with my child, he’s only in prep. I’m like, if he’s seen to, and gets the help and assistance and support he needs now, he will be much better off. But it’s through dealing with, it’s like there’s a section in Education Commons – I think both years I did it – where we looked at dealing with that scenario. So by sitting back and not pushing the parents, I’m taking constant observations on all the things that he does so that when the time comes, we can go, well look, this is the evidence that we have. We really need to get him to see a paediatrician. In small story 2, the early career teacher identified a particular issue in her class that she wanted to address. One of the children in the class had a pica, a compulsive eating of nonfood items, and the “mum’s not up for discussion yet.” The teacher’s description framed the issue in terms of the model of critical reflection. She began by deconstructing classroom observations: “putting particular textures into his mouth … rocking … he will repeat it over and over again.” She identified the child’s behaviours as “alarming signs of autism,” but realised that the child’s mother had not accepted that there might be something wrong with her child: “she’s just like, no there’s nothing wrong with my child.” In theorising an approach and coming up with an alternate plan (thinking otherwise), the teacher returned to her learnings about good teaching and research practices and found a way forward: “constant observations,” collecting and documenting evidence, while “not pushing the parents” until that evidence was collected. Australian and International Journal of Rural Education, Vol. 23 (1) 2013 CONCLUSION The two small stories told by the early career teacher as part of her discussion about the effect of Education Commons on her practice as a teacher demonstrated that she was using the model of critical reflection (Macfarlane et al., 2005) as a way of thinking through significant events and her responses to those events. In drawing on the model and its steps of deconstruct, confront, theorise and think otherwise, she was able to find a way forward when trying to manage challenging circumstances. While we cannot say that the teacher’s actions were a direct result of her participation in Education Commons, she attributed the program with giving her skills and confidence to cope with the unexpected. In examining the teacher’s small stories for evidence of career development learning, we found attributes that seemed to align with all four stages of MCEECDYA’s (2010) learning taxonomy. The teacher demonstrated that she had acquired knowledge (stage 1) and was able to apply that knowledge (stage 2) to particular situations. She personalised her understandings (stage 3) in a way that enabled her to assess her personal characteristics and draw on her past experiences and learnings. She transferred skills and knowledge (stage 4) from those previous learnings to find positive ways of addressing the issues that had presented. While the taxonomy indicates developmental stages and suggests that not all learners will achieve all stages, depending “on their motivation and the context in which they use the skill, knowledge or attitude they have developed” (MCEECDYA, 2010, p. 26), we regard Education Commons as facilitating an iterative process that can enable learners to acquire, apply, personalise and act. From the small data set that we have presented, we make no claims about the effect of Education Commons on all students who participate in the program. However, for the teacher who volunteered to share her experiences of the first few weeks of being a teacher, Education Commons seemed to play a significant role in her explanation of being able to cope with new situations and challenges. The teacher regarded as important the ability to reflect on practice, to identify potential solutions to identified problems or issues, and to be able to adapt and refine her actions and practice. Small story 2 The teacher’s acquisition of knowledge (stage 1) was evident in her discussion of autism and her understanding about how to work with the parent of the child she was concerned about. She had gathered relevant information and could give examples to illustrate her understandings. She showed that she could apply that knowledge to the situation (stage 2), by developing a plan Australian and International Journal of Rural Education, Vol. 23 (1) 2013 54 of action, practising her skills of observation and documentation, and finding a way that might persuade the parent that medical advice was required. The story also illustrated the teacher’s ability to personalise her learning (stage 3). This was evident in her analysis of the situation, her reflection on her decision, and her ability to evaluate the impact of her decisions on her actions in the classroom and on the parent. In acting on her concern for the child’s future, the teacher also demonstrated that she could act as a teacher in a responsible fashion (stage 4). Australian and International Journal of Rural Education, Vol. 23 (1) 2013 CONCLUSION We see these as developing a capacity for lifelong and lifewide learning, with learning continuing as new situations and contexts are encountered, and facilitating the transfer of learning from one situation and context to another. These are particularly important attributes for teachers in rural and remote areas, where 55 Australian and International Journal of Rural Education, Vol. 23 (1) 2013 opportunities for professional development and even talking with others working in a similar field can be limited. If we return to our initial discussion about APIS, the Additional Professional Induction Strategy, used in our faculty, we posit that the use of a model of critical reflection and a career development learning framework can provide all stakeholders with ways of understanding educational discourses and a process for problem-solving situations that arise on a daily basis. These also promote professional learning that is both lifelong and lifewide, thus assisting transition to the world of work. In engaging pre-service educators in induction to the education profession during the course of their tertiary study, the strategy offers the potential to retain early career educators in the profession, by enabling them to see themselves as teachers and to think like teachers well before they move into the education workforce. Australian and International Journal of Rural Education, Vol. 23 (1) 2013 ACKNOWLEDGEMENT We acknowledge Education Commons’ participants, past and present, particularly the early career teacher who so generously shared her experiences and fostered deep and meaningful discussion amongst a group of pre-service educators and academics. 56 Australian and International Journal of Rural Education, Vol. 23 (1) 2013 Australian and International Journal of Rural Education, Vol. 23 (1) 2013 57 REFERENCES Alsup, J. (2006). 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https://openalex.org/W4310207296	https://chemrxiv.org/engage/api-gateway/chemrxiv/assets/orp/resource/item/6383c95bebc1c7bb8dd4e17a/original/mapping-the-chemical-space-of-active-site-targeted-covalent-ligands-for-protein-tyrosine-phosphatases.pdf	English	null	Mapping the chemical space of active-site targeted covalent ligands for protein tyrosine phosphatases	null	2,022	cc-by	11,439	ORCIDs Suk ho Hong: 0000-0002-6024-0685 Sarah Y. Xi: 0000-0001-7590-3202 Andrew Johns: 0000-0002-3197-1390 Lauren C. Tang: 0000-0001-8786-5418 Allyson Li: 0000-0003-2359-7703 Marko Jovanovic: 0000-0001-9707-3377 Neel H. Shah: 0000-0002-1186-0626 Table of Contents Graphic Table of Contents Graphic Table of Contents Graphic Table of Contents Graphic Table of Contents Text The development of covalent chemical probes and inhibitors for tyrosine phosphatases has historically been challenging. We report the characterization of diverse cysteine-reactive electrophiles and fragment-like scaffolds as covalent ligands for tyrosine phosphatases. Our investigation reveals chemical constraints for tyrosine phosphatase covalent inhibitor design and sheds light on the structural features of phosphatases that govern their susceptibility to covalent inhibition. Table of Contents Text Table of Contents Text The development of covalent chemical probes and inhibitors for tyrosine phosphatases has historically been challenging. We report the characterization of diverse cysteine-reactive electrophiles and fragment-like scaffolds as covalent ligands for tyrosine phosphatases. Our investigation reveals chemical constraints for tyrosine phosphatase covalent inhibitor design and sheds light on the structural features of phosphatases that govern their susceptibility to covalent inhibition. The development of covalent chemical probes and inhibitors for tyrosine phosphatases has historically been challenging. We report the characterization of diverse cysteine-reactive electrophiles and fragment-like scaffolds as covalent ligands for tyrosine phosphatases. Our investigation reveals chemical constraints for tyrosine phosphatase covalent inhibitor design and sheds light on the structural features of phosphatases that govern their susceptibility to covalent inhibition. Hong, Xi, et al., 2022 – Main Text & Figures - page 1 of 19 Abstract Protein tyrosine phosphatases (PTPs) are an important class of enzymes that modulate essential cellular processes through protein dephosphorylation and are dysregulated in various disease states. There is demand for new compounds that target the active sites of these enzymes, for use as chemical tools to dissect their biological roles or as leads for the development of new therapeutics. In this study, we explore an array of electrophiles and fragment scaffolds to investigate the required chemical parameters for covalent inhibition of tyrosine phosphatases. Our analysis juxtaposes the intrinsic electrophilicity of these compounds with their potency against several classical PTPs, revealing chemotypes that inhibit tyrosine phosphatases while minimizing excessive, potentially non-specific reactivity. We also assess sequence divergence at key residues in PTPs to explain their differential susceptibility to covalent inhibition. We anticipate that our study will inspire new strategies to develop covalent probes and inhibitors for tyrosine phosphatases. Hong, Xi, et al., 2022 – Main Text & Figures - page 2 of 19 Introduction Protein tyrosine phosphorylation is a prevalent post-translational modification that can modulate enzyme activity, protein localization, protein stability, and protein-protein interactions. Protein tyrosine phosphatases (PTPs) make up a large family of over 100 enzymes in humans that catalyze the dephosphorylation of tyrosine residues on proteins.[1,2] These enzymes counteract protein tyrosine kinases, which phosphorylate proteins on tyrosine residues. Many diseases including cancers arise from the dysregulation of tyrosine phosphorylation, and tyrosine phosphatases have been proposed as candidate drug targets for the treatment of these diseases.[3] For example, the phosphatase PTP1B has been linked to diabetes and obesity, as it acts as a negative regulator of the insulin receptor signaling pathway by dephosphorylating the insulin receptor and its substrates.[4,5] The phosphatase SHP2 has been shown to promote oncogenic signaling downstream of receptor tyrosine kinases.[6,7] Both of these enzymes have been the targets of extensive preclinical drug discovery campaigns, and allosteric inhibitors of SHP2 are currently in clinical trials.[8,9] Despite their physiological importance, the regulatory mechanisms and biological roles of many PTPs remain poorly defined, in part due to the lack of robust chemical tools to selectively inhibit PTPs or monitor their activity in cells.[10] Most tyrosine phosphatases have a catalytic cysteine that is directly responsible for substrate dephosphorylation. They also have a catalytic arginine on the same loop as the cysteine residue (P loop), as well as conserved aspartic acid and glutamine residues on the WPD and Q loops, respectively. These residues make up the conserved catalytic core of classical PTPs (Figure 1A,B).[11,12] The WPD loop can adopt an open or closed conformation that is dependent on substrate or inhibitor binding (Figure 1A, left panel), and it has been suggested that this loop plays a regulatory role.[11–14] In some phosphatases, such as SHP2, auxiliary non-catalytic domains physically occlude the active site to regulate activity.[15–18] Furthermore, the catalytic cysteines of most PTPs are susceptible to reversible oxidation by reactive-oxygen species, and oxidation has been proposed as a dynamic regulatory mechanism.[19–21] Other uncharacterized regulatory mechanisms likely exist for PTPs, making these enzymes an opportune target class for activity-based profiling.[22] In this context, it is noteworthy that the catalytic cysteine in PTPs has a characteristically low pKa due to the active site environment,[23,24] which should make it a good target for covalent probes. There has been an exciting revolution in chemical biology to examine the proteome-wide reactivity and ligandability of cysteine residues using mass spectrometry proteomics. Keywords tyrosine phosphatase, covalent inhibition, cysteine labeling, enzymes, inhibitors, structure-activity relationships, protein structures Hong, Xi, et al., 2022 – Main Text & Figures - page 2 of 19 Introduction There have been several noteworthy efforts towards the development of activity-based covalent probes for PTPs over the past two decades, but these efforts have focused primarily on Hong, Xi, et al., 2022 – Main Text & Figures - page 3 of 19 phosphotyrosine isosteres (Figure 1C).[10,32] Early candidates included mechanism-based quinone methide generating probes[33–35] and α-bromobenzylphosphonates.[36] These molecules generally have poor membrane permeability, display nonspecific labeling, or are susceptible to solvolysis at physiological pH.[10] Aryl vinyl sulfones and sulfonates were presented as a promising class of molecules that alleviated previous shortcomings and posed potential for further development.[37] phosphotyrosine isosteres (Figure 1C).[10,32] Early candidates included mechanism-based quinone methide generating probes[33–35] and α-bromobenzylphosphonates.[36] These molecules generally have poor membrane permeability, display nonspecific labeling, or are susceptible to solvolysis at physiological pH.[10] Aryl vinyl sulfones and sulfonates were presented as a promising class of molecules that alleviated previous shortcomings and posed potential for further development.[37] In this study, we report that aryl vinyl sulfonates have exceedingly fast non-specific reactivity with cysteines at physiological pH, making them unsuitable for selective phosphatase labeling or inhibition in a cellular context. We present a broader exploration of chemical space beyond reactive phosphotyrosine isosteres for the covalent inhibition of tyrosine phosphatases. We assessed a variety of thiol-reactive groups and fragment scaffolds using biochemical assays on an array of purified PTPs. We combined this enzyme- targeted approach with compound reactivity assays and protein sequence/structure analyses to better understand how both ligand and protein structural features govern covalent PTP inhibition. Motivated by the lack of suitable covalent chemical tools to investigate PTPs, our studies identify lead compounds that will guide the future development of inhibitors and chemical probes for PTPs. Figure 1. Covalent labeling of protein tyrosine phosphatases. (A) Crystal structure of PTP1B bound to a substrate- mimetic inhibitor, highlighting key active site loops in red and catalytic residues in yellow (PDB code 1KAK). An alternative conformation of the WPD loop, derived from a loop-open structure (PDB code 2HNP), is shown in green on the left panel. (B) The conserved catalytic mechanism of classical protein tyrosine phosphatases (PTP1B residue numbering). (C) Previously reported activity-based probe classes for protein tyrosine phosphatases. Figure 1. Covalent labeling of protein tyrosine phosphatases. (A) Crystal structure of PTP1B bound to a substrate- mimetic inhibitor, highlighting key active site loops in red and catalytic residues in yellow (PDB code 1KAK). Introduction An alternative conformation of the WPD loop, derived from a loop-open structure (PDB code 2HNP), is shown in green on the left panel. (B) The conserved catalytic mechanism of classical protein tyrosine phosphatases (PTP1B residue numbering). (C) Previously reported activity-based probe classes for protein tyrosine phosphatases. Hong, Xi, et al., 2022 – Main Text & Figures - page 4 of 19 Results and Discussion Aryl vinyl sulfonates inhibit tyrosine phosphatases but are too reactive at physiological pH. Phenyl vinyl sulfone (PVS, 1) and phenyl vinylsulfonate (PVSN, 2) were previously reported as promising active site-directed covalent ligands for PTPs.[37] They were shown to be cell-permeable, owing to their relatively simple structures and lack of charged functional groups. An azide-tagged variant of PVSN was also reported as the starting point for activity-based profiling. We envisioned that a more elaborate scaffold structure, larger than a phenyl ring, might improve the potency of PVSN while retaining the potentially favorable features of the vinyl sulfonate moiety. Thus, we synthesized a series of aryl vinylsulfonates and assessed their ability to covalently label and inhibit tyrosine phosphatases using two complementary assays (Figure 2A,B). In the first assay, the residual activities of several tyrosine phosphatase catalytic domains were measured using the colorigenic substrate p-nitrophenyl phosphate (pNPP) after treatment with each covalent inhibitor.[38] In the second assay, we assessed the extent of labeling of the same PTPs by each vinyl numbering). (C) Previously reported activity based probe classes for protein tyrosine phosphatases. Introduction These studies have served as a starting point for identification of new ligandable protein targets and the development of covalent probes and drugs.[25,26] Cysteine reactivity across the proteome is generally assessed by measuring labeling efficiency with a highly reactive iodoacetamide probe.[27,28] Cysteine ligandability, on the other hand, is examined by treating the proteome with small-molecule covalent fragments bearing weaker electrophiles, like chloroacetamide or acrylamide, followed by treatment with an iodoacetamide probe. The loss of iodoacetamide labeling at specific cysteines reveals the selectivity profile of fragment electrophiles across the proteome.[29] Cysteine ligandability screens have the potential to simultaneously identify target proteins and lead compounds for covalent probe development. In general, these chemoproteomic efforts are not targeted – instead, they yield large datasets on the ligandability of thousands of cysteine residues. These datasets can be leveraged for the development of covalent inhibitors, probes, and degraders for a variety of protein targets, particularly for non-active site cysteine residues.[25] However, published proteome-wide ligandability screens have not yielded any substantial leads for PTP active site cysteine residues.[30] Rather, these screens suggest that PTP catalytic cysteines are not very reactive relative to the entire proteome, despite their low pKa values,[23,24] and they are apparently unliganded by most fragments used in existing screens. Consequently, it is difficult to extract information from these global ligandability screens on how different structures impact covalent inhibition of PTPs. In this study, we have taken a target-centric approach to identify structural leads for the development of covalent PTP inhibitors and probes. PTPs have historically been difficult to inhibit potently and selectively, due to their highly conserved and charged active site architectures.[31] Thus, we set out to understand the chemical parameters that govern covalent targeting of the conserved active-site cysteine across the classical PTP subfamily. onates inhibit tyrosine phosphatases but are too reactive at physiological pH Hong Xi et al 2022 Main Text & Figures page 4 of 19 Phenyl vinyl sulfone (PVS, 1) and phenyl vinylsulfonate (PVSN, 2) were previously reported as promising active site-directed covalent ligands for PTPs.[37] They were shown to be cell-permeable, owing to their relatively simple structures and lack of charged functional groups. An azide-tagged variant of PVSN was also reported as the starting point for activity-based profiling. We envisioned that a more elaborate scaffold structure, larger than a phenyl ring, might improve the potency of PVSN while retaining the potentially favorable features of the vinyl sulfonate moiety. Thus, we synthesized a series of aryl vinylsulfonates and assessed their ability to covalently label and inhibit tyrosine phosphatases using two complementary assays (Figure 2A,B). In the first assay, the residual activities of several tyrosine phosphatase catalytic domains were measured using the colorigenic substrate p-nitrophenyl phosphate (pNPP) after treatment with each covalent inhibitor.[38] In the second assay, we assessed the extent of labeling of the same PTPs by each vinyl sulfonate using intact protein mass spectrometry. sulfonate using intact protein mass spectrometry. As expected, aryl vinylsulfonates were able to inhibit several PTPs after treatment with 100 μM compound for one hour (Figure 2C). Phenyl vinyl sulfone (1) showed very little activity against every phosphatase except HePTP. In general, vinylsulfonates on bulkier scaffolds (compounds 3-6) showed increased potency. However, intact protein mass spectrometry revealed that these molecules were labeling other residues in addition to the catalytic cysteine, as demonstrated by the existence of protein species with more than one adduct (Figure 2D, top panel, Figure 2E, and Figure S1). Although the bulkier scaffolds were more potent inhibitors, they did not show enhanced selectivity for the active site, suggesting that the intrinsic more potent inhibitors, they did not show enhanced selectivity for the active site, suggesting that the intrinsic Figure 2. Characterization of aryl vinyl sulfonate inhibitors of tyrosine phosphatases. (A) Workflow for the analysis of PTP inhibition and covalent labeling. After treatment with a covalent ligand, phosphatases were characterized in two different assays. For enzyme activity assays, the protein sample was diluted and quenched with excess 1,4-dithiothreitol (DTT), then used to dephosphorylate pNPP. Alternatively, proteins were directly analyzed by intact mass spectrometry after ligand treatment. (B) Structures of the vinyl sulfone and vinylsulfonate compounds tested in this study. (C) Inhibition of various classical PTPs by vinyl sulfone (1) and vinylsulfonate (2-6) compounds. onates inhibit tyrosine phosphatases but are too reactive at physiological pH reactivity of the vinylsulfonate was obscuring any effects of scaffold structure on binding (Figure 2C,E). We note that off-target covalent labeling is more prevalent in our assays than in the original report of PVSN.[37] This can be attributed to the fact our assays were done at pH 7.5, whereas PVSN and PVS were previously characterized at pH 6. Labeling of PTP1B by compound 2 was greatly attenuated at pH 6 in our assay (Figure 2D, bottom panel). Lower pH allows for differentiation of the active site Cys residue from off-target Cys residues, due to its lower pKa,[23] but these conditions could limit labeling applications on intact cells. A survey of published reports showing electrophilic labeling of PTPs in cell lysates revealed that most experiments were conducted between pH 5.5 and 6.[36,39,40] These observations suggest that selective active site labeling of PTPs at physiological pH will require less reactive molecules with higher affinity for the active site. onates inhibit tyrosine phosphatases but are too reactive at physiological pH Enzymes were pre-treated with 100 μM compound for one hour prior to dilution, quenching, and measurement of enzyme activity. (D) Deconvoluted intact protein mass spectra of PTP1B after incubation with 100 μM vinyl sulfonate compound for one hour. (E) Quantification of multi-adduct formation for vinyl sulfone and sulfonate compounds by intact protein mass spectrometry after incubation with 100 μM compound for one hour. Figure 2. Characterization of aryl vinyl sulfonate inhibitors of tyrosine phosphatases. (A) Workflow for the analysis of PTP inhibition and covalent labeling. After treatment with a covalent ligand, phosphatases were characterized in two different assays. For enzyme activity assays, the protein sample was diluted and quenched with excess 1,4-dithiothreitol (DTT), then used to dephosphorylate pNPP. Alternatively, proteins were directly analyzed by intact mass spectrometry after ligand treatment. (B) Structures of the vinyl sulfone and vinylsulfonate compounds tested in this study. (C) Inhibition of various classical PTPs by vinyl sulfone (1) and vinylsulfonate (2-6) compounds. Enzymes were pre-treated with 100 μM compound for one hour prior to dilution, quenching, and measurement of enzyme activity. (D) Deconvoluted intact protein mass spectra of PTP1B after incubation with 100 μM vinyl sulfonate compound for one hour. (E) Quantification of multi-adduct formation for vinyl sulfone and sulfonate compounds by intact protein mass spectrometry after incubation with 100 μM compound for one hour. Hong, Xi, et al., 2022 – Main Text & Figures - page 5 of 19 reactivity of the vinylsulfonate was obscuring any effects of scaffold structure on binding (Figure 2C,E). We note that off-target covalent labeling is more prevalent in our assays than in the original report of PVSN.[37] This can be attributed to the fact our assays were done at pH 7.5, whereas PVSN and PVS were previously characterized at pH 6. Labeling of PTP1B by compound 2 was greatly attenuated at pH 6 in our assay (Figure 2D, bottom panel). Lower pH allows for differentiation of the active site Cys residue from off-target Cys residues, due to its lower pKa,[23] but these conditions could limit labeling applications on intact cells. A survey of published reports showing electrophilic labeling of PTPs in cell lysates revealed that most experiments were conducted between pH 5.5 and 6.[36,39,40] These observations suggest that selective active site labeling of PTPs at physiological pH will require less reactive molecules with higher affinity for the active site. Scaffold exploration reveals global trends in tyrosine phosphatase covalent inhibition. We next sought to explore the scope of scaffold chemical space that is suitable for covalent inhibition of classical tyrosine phosphatases. We selected chloroacetamide as our electrophilic warhead given its precedent in fragment electrophile screens and because the phenyl species showed sufficiently detectable phosphatase inhibition to build upon without the significant multi-site labeling seen for aryl vinyl sulfonates at physiological pH (Figure S2). We constructed a small library containing 65 chloroacetamide-functionalized fragments bearing diverse aryl and alkyl substituents (Figure 4A and Table S1). These molecules were tested in the same pNPP dephosphorylation assay described above against seven classical human tyrosine phosphatases. Inhibition was assessed over a range of concentrations and time points to obtain time- dependent IC50 values (Figure 4B, Figure S3, and Table S3,4). The chemically diverse scaffolds in our library are likely to impact both active site binding affinity, as well as intrinsic reactivity of the chloroacetamide warhead. To account for changes in intrinsic reactivity, we measured the rate at which each chloroacetamide reacted with TNB2-, a reduced form of Ellman’s reagent (5,5-dithio-bis-2-nitrobenzoic acid, DTNB) using a previously reported colorimetric assay.[45] The compounds in our library showed a wide range of reactivities, from compounds with no discernable reaction to rates around 1 M-1s-1 (Figure 4C and Table S5). Juxtaposition of enzyme inhibition data from the pNPP dephosphorylation assay with thiol reactivity from the DTNB assay demonstrated that there was no significant correlation between reactivity towards PTPs and intrinsic thiol reactivity (Figure 4D and Figure S4). Thus, we reasoned that we could identify covalent fragments that inhibit PTPs without having excessive intrinsic reactivity that could undermine the downstream development of a drug molecules or chemoproteomic probe. The chloroacetamide fragment survey revealed a series of informative general trends, both with respect to the enzymes and the scaffold structures. As seen in our warhead survey, we observed substantially different degrees of inhibition of each phosphatase that was roughly inversely correlated with intrinsic catalytic activity (Figure 4E). On average across the library, HePTP and SHP1 were inactivated at a faster rate than other phosphatases, and both CD45 and CD148 were hard to inhibit. Notably, we observed unexpected correlations between the inhibition profiles of different phosphatase pairs (Figure 4F). Thiol-reactive groups display a wide range of PTP inhibition. Substitution of ester linkages with amides resulted in lower reactivity for all cases (compare 2 to 10, 9 to 12, and 13 to 15), as anticipated.[23] Chloroacetamide (compound 16), which is commonly used in fragment electrophile screening libraries,[29] showed a small but detectable amount of phosphatase inhibition under these conditions (Figure 3B and Table S2). We reasoned that the chloroacetamide warhead would be a good starting point for fragment scaffold screening, and that the electrophile survey presented here could be used to guide late-stage tuning of compound reactivity. warheads (Figure 3B). Given the hyper-reactivity of vinylsulfonates observed at physiological pH, we conclude that electrophiles with similar reactivity may also be unsuitable for PTP inhibitor or probe design. Substitution of ester linkages with amides resulted in lower reactivity for all cases (compare 2 to 10, 9 to 12, and 13 to 15), as anticipated.[23] Chloroacetamide (compound 16), which is commonly used in fragment electrophile screening libraries,[29] showed a small but detectable amount of phosphatase inhibition under these conditions (Figure 3B and Table S2). We reasoned that the chloroacetamide warhead would be a good starting point for fragment scaffold screening, and that the electrophile survey presented here could be used to guide late-stage tuning of compound reactivity. We were surprised to observe drastically different degrees of covalent inhibition across the eight classical PTP catalytic domains included in our experiments, despite the use of a simple phenyl scaffold (Figure 3B). Whereas HePTP, SHP1, and TCPTP were generally inhibited the most by compounds in the phenyl-electrophile series, other PTPs such as CD45, CD148, and YopH were not readily inhibited. These data suggest that the catalytic cysteine residues of individual phosphatases either have different reactivity or accessibility. These phosphatases also showed significantly different intrinsic catalytic activities in the absence of any inhibitor, consistent with previous observations (Figure 3C).[13,41] The susceptibility to covalent inhibition showed a roughly inverse correlation with intrinsic catalytic activity (Figures 3B,C). The underlying molecular basis for this inverse correlation between covalent inhibition and catalytic activity is currently unclear. It could reflect the possibility that the rate-limiting step in the tyrosine phosphatase catalytic cycle is hydrolysis of the phospho-enzyme intermediate, as opposed to the initial nucleophilic attack by the catalytic cysteine residue, as suggested previously (Figure 1B).[42,43] Alternatively, the ligand on-rates may be too slow to efficiently access the active sites of the highly active phosphatases, which have very fast WPD-loop closure rates.[44] Thiol-reactive groups display a wide range of PTP inhibition. We assessed a broad range of thiol-reactive electrophilic “warheads” for their ability to target the active site cysteine of PTPs (Figure 3A and Table S1). To isolate the effect of the reactive group, we focused on compounds with a simple phenyl scaffold and assessed phosphatase inhibition using the pNPP dephosphorylation assay described above (Figure 2A). These experiments revealed a general rank-order for the reactivity of different warheads towards PTPs (Figure 3B and Table S2). As expected, there was a wide range of potency across different warheads. Vinylsulfonate (compound 2) was one of the most reactive Hong, Xi, et al., 2022 – Main Text & Figures - page 6 of 19 Figure 3. Profiling electrophiles for compatibility with tyrosine phosphatase inhibition. (A) Chemical structures of phenyl-derived electrophilic compounds used in this study. (B) Heat map showing inhibition of PTPs, as measured by pNPP dephosphorylation rates after one hour incubation with 100 µM compound. Rates after electrophile treatment are normalized to the uninhibited rate for each enzyme. (C) Intrinsic catalytic activity of different PTPs in the pNPP dephosphorylation assay after one hour incubation with DMSO. All phosphatases were assayed at 25 nM final enzyme concentration. 20 mM pNPP was used with all enzymes except CD148, for which 2 mM pNPP was used due to the exceedingly fast dephosphorylation rate. Figure 3. Profiling electrophiles for compatibility with tyrosine phosphatase inhibition. (A) Chemical structures of phenyl-derived electrophilic compounds used in this study. (B) Heat map showing inhibition of PTPs, as measured by pNPP dephosphorylation rates after one hour incubation with 100 µM compound. Rates after electrophile treatment are normalized to the uninhibited rate for each enzyme. (C) Intrinsic catalytic activity of different PTPs in the pNPP dephosphorylation assay after one hour incubation with DMSO. All phosphatases were assayed at 25 nM final enzyme concentration. 20 mM pNPP was used with all enzymes except CD148, for which 2 mM pNPP was used due to the exceedingly fast dephosphorylation rate. Hong, Xi, et al., 2022 – Main Text & Figures - page 6 of 19 Hong, Xi, et al., 2022 – Main Text & Figures - page 6 of 19 warheads (Figure 3B). Given the hyper-reactivity of vinylsulfonates observed at physiological pH, we conclude that electrophiles with similar reactivity may also be unsuitable for PTP inhibitor or probe design. Scaffold exploration reveals global trends in tyrosine phosphatase covalent inhibition. PTP1B and SHP2 displayed the highest correlation, which was surprising, given the close sequence and structural homology between PTP1B and TCPTP and between SHP2 and SHP1.[2,13] More nuanced enzyme-differentiating trends are discussed in the subsequent sections. Hong, Xi, et al., 2022 – Main Text & Figures - page 7 of 19 Figure 4. Exploring diverse chloroacetamide-based fragments for tyrosine phosphatase inhibition. (A) Structures of the core chemotypes found in our chloroacetamide library. For the thiazole category, there is one 4,5- dihydrothiazole. (B) Schematic diagram of the inhibition assay (top) and distributions of HePTP inhibition values for molecules bearing different core chemotypes (bottom). (C) Schematic diagram of the DTNB reactivity assay (top) and distributions of reaction rates with the reduced form of DTNB for molecules bearing different core chemotypes (bottom). (D) Scatterplot juxtaposing representative IC50 values for HePTP at one inhibition time point with intrinsic reactivity data obtained using the DTNB assay. (E) Distribution of IC50 values across the library for each tyrosine phosphatase after one hour of compound treatment. Phosphatases are ranked as shown in Figure 3C. Compounds denoted “poor fit” had data that could not be fit well to a dose-response curve, due to low degrees of inhibition. (F) Pearson’s correlation coefficients for inhibition datasets of each pair of phosphatases tested against the chloroacetamide library. C ff C f f Figure 4. Exploring diverse chloroacetamide-based fragments for tyrosine phosphatase inhibition. (A) Structures of the core chemotypes found in our chloroacetamide library. For the thiazole category, there is one 4,5- dihydrothiazole. (B) Schematic diagram of the inhibition assay (top) and distributions of HePTP inhibition values for molecules bearing different core chemotypes (bottom). (C) Schematic diagram of the DTNB reactivity assay (top) and distributions of reaction rates with the reduced form of DTNB for molecules bearing different core chemotypes (bottom). (D) Scatterplot juxtaposing representative IC50 values for HePTP at one inhibition time point with intrinsic reactivity data obtained using the DTNB assay. (E) Distribution of IC50 values across the library for each tyrosine phosphatase after one hour of compound treatment. Phosphatases are ranked as shown in Figure 3C. Compounds denoted “poor fit” had data that could not be fit well to a dose-response curve, due to low degrees of inhibition. (F) Pearson’s correlation coefficients for inhibition datasets of each pair of phosphatases tested against the chloroacetamide library. Correlation coefficients were calculated using IC50 values after one hour of compound treatment. Scaffold exploration reveals constraints on fragment structure for tyrosine Most efforts to covalently inhibit tyrosine phosphatases have focused on phenyl-based scaffolds, building on the structure of the endogenous substrate phosphotyrosine.[32] As noted above, our assessment of chloroacetamides suggests that some heterocyclic scaffolds may be preferable over a phenyl scaffold (Figure 4A-C). A comparison of phenyl, benzyl, phenylethyl, and cyclohexyl chloroacetamides showed that an aromatic ring is beneficial near the warhead, mostly likely both for active site binding as well as chloroacetamide activation through inductive effects (Figure 5A). Our data also revealed that the classical PTP active site cannot tolerate steric bulk close to the chloroacetamide warhead. Substitution of the amide nitrogen with large substituents diminished activity in a variety of contexts (Figure 5B and Figure S6A), as did introduction of large functional groups at the ortho-position relative to the chloroacetamide (Figure S6B). For example, we examined a few tertiary alkyl amides that have been used in protein-wide cysteine ligandability studies (compounds 31 and 32) and found that they were not as potent as structurally similar secondary aryl amides, despite their high intrinsic reactivity (Figure 5B).[28,29,48] Recent work in this realm has focused on the development of fragment libraries with even more sterically crowded electrophiles to yield lead fragments with proteome-level selectivity.[49] Our data suggest that the compounds in these libraries may not be optimal for targeting the active sites of tyrosine phosphatases. We were able to extract other useful structure-activity relationships in relation to previously reported non-covalent inhibitors. For example, in the context of a phenyl scaffold, we found that a carboxy substitution at the 2-position reduced intrinsic reactivity, which could be useful for probe design. However, this substitution did not improve potency (Figure 5C). Previous reports have shown that 2-carboxy substitutions are favorable in unnatural phosphotyrosine-like substrates[50] and non-covalent inhibitors,[51,52] due to an intimate electrostatic interaction with a conserved lysine residue in PTPs. In the context of covalent inhibition, the significant decrease in chloroacetamide reactivity caused by the 2-carboxy substituent likely negates beneficial binding effects of this appendage. By contrast, a 2-hydroxy substitution both modestly reduced reactivity and enhanced potency (Figure 5C). Scaffold exploration reveals global trends in tyrosine phosphatase covalent inhibition. Hong, Xi, et al., 2022 – Main Text & Figures - page 8 of 19 In terms of overall fragment structure, our data revealed that aliphatic chloroacetamides were generally poor inhibitors of all PTPs tested, irrespective of their intrinsic reactivity (Figure 4A-C). Of the aryl chloroacetamides, thiazole-containing compounds, including those with relatively average intrinsic reactivity, showed significant phosphatase inhibition when compared to most other chemotypes (Figure 4A-D and Figure S4). This is particularly noteworthy, given recent reports of thiazole-derived sulfophenyl acetic amides as non-covalent inhibitors of the low molecular weight protein tyrosine phosphatase (LMW-PTP).[46,47] Although the architecture of the LMW-PTP active site is distinct from that of classical PTPs, there are some structurally conserved features, including a catalytic loop and pTyr loop. We compared a crystal structure of LMW-PTP bound to a benzothiazole-containing non-covalent inhibitor[47] with a covalent docking model of PTP1B bound to a benzothiazole-derived chloroacetamide (compound 74). Both proteins engage the benzothiazole in a similar orientation, suggesting that it may be a preferred scaffold for diverse tyrosine phosphatases (Figure S5). Scaffold exploration reveals constraints on fragment structure for tyrosine Non-covalent inhibitors of TCPTP and PTP1B have recently entered the clinical pipeline, and these molecules have a 2-hydroxy substitution on an aryl ring, adjacent to the phospho-mimetic “head group” that lies in the same position as the electrophile in our molecules (Figure 5C).[53,54] Our fragment library contains several thiazole-derived molecules that also reveal informative structure- activity relationships (Figure 5D). Compound 65, comprised of a simple thiazole scaffold, shows moderate potency towards PTPs relative to our entire library. However, amongst thiazoles, it was one of the weakest inhibitors of PTPs and showed high intrinsic thiol reactivity. Modifications to this base scaffold generally imparted increased potency against PTPs and in most cases reduced intrinsic thiol reactivity. Benzothiazole (compound 74), was both more reactive and more potent than the unsubstituted thiazole (compound 65). However, further substitution off the benzothiazole decreased intrinsic reactivity, in some cases with mild or even favorable impact on potency against PTPs (Figure S7). As with phenyl-based compounds, aromatic substitutions on the thiazole resulted in increased potency (Figure 5D). Altering the substituent on the coupled aromatic ring further modulated PTP inhibition and thiol reactivity, as seen with compounds 71 and 73. These distal substituents also had an impact on phosphatase selectivity, as exemplified by the biased inhibition of HePTP, and to a lesser extent SHP1, by compound 71, the 4-hydroxy derivative (Figure 5D). Hong, Xi, et al., 2022 – Main Text & Figures - page 9 of 19 Hong, Xi, et al., 2022 – Main Text & Figures - page 10 of 19 Figure 5. Structure-activity relationships for covalent tyrosine phosphatase inhibition. (A) Inhibition of PTPs by phenyl, cyclohexyl, benzyl, and phenylethyl chloroacetamide. (B) The effect of chloroacetamide-proximal steric bulk on phosphatase inhibition. (C) The effects of 2-carboxy and 2-hydroxy substituents on covalent inhibition. Known phosphatase inhibitors with the same functional groups are shown. (D) Potency and reactivity effects of substituents on a thiazole scaffold. (E) Biased inhibition of SHP1 by isoxazole chloroacetamides. Figure 5. Structure-activity relationships for covalent tyrosine phosphatase inhibition. (A) Inhibition of PTPs by phenyl, cyclohexyl, benzyl, and phenylethyl chloroacetamide. (B) The effect of chloroacetamide-proximal steric bulk on phosphatase inhibition. (C) The effects of 2-carboxy and 2-hydroxy substituents on covalent inhibition. Known phosphatase inhibitors with the same functional groups are shown. (D) Potency and reactivity effects of substituents on a thiazole scaffold. (E) Biased inhibition of SHP1 by isoxazole chloroacetamides. Figure 5. Hong, Xi, et al., 2022 – Main Text & Figures - page 10 of 19 sphatase covalent inhibition are dictated by residues beyond the active site Our experiments have revealed significant differences in susceptibility to covalent inhibition between tyrosine phosphatases, as well as more granular effects of ligand structure on potency and selectivity. We sought to identify divergent structural features of these enzymes that might explain their differences in covalent inhibition. We first used covalent docking to predict plausible binding poses for a few select compounds against four phosphatases: PTP1B, HePTP, SHP1, and SHP2 (Figure S10). Based on these models, we identified residues that made direct contact with the molecules and examined conservation across the panel of phosphatases used in this study. Most first-shell residues were conserved, ruling out these residues as differentiating factors in our analysis (Figure 6A). As noted above, differences in overall covalent inhibition across the tested phosphatases showed an approximately inverse correlation with the catalytic activity of those phosphatases. Thus, we hypothesize that divergent structural features which affect catalytic activity may also affect covalent inhibition. The WPD loop of phosphatases is the active site feature that has been most directly connected to differences in catalytic activity between phosphatases.[43,57–61] Sequence divergence in this region can lead to differences loop dynamics, which in turn impacts catalysis. Two proline residues found in PTP1B (Pro 185 and Pro 188), but not YopH, are reported to constrain the dynamics of the PTP1B WPD loop, potentially leading to its lower enzyme activity.[59] While Pro 185 is invariant in the human PTPs, Pro 188 is replaced by threonine in CD148, and alanine in HePTP, the least and most inhibitable human phosphatases examined in our study, respectively (Figure 6A). Previous studies have shown that the P188A mutation in PTP1B alters WPD loop structure and dynamics and also impacts the rates of multiple steps in the phosphatase catalytic cycle.[59] Based on these observations, we expect that the identity of the P188-analogous residue in other phosphatases is likely to also impact their rates of covalent inhibition. Our sequence and structure analysis revealed several additional residues that are not conserved across the phosphatases and may impact covalent inhibition. Arg 47 and Asp 48 in PTP1B lie on the pTyr loop, alongside Tyr 46, which forms the conserved hydrophobic floor of the active site (Figure 6C). These two residues diverge across the phosphatases tested and are likely to impact selectivity for the multi-ring molecules in our library (Figure 6A). Scaffold exploration reveals constraints on fragment structure for tyrosine Structure-activity relationships for covalent tyrosine phosphatase inhibition. (A) Inhibition of PTPs by phenyl, cyclohexyl, benzyl, and phenylethyl chloroacetamide. (B) The effect of chloroacetamide-proximal steric bulk on phosphatase inhibition. (C) The effects of 2-carboxy and 2-hydroxy substituents on covalent inhibition. Known phosphatase inhibitors with the same functional groups are shown. (D) Potency and reactivity effects of substituents on a thiazole scaffold. (E) Biased inhibition of SHP1 by isoxazole chloroacetamides. Hong, Xi, et al., 2022 – Main Text & Figures - page 10 of 19 As with thiazole-based compounds, isoxazole-based compounds also offered improvements over phenyl-based compounds. In general, modifications to this base scaffold similarly improved potency against PTPs, particularly with aromatic substitutions from the isoxazole ring. Notably, SHP1 showed a distinct preference for isoxazole-based compounds over analogous thiazole-based compounds when compared to other PTPs (Figure 5E and Figure S8). For example, benzothiazole 74 is preferred over benzisoxazole 39 for every PTP except for SHP1. Moreover, while compounds 37 and 73 perform essentially identically for most other PTPs, compound 37, the isoxazole, is a much more potent inhibitor of SHP1, and compound 36 is the most potent inhibitor of SHP1 (Figure 5E). During these assays, compound 66, a tetralin-substituted benzothiazole, showed visible aggregation at high concentrations, raising concern that this compound may be partially inhibiting the enzymes by inducing aggregation. To investigate the risk of aggregation, we assessed some compounds with and without the addition of Triton X-100, a nonionic surfactant which has been previously used in enzymatic assays to prevent colloidal aggregation.[55,56] When assayed with Triton X-100, compound 66 showed a noticeable reduction in inhibition, though it still showed potent inhibition of PTPs, suggesting that aggregation is partially contributing to the inhibition with this compound. By comparison, compound 74 showed no visible aggregation and likewise retained inhibitory activity towards PTPs in the presence of Triton X-100 (Figure S9). These results indicate that future screening and optimization of this class of inhibitors should include a critical evaluation of misleading aggregate-based inhibition. sphatase covalent inhibition are dictated by residues beyond the active site For example, we observed a distinctive improvement in potency against HePTP for compound 71 (Figure 5D). This molecule has a hydroxy group that is well-positioned to engage the unique Thr 106 on the HePTP pTyr loop (Asp 48 in PTP1B). A previous investigation exploited an electrostatic interaction with Asp 48 to design selective non-covalent inhibitors of PTP1B, as this charged residue is not present in all classical tyrosine phosphatases.[52] We also identified two residues with modest conservation (Leu 110 and Asn 111) that buttress the catalytic cysteine (Cys 215 in PTP1B) (Figure 6A,D). A recent investigation showed that the dynamics of Leu 110 in PTP1B play a role in enzyme catalysis.[62] Hong, Xi, et al., 2022 – Main Text & Figures - page 11 of 19 SHP1 and SHP2 both have a unique Thr residue in place of Leu 110, and this Thr residue hydrogen-bonds to the backbone carbonyl preceding the catalytic cysteine in those enzymes. Thus, although Leu 110 and Asn 111 do not make direct contact with substrates or covalent ligands, we hypothesize that changes at these positions will impact the orientation, dynamics, and reactivity of the catalytic cysteine. SHP1 and SHP2 both have a unique Thr residue in place of Leu 110, and this Thr residue hydrogen-bonds to the backbone carbonyl preceding the catalytic cysteine in those enzymes. Thus, although Leu 110 and Asn 111 do not make direct contact with substrates or covalent ligands, we hypothesize that changes at these positions will impact the orientation, dynamics, and reactivity of the catalytic cysteine. Perhaps the most surprising difference between phosphatases observed in our screens was the divergence in ligand engagement by SHP1 and SHP2. The catalytic domains of these enzymes have 59% sequence identity, which increases to 88% identity within 10 Å of the active site, and both proteins share a common domain architecture and regulatory mechanism.[16,17,63,64] Despite this high degree of structural homology, SHP1 is more readily inhibited by aryl chloroacetamides than SHP2, and it has a unique preference for isoxazole-containing molecules (Figure 5E). Based on docking models and sequence alignments, there are no ligand-proximal residues that differentiate SHP1 and SHP2. The only potentially significant active site difference we identified between the two enzymes is the first residue of the WPD loop (Figure 6A,B, Thr 177 in PTP1B, Leu 415 in SHP1, and Arg 421 in SHP2). Conclusions A handful of protein tyrosine phosphatases are well-characterized and have been identified as potential therapeutic targets for the treatment of cancer, metabolic diseases, and immune disorders. Nevertheless, the biological roles and regulatory mechanisms of most members of this enzyme family remain poorly characterized. Given their promise as therapeutic targets and the need for more phosphatase-focused discovery biology, there have been extensive efforts to design new chemical tools for tyrosine phosphatases. These can be broadly classified into: (1) non-covalent orthosteric and allosteric inhibitors that serve as both chemical probes and lead compounds for drug discovery,[31,65] (2) fluorescent substrates that report on the activity of tyrosine phosphatases in cells,[66,67] and (3) covalent probes to profile tyrosine phosphatase activity and regulation in the proteome.[10] On the one hand, the design of drug molecules and enzyme-specific reporters requires selectivity for individual phosphatases. On the other hand, probes for activity-based profiling of tyrosine phosphatases would ideally be family-wide, and the identification of labeled phosphatases would be enabled by mass spectrometry proteomics. Tackling either of these goals requires a comprehensive view of the small molecule chemotypes that are compatible with tyrosine phosphatases, as well as a deep understanding of the features of tyrosine phosphatases that govern their selective ligandability. Inspired by a recent resurgence in covalent inhibitors for challenging therapeutic targets, we set out to explore the chemical parameters required for active site covalent inhibition of phosphatases. While covalent ligands for tyrosine phosphatases have been previously identified, most studies have focused on phosphotyrosine isosteres and a handful of reactive warheads.[32] These past efforts have identified promising molecules but have not yielded significant drug leads or probes that are compatible with mass spectrometry proteomics. As an alternative approach, we explored a broad range of electrophiles and compound structures that deviate from previously characterized chemotypes. Our focused screening yielded a series of guiding principles for future tyrosine phosphatase inhibitor and probe design: (1) we have determined which of the common electrophilic warheads are compatible with tyrosine phosphatase inhibition and identified chloroacetamide as useful starting point for ligand design, (2) our study points to thiazoles and isoxazoles as unique core scaffolds for tyrosine phosphatase inhibitors, and (3) our data show that aliphatic scaffolds and bulky tertiary amide-derived electrophiles have uniquely low potency against all phosphatases that we tested. sphatase covalent inhibition are dictated by residues beyond the active site A previous study showed that the T177A mutation in PTP1B stabilizes a closed state of the WPD loop but only marginally impacts catalytic activity.[59] Mutations to the bulkier Leu or Arg found in SHP1 and SHP2 have not been examined, but we anticipate that they will have a substantial impact on loop dynamics and covalent inhibition. Figure 6. Structural features of tyrosine phosphatases that may govern covalent inhibition. (A) Sequence alignment of the eight tyrosine phosphatases used in this study, highlighting conservation in the loops that line the active site. Residues that directly contact active site ligands are indicated with a black asterisk below the alignment. Six positions with noteworthy sequence divergence, as discussed in the main text, are indicated with a red arrow below the alignment. (B) Potential reactivity- and specificity-defining residues on the WPD loop, mapped onto a model of PTP1B bound to compound 74. (C) Residues on the pTyr loop that can engage the side of covalent fragments that is distal to the electrophile, highlighted on a model of PTP1B bound to compound 73. (D) A modestly conserved buttress for the catalytic cysteine residue on the E loop of PTPs, highlighted on a model of PTP1B bound to compound 74. Figure 6. Structural features of tyrosine phosphatases that may govern covalent inhibition. (A) Sequence alignment of the eight tyrosine phosphatases used in this study, highlighting conservation in the loops that line the active site. Residues that directly contact active site ligands are indicated with a black asterisk below the alignment. Six positions with noteworthy sequence divergence, as discussed in the main text, are indicated with a red arrow below the alignment. (B) Potential reactivity- and specificity-defining residues on the WPD loop, mapped onto a model of PTP1B bound to compound 74. (C) Residues on the pTyr loop that can engage the side of covalent fragments that is distal to the electrophile, highlighted on a model of PTP1B bound to compound 73. (D) A modestly conserved buttress for the catalytic cysteine residue on the E loop of PTPs, highlighted on a model of PTP1B bound to compound 74. Hong, Xi, et al., 2022 – Main Text & Figures - page 12 of 19 Conclusions A unique feature of our study is the parallel characterization of covalent inhibition for several members of the classical protein tyrosine phosphatase family. This comparative approach revealed unexpected trends in reactivity and selectivity across different phosphatases, even for a relatively small compound library comprised entirely of fragment-like molecules. Most notably, we observed that tyrosine phosphatases with high catalytic activity are difficult to inhibit covalently, and we found that the closely related enzymes SHP1 and SHP2 have distinct fragment preferences. Our study provides new insights into sequence-structure- activity relationships in PTPs by analyzing these enzymes through the lens of covalent inhibition. Altogether, we anticipate that our findings will guide future ligand design efforts and will fuel new biophysical and biochemical investigations into tyrosine phosphatase activity and regulation. Intact protein mass spectrometry Each enzyme was diluted in pH 7.5 Tris buffer to achieve a final concentration 500 nM after addition of compound. Enzymes were pretreated by adding 2 μL of compound stock to 198 μL of enzyme solution. Unless otherwise specified, the final concentration of each compound was 100 μM. After one hour, the mixture was injected onto a BEH C8 column (Waters) on a UPLC-MS system (Xevo QToF, Waters). Reverse-phase liquid chromatography was carried out with gradient of 5% to 95% of acetonitrile (with 0.02% formic acid) for 8.5 min. The protein typically eluted around 4 min; this peak on the chromatogram was integrated and deconvoluted using the MaxEnt1 algorithm. Subsequently, peaks were chosen according to the theoretical MW of each adduct, within a range of 5 Da, and integrated for the signal intensity ± 1 Da. The abundance of each detectable enzyme species was normalized to the total intensity of all enzyme species. Expression and purification of tyrosine phosphatases For YopH, which was tagless, the protein was purified by cation exchange of the soluble fraction of the cell lysate over a 5 mL HiTrap SP column (Cytiva), followed by size-exclusion chromatography as described for the other phosphatases. Ni-NTA column (Cytiva). The resin was washed with lysis buffer and wash buffer (50 mM Tris, pH 8.5, 50 mM NaCl, 10 mM imidazole, 2 mM BME, 10 % glycerol). The protein was eluted with pH 8.5 buffer (50 mM Tris, 50 mM NaCl, 500 mM imidazole, 2 mM BME, and 10% glycerol) and passed through a 5 mL HiTrap Q anion exchange column. The His6-tag of the collected fractions was cleaved by incubating with TEV protease or Ulp1 (SUMO protease) overnight. The reaction mixture was subsequently flowed through 2 mL of Ni-NTA resin (ThermoFisher). The cleaved protein was collected in the flow-through and washes. Proteins were further purified using a Superdex 75 or 200 16/600 gel filtration column (Cytiva) in 10 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM TCEP, 10 % glycerol. Pure fractions were pooled then concentrated with centrifugal filters (Millipore). The resulting solution was aliquoted, and flash frozen in liquid N2 for long-term storage at - 80 °C. For YopH, which was tagless, the protein was purified by cation exchange of the soluble fraction of the cell lysate over a 5 mL HiTrap SP column (Cytiva), followed by size-exclusion chromatography as described for the other phosphatases. Chemical synthesis of electrophilic compounds Detailed experimental procedures for the synthesis of compounds is provided in the Supporting Information, along with NMR and MS characterization data. Expression and purification of tyrosine phosphatases Constructs bearing the catalytic domains of PTP1B, SHP1, SHP2, HePTP, TCPTP, and CD148 were prepared by over-expression in E. coli (see Supporting Information for protein sequences). All of these constructs had an N-terminal His6-tag followed by a TEV protease cleavage site. For CD45, our construct contained the tandem phosphatase and pseudophoshatase domains and an N-terminal His6-SUMO tag, and for YopH, a tagless version of the full-length protein was expressed. BL21(DE3) cells transformed with a phosphatase-encoding plasmid were grown at 37 °C in 2 L of terrific broth (TB), supplemented with the appropriate antibiotic. After cells reached an optical density at 600 nm of 0.5, isopropyl-β-D-1- thiogalactopyranoside (IPTG) was added for a final concentration of 0.5 mM to induce the expression of proteins, and the cultures were incubated at 18 °C overnight. Cells were harvested by centrifugation (7800 rcf at 4 °C for 30 min), resuspended in lysis buffer (pH 8, 50 mM Tris, 300 mM NaCl, 20 mM imidazole, 2 mM 2-mercaptoethanol (BME), 10 % glycerol), and lysed by sonication on ice. After separation of insoluble material by centrifugation (33,000 rcf at 4 °C for 45 minutes), the supernatant was applied to a 5 mL HisTrap Hong, Xi, et al., 2022 – Main Text & Figures - page 13 of 19 Ni-NTA column (Cytiva). The resin was washed with lysis buffer and wash buffer (50 mM Tris, pH 8.5, 50 mM NaCl, 10 mM imidazole, 2 mM BME, 10 % glycerol). The protein was eluted with pH 8.5 buffer (50 mM Tris, 50 mM NaCl, 500 mM imidazole, 2 mM BME, and 10% glycerol) and passed through a 5 mL HiTrap Q anion exchange column. The His6-tag of the collected fractions was cleaved by incubating with TEV protease or Ulp1 (SUMO protease) overnight. The reaction mixture was subsequently flowed through 2 mL of Ni-NTA resin (ThermoFisher). The cleaved protein was collected in the flow-through and washes. Proteins were further purified using a Superdex 75 or 200 16/600 gel filtration column (Cytiva) in 10 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM TCEP, 10 % glycerol. Pure fractions were pooled then concentrated with centrifugal filters (Millipore). The resulting solution was aliquoted, and flash frozen in liquid N2 for long-term storage at - 80 °C. Analysis of compound intrinsic reactivity using a colorimetric assay Thiol reactivity assays were carried out based on a previously reported assay by Resnick et al.[45] All assays were performed in buffer containing 20 mM sodium phosphates and 150 mM NaCl at pH 7.4. All measurements were taken at 37 ºC. 50 µM DTNB and 200 µM TCEP were incubated in sodium phosphate buffer for 5 min at 37 ºC to obtain TNB2-. Separately, 200 µM TCEP was incubated without DTNB in sodium phosphate buffer for 5 min at 37 ºC. For each compound, TNB2- solution (73.5 µL) was treated with 200 µM compound (1.5µL of 10mM stock in DMSO) in triplicate in a 384-well plate. Additionally, TCEP solution without TNB2- was treated with 200µM compound in triplicate to control for background absorbance. A DMSO group was included to serve as a negative control. UV absorbance measurements were acquired every 7.5 min for 7 h at 412 nm on a microplate reader. For each compound, the background absorbance without TNB2- was subtracted from the absorbance with TNB2-. The initial inhibitor concentration, [I]0, and TNB2- concentration, [TNB2-]0, were 200 µM and 100 µM, respectively. The remaining concentrations [I] and [TNB2-] were determined as a function of time from absorbance data using absorbance of the DMSO group as a reference for 100 µM TNB2-. Subtracted reaction progress curves were directly fit to the second-order rate equation below, using GraphPad Prism, to extract a second-order rate constant.[68,69] [TNB!"] = [TNB!"]#([I]# −[TNB!"]#) [I]#𝑒([&]!"[()"#]!),- −[TNB!"]# [TNB!"] = [TNB!"]#([I]# −[TNB!"]#) [I]#𝑒([&]!"[()"#]!),- −[TNB!"]# In the original report for this assay, data were fit by linear least squares to obtain the reaction rate constant, where k([I]0-[TNB2-]0) is the slope of ln([I][ TNB2-]0/[ TNB2-][I]0) plot versus time.[45] We also analyzed our data using this method, and the rate constants from both analyses were in good agreement (Table S5). In the original report for this assay, data were fit by linear least squares to obtain the reaction rate constant, where k([I]0-[TNB2-]0) is the slope of ln([I][ TNB2-]0/[ TNB2-][I]0) plot versus time.[45] We also analyzed our data using this method, and the rate constants from both analyses were in good agreement (Table S5). Covalent docking of compounds to tyrosine phosphatase All docking was performed in Maestro by Schrödinger[70] according to the “Covalent Docking for Virtual Screening and Pose Prediction” and “Introduction to Structure Preparation and Visualization” tutorials published by Schrödinger.[71] Briefly, each compound was prepared using LigPrep to generate possible tautomers, conformations, and ionization states between pH 5-9.[72] Structures for PTP1B (1KAK), SHP1 (4HJQ), SHP2 (6CMQ), and HePTP (3D42) were downloaded from the Protein Data Bank.[73] These structures were prepared using the Protein Preparation Wizard[74] to optimize charge, add hydrogens, and remove water molecules. All structures were aligned to PTP1B (1KAK), and the receptor grid box for docking calculations was centered on the co-crystalized ligand in 1KAK. Covalently docked complexes were generated using the CovDock module in the Pose Prediction docking mode, and the top scoring complex for each compound was selected as a representative pose for further structural analysis.[72] Analysis of phosphatase activity using a colorimetric assay Phosphatase catalytic activity was measured by monitoring dephosphorylation of p-Nitrophenyl phosphate (pNPP). All assays were performed in Tris buffer containing 50 mM Tris adjusted to an ionic strength of 150 mM with NaCl at pH 7.5. All measurements were taken at 30 ºC. Each enzyme was diluted to 250 nM in Tris buffer. Enzymes were pretreated with compound: for each enzyme, an aliquot (2.2 µL) of each compound in DMSO stock was dispensed into a well of a 96-well plate. A DMSO group was included to serve as an uninhibited control. Subsequently, enzyme solution (217.8 µL, final DMSO concentration: 1% v/v) was dispensed and mixed in each well. Similarly, a well was prepared with DMSO and Tris buffer without enzyme to serve as a substrate-only control. At 1, 2, and 4 hour time points, the pretreated enzyme solutions were aliquot (20 µL) in triplicate to a 96-well plate, and pNPP solution (180 µL) was added to each well. pNPP solution was prepared fresh with 100 mM DTT, and 0.1% (w/v) BSA in Tris buffer for a final pNPP concentration of 20mM during measurement. For CD148 only, this solution was diluted 10x for a final pNPP concentration of 2 mM. Absorbance measurements at 405nm were acquired every 30 seconds for 10 min in a microplate reader. The data were fit to a linear regression to obtain an initial rate from the slope, and the slope of the substrate-only control was subtracted from each group. The residual activity of each enzyme after incubation with compound was calculated from the slope of the compound-treated group as a percentage of the slope of the uninhibited group. pNPP dephosphorylation assays were conducted against each enzyme for each compound across 6 concentrations (3.3 µM, 10 µM, 33 µM, 100 µM, 330 µM, 1 mM). The IC50 value for the inhibition of phosphatase activity was determined by fitting the plot of residual enzyme activity against the inhibitor concentrations using a four-parameter dose-response nonlinear regression in GraphPad Prism.[68] Hong, Xi, et al., 2022 – Main Text & Figures - page 14 of 19 Acknowledgements We thank members of the Shah and Jovanovic labs for their guidance throughout this project; Fereshteh Zandkarimi and Brandon Fowler from the Columbia Chemistry mass spectrometry facility for their assistance with mass spectrometry; and Daniel Keedy for critically reading the manuscript. Bacterial expression vectors for HePTP and SHP2 were gifts from Nicola Burgess-Brown (Addgene plasmid #s 38945 and 38965). Plasmids encoding PTP1B, TCPTP, and SHP1 were gifts from Pau Creixell. Plasmids encoding CD45 and CD148 were gifts from Arthur Weiss. This work was funded in part by NIH grant R35 GM128014 awarded to NHS, and NIH grant R35 GM128802 awarded to MJ. Experiments involving cryoprobe NMR data were supported by funding from the NIH Office of the Director (award # S10OD026749). SYX is supported by a Barry Goldwater Scholarship. LCT is supported by an NSF Graduate Research Fellowship (award # 2036197). Conflict of Interest The authors declare no conflict of interest. References [1] A. Alonso, J. Sasin, N. Bottini, I. Friedberg, I. Friedberg, A. Osterman, A. Godzik, T. Hunter, J. Dixon, T. Mustelin, Cell 2004, 117, 699–711. M. J. Chen, J. E. Dixon, G. Manning, Sci. Signal. 2017, 10, DOI 10.1126/scisigna [2] M. J. Chen, J. E. Dixon, G. Manning, Sci. 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https://openalex.org/W4256071388	https://www.qeios.com/read/K50J2Y/pdf	English	null	Synpolydactyly	Definitions	2,020	cc-by	72	Qeios · Definition, February 7, 2020 Open Peer Review on Qeios Open Peer Review on Qeios Synpolydactyly National Cancer Institute National Cancer Institute Qeios ID: K50J2Y · https://doi.org/10.32388/K50J2Y Source National Cancer Institute. Synpolydactyly. NCI Thesaurus. Code C75003. A rare genetic disorder characterized by malformations in the hands and feet. The abnormalities include increased number of fingers and toes and fusion of digits into one large digit. Qeios ID: K50J2Y · https://doi.org/10.32388/K50J2Y 1/1
https://openalex.org/W4319151211	https://fjs.fudutsinma.edu.ng/index.php/fjs/article/download/1066/803	English	null	COMPARATIVE ASSESSMENT OF PARTICULATE MATTER USING LOW COST SENSOR: A CASE STUDY OF ABUJA AND KANO, NIGERIA	Fudma Journal of Sciences	2,022	cc-by	7,828	ABSTRACT Inhaling excessive amounts of Particulate Matter (PM) which can be blown over great distances by the wind and then settle in the ground, water, or in the air we breathe, can be hazardous to both sensitive and non- sensitive persons. The study investigates the mass concentration of particulate matter (PM) in Kano and Abuja. Utilizing a purple air sensor, PM1.0, PM2.5, and PM10.0 were all examined along with some climatic variables including temperature and relative humidity. In Kano and Abuja, monitoring took place between January 2021 and December 2021. Results indicate that the monthly PM exceeds the WHO 24-hour limit for the two locations. When the standard limit of the Air Quality Index (AQI) is taken into consideration, the mean value of PM2.5 shows that the air quality in both locations is dangerous for sensitive persons, such as those who have respiratory ailments while the mean value of PM10.0 shows that the air quality in both locations was moderate for both sensitive and non- sensitive person. The results in this study suggests that government should enhance its current air quality regulations and install new air quality sensors in sufficient locations in Nigeria so that additional research may be done on such regions. The results of a Pearson correlation analysis show that PMs and relative humidity have substantial negative correlations indicating that as relative humidity rises in either locations, PMs mass concentration would decrease as well. A relatively high correlation existed between PMs and temperature for both locations. Keywords: Air Quality Index (AQI), Correlation coefficient, Kurtosis, Particulate Matter (PM), Purple Air, Skewness soil debris, and airborne particles (EPA, 2020). The concentration of PM differs according to but not limited to the following factors wind speed, precipitation, weather conditions and relative humidity (Ghim, 2001). It appears in a variety of sizes and forms which are made up of different chemicals (US-EPA, 2017). F PARTICUL… Meseke et al., 8 FUDMA Journal of Sciences (FJS) ISSN online: 2616-1370 ISSN print: 2645 - 2944 Vol. 6 No. 4, August, 2022, pp 203 - 211 DOI: https://doi.org/10.33003/fjs-2022-0604-1 F PARTICUL… Meseke et al., 8 FUDMA Journal of Sciences (FJS) ISSN online: 2616-1370 ISSN print: 2645 - 2944 Vol. 6 No. 4, August, 2022, pp 203 - 211 DOI: https://doi.org/10.33003/fjs-2022-0604-1 PARTICUL… Meseke et al., 8 FUDMA Journal of Sciences (FJS) ISSN online: 2616-1370 ISSN print: 2645 - 2944 Vol. 6 No. 4, August, 2022, pp 203 - 211 DOI h //d i /10 33003/fj 2022 0604 Corresponding authors’ email: naomiomeseke@gmail.com Corresponding authors’ email: naomiomeseke@gmail.com 1Meseke, N. O., 2Akpootu, D. O., 1Falaiye, O. A. and 3Targema, T. V. 1Meseke, N. O., 2Akpootu, D. O., 1Falaiye, O. A. and 3Targema, T. V. 1Department of Physics, University of Ilorin, Nigeria 2Department of Physics, Usmanu Danfodiyo University Sokoto, Nigeria 3Department of Physics, Taraba State University, Nigeria COMPARATIVE ASSESSMENT OF PARTICUL… Meseke et al., FJS 2021). With over 16,000 devices in use, Purple Air is one of the most widely utilized monitors. Fine particles contain secondary produced aerosols, combustion particles, recompensed organic and metallic vapor (Amato et al., 2016), while coarse particles generally contain components from the earth's crust, dust from cars and industrial facilities (Akpan, & William, 2014) whereas ultrafine particles is made up of a variety of hazardous chemicals produced as primary emissions, such as trace metals and diesel black carbon [BC] or as secondary aerosols formed by deposition on existing particles from gaseous progenitors (E.S.T, 2021), because it reduces interference from natural sources. In Sub-Saharan Africa (SSA), the viability and applicability of measuring air quality for PM2.5 using a low-cost sensor (purpleair), with the goal of evaluating the effectiveness of its data recovery rate and identification of difficulties faced by users in each region has been determined (Awokola et al., 2020). Their investigation showed that, despite certain operational difficulties, it is rationally practicable and possible to set up a network of low cost devices to provide data on local PM2.5 concentrations in SSA nations. Such information is essential for increasing public awareness of air pollution throughout SSA. The PM2.5 has been recognized to have lesser indication for automotive emissions occurring in roadside than the PM1.0 (E.S.T., 2021). PM is discharged into the air by a variety of natural (lower ratio) and man-made sources (higher ratio) (Miranda & Tomaz, 2008). They are released from smoke, dirt, dust or construction sites, while others are formed in the atmosphere due to complicated chemical reactions, such as nitrogen oxides and sulfur (iv) oxide—pollutants produced by factories and power plants. PM emissions are stated to be causing increasing issue around the globe due to its tremendous influence on man and the environment (Duan et al., 2015). PM10.0 (smaller than one tenth the breadth of a human hair) has health implications since it generates noise and throat discomfort when inhaled, which might result in high blood pressure (HBP), stroke, lung cancer, heart attack, bronchitis, and other health problems (Zhao et al., 2020). PM2.5 enters the blood as well as the lungs. It causes inflammation and harm, such as respiratory sickness, a lowered immune response, congenital defects, and diabetes, among other things (Feng et al., 2016). Study Area Ni i ' li Nigeria's climate is tropical, with variation of rainy and dry seasons based on location. For the most part, the south is damp, whilst the north is usually dry. The rainy season in th e south lasts from March to November, but only from mid-May to September in the far north. The dry season is referred to as the harmattan season. Harmattan is a cool, dry breeze that sweeps from the northeast or east across the Western Sahara. Up to half of the world's substantial dust emissions come from the Sahara Desert (Ogunjo et al., 2022). Generally it has been reported that in Nigeria the rainy season falls between the months of April through October and the dry season in between November through March (Akpootu et al., 2017; Akpootu et al., 2019a) Statistics from the World Health Organization showed that, each year over seven million people die as a result of air pollution, 70% of people breathe air with PM levels that are higher than the guidelines of Air quality recommended by WHO (PM2.5 should not exceed 10 𝜇𝑔/𝑚3 annual mean and 25 𝜇𝑔/𝑚3 24 hours mean: PM10.0 should not exceed 20 𝜇𝑔/𝑚3 annual mean and 50 𝜇𝑔/𝑚3 24 hours mean) (WHO, 2021). Several research reveals those who live in areas with poor air quality are more prone to develop respiratory disorders, as well as cardiovascular and circulation problem. Kano state (Latitude: 12° 40′ and 10° 30′N, and longitude: 7° 40′ and 9° 30′ E). The climate is divided into two seasons: dry and wet. The dry season usually start in November and lasts until March, with the rainy season beginning in May and ending in September. The average annual rainfall is approximately 690 mm, and the average annual temperature swings between a maximum of 33°C and a minimum of 19°C. The vegetation is primarily Savanna, which is classified climatically as Northern Guinea savanna and Sudan savanna (Wakawa et al., 2016). Having a population of more than 15 million people Kano State is consider as the second-largest industrial center in Nigeria, after Lagos State, and the largest in Northern Nigeria, with industries including textile, tanning, footwear, cosmetics, plastics, enamelware, pharmaceuticals, ceramics, and furniture. Other commodities include agricultural instruments, soft drinks, food and beverages, dairy products, vegetable oil, animal feeds and it has an altitude of 360 m Owoade et al. (2012); Nwaogazie and Zagha, (2015); Akin- folarin et al. COMPARATIVE ASSESSMENT OF PARTICUL… Meseke et al., Scientists have yet to discover any health implications for PM1.0, but it is thought to have a larger impact (WHO, 2006; Polichetti et al., 2009, U.S. EPA, 2012; Health Effects Institute, 2020). Ogunjo et al. (2022) conducted a study to analyze PMs with a view to establish the places with the strongest connections among COVID-19 cases, In their study they utilized low-cost air quality monitors (Purple Air sensor) across seven administrative states in Nigeria. According to them, strong positive correlation values exits between COVID-19 cases and PMs. They believe that the considerable positive correlation between PMs and COVID-19 cases and fatalities will help control and mitigate pandemic spread within a group of people. The purpose of this study is to investigate the variability of PMs over Abuja and Kano with a view to assess the environmental impacts. INTRODUCTION The introduction of dangerous items into the environment is referred to as pollution. Pollutants are the name given to these dangerous substances which have negative consequences on the quality of the air, water, and land (West et al., 2020). The release of pollutants into the atmosphere is classified as air pollution, these pollutants are damaging human health and the environment as a whole (Abulude & Abulude, 2021). Duan et al. (2015) has pointed out that PM is divided into three size fractions: Ultrafine, Fine, and Coarse, each with its own set of physiologic and source features. PM1.0 or Ultrafine particle, with aerodynamic diameter (Di) less than 0.1 𝜇𝑚, PM2.5 or Fine particle (Di ≤2.5 𝜇𝑚) and PM10.0 or Coarse particle (Di< 10 𝜇𝑚). The particles size distribution and its content are determined by their production processes, which include their source, which has been studied extensively (Tsai et al., 2015). The Smog (also known as ground-level ozone) and Soot (also known as particulate matter) are the two most common forms of air pollution. The gaseous combination of solid and liquid particles suspended in air is known as Particulate matter (PM) (Dockery et al., 1997; WHO, 2013; Adams et al., 2015; Istiqomah et al., 2020). It is also a complex mixture of liquid droplets made up of acids (such as nitrates and sulfates), ammonium, water, black carbon, organic compounds, metals, Figure 1: Types of PM Source: (Slezakova et al., 2013) Figure 1: Types of PM Source: (Slezakova et al., 2013) FUDMA Journal of Sciences (FJS) Vol. 6 No. 4, August, 2022, pp 203 - 211 203 203 COMPARATIVE ASSESSMENT OF PARTICUL… Meseke et al., 5-second readings averaged over 120 seconds (http://www.plantower.com/en/). It measures the size of particles suspended in increments of 0.3, 0.5, 1.0, 2.5, 5.0, and 10 𝜇m (Plantower User Manual, 2016).The sensor processes these particle counts using a complicated algorithm to compute the mass concentrations of PM1.0, PM2.5, and PM10.0 in 𝜇g/m3 (microgram of gaseous pollutant per cubic meter of ambient air) for standard indoor (CF-1 i.e for laboratory use) and outdoor particles (ATM i.e for Atmospheric condition) (Plantower User Manual, 2016). PurpleAir's website displays real-time data in the color-coded air quality index (AQI) form and actual PM concentrations (PurpleAir, 2019).The Purple Air-II-SD has an in-built when connected to the internet, a Real-Time Clock (RTC) sets itself. 3,564,126. Agriculture is one of the economic foundations. Millet, corn (maize), yams, sorghum and beans are produced in this region. Mineral resources include clay, tin, feldspar, gold, iron ore, lead, marble, and talc. Abuja and Kano were chosen because they have comparable vegetation and weather features. 3,564,126. Agriculture is one of the economic foundations. Millet, corn (maize), yams, sorghum and beans are produced in this region. Mineral resources include clay, tin, feldspar, gold, iron ore, lead, marble, and talc. Abuja and Kano were chosen because they have comparable vegetation and weather features. Study Area Ni i ' li (2017); Osimobi et al. (2019); Abulude et al. (2021); Falaiye et al. (2021) are some of Nigeria researchers out of many researchers who have carried out studies in Nigeria on the evaluation of air quality using instrument, while many others have not due to the cost of these instruments, the time required, and the work required in executing the study hence it has limited certain study due to a lack of PM1.0, PM 2.5, and PM10.0 monitoring sites. To address the issue, less expensive air quality instrument that measure PM are made readily available, making it simple to provide safety information on the air we breathe in. Williams et al. (2014) and US EPA (2017) have noted that regions like Asia and the United States, the use of low-cost air quality sensors that directly send PM concentrations to the internet is increasing rapidly. A lot of enterprises are developing tiny electronic sensors for the purpose of monitoring, which use lasers to scatter light off the particles into detectors. The scattered light during this process are studied to measure number, particle size, and mass concentration using Mie scattering theory (Wallace et al., Abuja is an administrative territory central Nigeria. It is bordered by the states of Niger to the west and northwest Kaduna to the northeast Nassarawa to the east and south and Kogi to the southwest. It is located between latitude 9.0765° N and longitude 7.3986° E. Guinea Savannah is the predominant vegetation type in Abuja, and it is made up of tall grasses that are sprinkled with various species (Ahmad et al., 2017). With an altitude of 456m and a population of about FUDMA Journal of Sciences (FJS) Vol. 6 No. 4, August, 2022, pp 203 - 211 204 FJS MATERIALS AND METHOD Low-cost air quality stations (called Purple Air stations) were established in Nigeria to address the absence of PM concentrations. The stations are strategically positioned in some states in Nigeria. This information can be used to assess the potential links between Particulate matter ( 𝜇𝑔/𝑚3 ), temperature (℉), and relative humidity (%). Ardon-Dryer et al. (2020) and Liu et al. (2020) have evaluated data from Plantower sensors' low-cost optical devices for monitoring particulate matter. Data were collected for particulate matter (PM1.0, PM2.5 and PM10.0) from two PurpleAir sensors: Sensor 1 Space Weather and Atmospheric Physics Laboratory, Center for Atmospheric Research Bayero University Campus, Kano State and Sensor 2 Space Environment Research Laboratory, Centre for Atmospheric Research, Abuja. Measurements of particulate matter concentrations, temperature and relative humidity were collected for 1 year (1st January, 2021 to 31st December 2021). Purple air stations were assess to acquire PM readings in this investigation. The program is linked to a sensor, which communicates with the particle counter via an ESP8266 microcontroller chip, which also provides complete capabilities, including connecting to a WiFi network and sending data to the cloud (https://www.purpleair.com/). The sensor uses PMS5003 and PMS1003 laser counters to detect particle matter in real time, with each laser counter alternating Figure 2: Map of Nigeria showing the states where data were obtained Figure 2: Map of Nigeria showing the states where data were obtained FUDMA Journal of Sciences (FJS) Vol. 6 No. 4, August, 2022, pp 203 - 211 205 205 FJS COMPARATIVE ASSESSMENT OF PARTICUL… Meseke et al., a. b. Figure 3: a. Showing various PurpleAir sensors in Nigeria b. Sensor in Bayero University, Kano b. a. b. Figure 3: a. Showing various PurpleAir sensors in Nigeria b. Sensor in Bayero University, Kano Table 1: Air quality index and health effects (Nathaniel and Xiaoli, 2020) Table 1: Air quality index and health effects (Nathaniel and Xiaoli, 2020) Table 1: Air quality index and health effects (Nathaniel and Xiaoli, 2020) Correlation Analysis The Pearson Coefficient Correlation (PCC) was used as a comparison. It is defined as 𝑟= ∑{(𝑥𝑖−𝑥̅)×(𝑦𝑖−𝑦̅)} √∑{(𝑥𝑖−𝑥̅)2×(𝑦𝑖−𝑦̅)2} (1) where 𝑟 𝑟𝑒𝑝𝑟𝑒𝑠𝑒𝑛𝑡 𝑐𝑜𝑟𝑟𝑒𝑙𝑎𝑡𝑖𝑜𝑛 𝑐𝑜𝑒𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑡 𝑥𝑖 𝑟𝑒𝑝𝑟𝑒𝑠𝑒𝑛𝑡 𝑣𝑎𝑙𝑢𝑒𝑠 𝑜𝑓 𝑡ℎ𝑒 𝑥−𝑣𝑎𝑟𝑖𝑎𝑏𝑙𝑒 indicate negative linear correlation (Akpootu and Iliyasu, 2017). Descriptive Statistics The key components that are looked at in the descriptive statistical analysis are Skewness and Kurtosis. The asymmetry in the data surrounding the mean value of the independent meteorological parameters is measured and the comparison. It is defined as 𝑟= ∑{(𝑥𝑖−𝑥̅)×(𝑦𝑖−𝑦̅)} √∑{(𝑥𝑖−𝑥̅)2×(𝑦𝑖−𝑦̅)2} (1) where 𝑟 𝑟𝑒𝑝𝑟𝑒𝑠𝑒𝑛𝑡 𝑐𝑜𝑟𝑟𝑒𝑙𝑎𝑡𝑖𝑜𝑛 𝑐𝑜𝑒𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑡 𝑥𝑖 𝑟𝑒𝑝𝑟𝑒𝑠𝑒𝑛𝑡 𝑣𝑎𝑙𝑢𝑒𝑠 𝑜𝑓 𝑡ℎ𝑒 𝑥−𝑣𝑎𝑟𝑖𝑎𝑏𝑙𝑒 𝑥̅ 𝑟𝑒𝑝𝑟𝑒𝑠𝑒𝑛𝑡 𝑡ℎ𝑒 𝑚𝑒𝑎𝑛 𝑣𝑎𝑙𝑢𝑒𝑠 𝑜𝑓 𝑥−𝑣𝑎𝑟𝑖𝑎𝑏𝑙𝑒 𝑦𝑖 𝑟𝑒𝑝𝑟𝑒𝑠𝑒𝑛𝑡 𝑣𝑎𝑙𝑢𝑒𝑠 𝑜𝑓 𝑡ℎ𝑒 𝑦−𝑣𝑎𝑟𝑖𝑎𝑏𝑙𝑒 𝑦̅ 𝑟𝑒𝑝𝑟𝑒𝑠𝑒𝑛𝑡 𝑡ℎ𝑒 𝑚𝑒𝑎𝑛 𝑣𝑎𝑙𝑢𝑒𝑠 𝑜𝑓 𝑦−𝑣𝑎𝑟𝑖𝑎𝑏𝑙𝑒 Air Quality Index (AQI) The study relied on PM1.0, PM2.5, PM10.0, temperature, and relative humidity measurements, the data were obtained at 60 minutes intervals. A thorough data check on the raw data is undertaken to limit the effect of problematic data points such as duplicated datasets, incomplete measurements, odd zeros, and so on. Following the data quality check, measurement data for PM1.0, PM2.5, PM10.0, temperature, and relative humidity were combined into one average. The data was downloaded as a CSV file, and Excel was used to run statistical analyses (version 2013). The Air Quality Index, or AQI, is the system used to monitor air pollution by telling the general public how clean or polluted the air we breathe and how the air we breathe can be dangerous to both sensitive and non sensitive people. The AQI tracks ozone (smog) and particle pollution (tiny particles from ash, power plants and factories, vehicle exhaust, soil dust, pollen, and other pollution). The table below shows the standard limit for air quality set aside by the US, informations are available for PM2.5 and PM10.0. From the table below, we can deduce that the lower the values of PM the better the air quality for both sensitive and non sensitive people. COMPARATIVE ASSESSMENT OF PARTICUL… Meseke et al., and Abuja. The increase in the PM values during Harmattan season can be attributed to anthropogenic sources which bring about the formation of fog/haze and the dust from the Sahara desert since Kano and Abuja has a close proximity with it. The increase in PM from January to March can be associated with tiling of land, bush burning as farmer will be getting their land ready for farming as we approach rainy season and this is in line with the study reported by Abulude & Abulude (2021) and Ogunjo et al. (2022). The decrease in PM from May to October can be trace to wet season as precipitation helps to lower the amount of PM in the atmosphere as the particles drop together with rain to the ground. The PM concentrations are generally higher in the dry season than that of the rainy season. measures a distribution's relative peak or flatness in relation to the normal distribution, which illustrates the general form of a random variable. It measures the consistency of each climate parameter for the research locations (Hejase and Assi, 2011; Akpootu et al., 2019b). Descriptive Statistics The key components that are looked at in the descriptive statistical analysis are Skewness and Kurtosis. The asymmetry in the data surrounding the mean value of the independent meteorological parameters is measured and the direction of variation in the dataset is revealed using the skewness tests. If the data have a Gaussian distribution, this implies normal distribution; if the data are spread out more to the left of the mean value than to its right, it means negatively skewed and if the data are spread out more to the right than to its left, this implies positively skewed. The Kurtosis test The correlation coefficient values lies between -1 and + 1. The + sign indicate positive linear correlation while the – signs FUDMA Journal of Sciences (FJS) Vol. 6 No. 4, August, 2022, pp 203 - 211 206 FJS COMPARATIVE ASSESSMENT OF PARTICUL… Meseke et al., Table 2 indicates that temperature have a low correlation with PM concentration (PM1.0, 2.5, 10) while relative humidity has a relatively strong negative correlation in table 2 and 3.We observed a strong positive correlation between the three PM which reveals that an increase in PM1.0 directly increase in the remaining PMs (PM1.0, 2.5, 10). The findings in this study are in agreement with those of Obioh, et al (2013), Nathaniel and Xiaoli (2020) and Abulude and Abulude (2021). Generally, the Pearson correlation coefficients analysis shows that PM concentrations are strongly negatively correlated with relative humidity that is as relative humidity increases PM decreases in the atmosphere. Study has shown that as humidity increase PM becomes too large and can no longer remains in the atmosphere hence began to fall a process known as dry deposition of particulate matter (Wang and Ogawa 2015). While a positive correlation of temperature with PMs was recorded indicating that as the temperature increase PM concentration also increases and vice versa. From Table 2 PM1.0 has a strongly positive correlation with PM2.5 (r = 0.996), strong positively correlation was also found with PM10.0 (r = 0.989) while low positive correlation coefficient value with temperature (r = 0.499) and a strong negative correlation with relative humidity (r = -0.856). Meanwhile, PM2.5 has a strong positive correlation coefficient value with PM10.0, low positive correlation coefficient value with temperature and a strong negative correlation with relative humidity. PM10.0 has a positive correlation coefficient value (r = 0.531) with temperature and a strong negative correlation with relative humidity. A negative correlation of 0.714 existed between temperature and relative humidity. COMPARATIVE ASSESSMENT OF PARTICUL… Meseke et al., A negative c temperature and relati Table 2 indicates that PM concentration (PM relatively strong neg observed a strong pos which reveals that an i remaining PMs (PM1.0 agreement with those Xiaoli (2020) and Ab the Pearson correlatio concentrations are stro humidity that is as rel in the atmosphere. Stu PM becomes too lar for Abuja ) PM2.5 (μg/m3) PM10.0 (μg/m3) Temperature (℉) 1 3 1 4 0.992711 1 5 0.456857 0.530721 1 0 -0.860940 -0.905240 -0.713630 for Kano state PM2.5 (μg/m3) PM10.0 (μg/m3) Temperature (℉) Re 1 0.990974 1 0.596786 0.601484 1 -0.825460 -0.865330 -0.546041 ating the change of wn to be weather e consequences of rations in Tables 2 onitors temperature rrelation between d relative humidity ve correlation with tion was also found ositive correlation .499) and a strong dity (r = -0.856). relation coefficient n coefficient value e correlation with relation coefficient a strong negative ative correlation of humidity (r = -0.863). Similarly, PM correlated with PM10.0, positive correla with temperature and a strong nega relative humidity. PM10.0 is strongly temperature and a strong negative correl humidity. A negative correlation of 0.54 temperature and relative humidity. Table 2 indicates that temperature have PM concentration (PM1.0, 2.5, 10) while re relatively strong negative correlation observed a strong positive correlation b which reveals that an increase in PM1.0 d remaining PMs (PM1.0, 2.5, 10). The findin agreement with those of Obioh, et al ( Xiaoli (2020) and Abulude and Abulu the Pearson correlation coefficients an concentrations are strongly negatively c humidity that is as relative humidity in in the atmosphere. Study has shown tha One of the most important elements regulating the change of PM concentration has long been known to be weather conditions. As a result, we looked into the consequences of meteorological conditions on PM concentrations in Tables 2 and 3. Since the PurpleAir sensor also monitors temperature and relative humidity, the Pearson correlation between average PM concentration, temperature, and relative humidity was investigated in this table. humidity (r = -0.863). Similarly, PM2.5 is strong positive correlated with PM10.0, positive correlation coefficient value with temperature and a strong negative correlation with relative humidity. PM10.0 is strongly correlated with the temperature and a strong negative correlation with the relative humidity. A negative correlation of 0.546 existed between the temperature and relative humidity. FUDMA Journal of Sciences (FJS) Vol. 6 No. 4, August, 2022, pp 203 - 211 COMPARATIVE ASSESSMENT OF PARTICUL… Meseke et al., COMPARATIVE ASSESSMENT OF PARTICUL… Meseke et al., COMPARATIVE ASSESSMENT OF PARTICUL… Meseke et al., FJS Coeffient of Correlation Table 2: Pearson coeficient correlation for Abuja PM1.0 (μg/m3) PM2.5 (μg/m3) PM10.0 (μg/m3) Temperature (℉) Relative humidity (%) PM1.0 (μg/m3) 1 PM2.5 (μg/m3) 0.995843 1 PM10.0 (μg/m3) 0.988614 0.992711 1 Temperature (℉) 0.498995 0.456857 0.530721 1 Relative humidity (%) -0.856120 -0.860940 -0.905240 -0.713630 1 Table 3: Pearson coefficient correlation for Kano state PM1.0 (μg/m3) PM2.5 (μg/m3) PM10.0 (μg/m3) Temperature (℉) Relative humidity (%) PM1.0 (μg/m3) 1 PM2.5 (μg/m3) 0.979472 1 PM10.0 (μg/m3) 0.964921 0.990974 1 Temperature (℉) 0.606584 0.596786 0.601484 1 Relative humidity ( %) -0.862820 -0.825460 -0.865330 -0.546041 1 One of the most important elements regulating the change of PM concentration has long been known to be weather conditions. As a result, we looked into the consequences of meteorological conditions on PM concentrations in Tables 2 and 3. Since the PurpleAir sensor also monitors temperature and relative humidity, the Pearson correlation between average PM concentration, temperature, and relative humidity was investigated in this table. From Table 2 PM1.0 has a strongly positive correlation with PM2.5 (r = 0.996), strong positively correlation was also found with PM10.0 (r = 0.989) while low positive correlation coefficient value with temperature (r = 0.499) and a strong negative correlation with relative humidity (r = -0.856). Meanwhile, PM2.5 has a strong positive correlation coefficient value with PM10.0, low positive correlation coefficient value with temperature and a strong negative correlation with relative humidity. PM10.0 has a positive correlation coefficient value (r = 0.531) with temperature and a strong negative correlation with relative humidity. A negative correlation of 0.714 existed between temperature and relative humidity. Table 3 indicate that PM1.0 has strongly high positive correlation coefficient value with PM2.5 (r = 0.979), strong humidity (r = -0.863). Similarly, PM2.5 is strong positive correlated with PM10.0, positive correlation coefficient value with temperature and a strong negative correlation with relative humidity. PM10.0 is strongly correlated with the temperature and a strong negative correlation with the relative humidity. A negative correlation of 0.546 existed between the temperature and relative humidity. Monthly Variation Monthly Variation The monthly variations of PM are shown for one year (January 2021- December 2021) as depicted in Figure 4a and 4b. It can be seen that the PM concentrations were relatively high from January, February, March, and gradually decrease from May to October then it start to rise again as we get into the dry season from November to December for both Kano FUDMA Journal of Sciences (FJS) Vol. 6 No. 4, August, 2022, pp 203 - 211 high from January, February, March, and gradually decrease from May to October then it start to rise again as we get into the dry season from November to December for both Kano particles drop together with rain to the ground. The PM concentrations are generally higher in the dry season than that of the rainy season. Figure 4a: Monthly variation of PM values in Abuja Figure 4b: Monthly variation of PM values in Kano State 0 20 40 60 80 100 120 14021_Jan21_Feb21_Mar21_Apr21_May21_Jun21_Jul21_Aug21_Sep21_Oct21_Nov21_Dec PM concentration (μg/m3) Monitoring Months PM 1.0 μg/m^3 PM 2.5 μg/m^3 PM 10 μg/m3 -10 10 30 50 70 90 110 13021_Jan21_Feb21_Mar21_Apr21_May21_Jun21_Jul21_Aug21_Sep21_Oct21_Nov21_Dec PM Concentration (μg/m3) Monitoring Months Kano PM 1.0 μg/m^3 Kano PM 2.5 μg/m^3 Kano PM 10 μg/m^3 Figure 4a: Monthly variation of PM values in Abuja 0 20 40 60 80 100 120 14021_Jan21_Feb21_Mar21_Apr21_May21_Jun21_Jul21_Aug21_Sep21_Oct21_Nov21_Dec PM concentration (μg/m3) Monitoring Months PM 1.0 μg/m^3 PM 2.5 μg/m^3 PM 10 μg/m3 PM 1.0 μg/m^3 PM 2.5 μg/m^3 PM 10 μg/m3 PM 1.0 μg/m^3 PM 2.5 μg/m^3 PM 10 μg/m3 Monitoring Months Figure 4a: Monthly variation of PM values in Abuja Figure 4a: Monthly variation of PM values in Abuja -10 10 30 50 70 90 110 13021_Jan21_Feb21_Mar21_Apr21_May21_Jun21_Jul21_Aug21_Sep21_Oct21_Nov21_Dec PM Concentration (μg/m3) Monitoring Months Kano PM 1.0 μg/m^3 Kano PM 2.5 μg/m^3 Kano PM 10 μg/m^3 Kano PM 1.0 μg/m^3 Kano PM 2.5 μg/m^3 Kano PM 10 μg/m^3 Monitoring Months Figure 4b: Monthly variation of PM values in Kano State FUDMA Journal of Sciences (FJS) Vol. 6 No. 4, August, 2022, pp 203 - 211 207 207 COMPARATIVE ASSESSMENT OF PARTICUL… Meseke et al., Table 2 indicates that temperature have a low correlation with PM concentration (PM1.0, 2.5, 10) while relative humidity has a relatively strong negative correlation in table 2 and 3.We observed a strong positive correlation between the three PM which reveals that an increase in PM1.0 directly increase in the remaining PMs (PM1.0, 2.5, 10). The findings in this study are in agreement with those of Obioh, et al (2013), Nathaniel and Xiaoli (2020) and Abulude and Abulude (2021). Generally, the Pearson correlation coefficients analysis shows that PM concentrations are strongly negatively correlated with relative humidity that is as relative humidity increases PM decreases in the atmosphere. Study has shown that as humidity increase PM becomes too large and can no longer remains in the atmosphere hence began to fall a process known as dry deposition of particulate matter (Wang and Ogawa 2015). Coeffient of Correlation Table 2: Pearson coeficient correlation for Abuja PM1.0 (μg/m3) PM2.5 (μg/m3) PM10.0 (μg/m3) Temperature (℉) Relative humidity (%) PM1.0 (μg/m3) 1 PM2.5 (μg/m3) 0.995843 1 PM10.0 (μg/m3) 0.988614 0.992711 1 Temperature (℉) 0.498995 0.456857 0.530721 1 Relative humidity (%) -0.856120 -0.860940 -0.905240 -0.713630 1 Table 3: Pearson coefficient correlation for Kano state PM1.0 (μg/m3) PM2.5 (μg/m3) PM10.0 (μg/m3) Temperature (℉) Relative humidity (%) PM1.0 (μg/m3) 1 PM2.5 (μg/m3) 0.979472 1 PM10.0 (μg/m3) 0.964921 0.990974 1 Temperature (℉) 0.606584 0.596786 0.601484 1 Relative humidity ( %) -0.862820 -0.825460 -0.865330 -0.546041 1 Coeffient of Correlation Table 2: Pearson coeficient correlation for Abuja Coeffient of Correlation relation for Abuja 1.0 (μg/m3) PM2.5 (μg/m3) PM10.0 (μg/m3) Te 1 0.995843 1 0.988614 0.992711 1 0.498995 0.456857 0.530721 -0.856120 -0.860940 -0.905240 rrelation for Kano state PM1.0 (μg/m3) PM2.5 (μg/m3) PM10.0 (μg/m3) Tem 1 .979472 1 .964921 0.990974 1 .606584 0.596786 0.601484 .862820 -0.825460 -0.865330 nts regulating the change of een known to be weather ed into the consequences of M concentrations in Tables 2 r also monitors temperature arson correlation between rature, and relative humidity gly positive correlation with y correlation was also found e low positive correlation re (r = 0.499) and a strong ve humidity (r = -0.856). sitive correlation coefficient correlation coefficient value negative correlation with sitive correlation coefficient ture and a strong negative y. A negative correlation of re and relative humidity humidity (r = -0.863 correlated with PM10. with temperature an relative humidity. PM temperature and a stro humidity. COMPARATIVE ASSESSMENT OF PARTICUL… Meseke et al., COMPARATIVE ASSESSMENT OF PARTICUL… Meseke et al., Research-National Space Research and Development Agency (CAR-NASRDA). Aerosol Science and Engineering https://doi.org/10.1007/s41810-021-00116-3 Table 4 shows the descriptive statistics for both locations. The Air quality index (AQI) standard table in table 1 above was compared with descriptive summary in table 4, AQI provides adequate information about the air we breathe. The result shows that PM2.5 concentration for Abuja and Kano is unhealthy for sensitive group and falls under level 3 in air pollution, sensitive people are those people who are suffering from any respiratory and cardiovascular illness while PM10.0 concentration for both states falls in the moderate, that is the air in both regions is good for both the sensitive people and the non sensitive people which is in the level 2 category. PM1.0, PM2.5, and PM10.0 concentrations in Kano State were 37.35, 49.01 and 59.20 𝜇𝑔/𝑚3 respectively while for Abuja are 35.40, 50. 34 and 56.76 𝜇𝑔/𝑚3 respectively. The WHO 24-hour guideline limits of 25 𝜇𝑔/𝑚3 and 50 𝜇𝑔/𝑚3 were exceeded in Kano and Abuja respectively. Adams, K., Greenbaum, D. S., Shaikh, R.,van Erp, A. M. and Russell, A.G. (2015). Particulate matter components, sources, and health: Systematic approaches to testing effects, Journal of the Air & Waste Management Association, 65:5, 544- 558, DOI: 10.1080/10962247.2014.1001884 Ahmed, Y.A., Aderonke, M. and Oyewo, S.O. (2017). Health Impact of Leachates from Illegal Dumpsites: Case Study Of Kubwa Abuja, Nigeria. Ethiopian Journal of Environmental Studies & Management 10(1): 125 – 136, ISSN: 1998-0507 doi: http://dx.doi.org/10.4314/ejesm.v10i1.12 Akinfolarin, O.M., Boisa, N. and Obunwo, C.C. (2017). Assessment of particulate matter-based air quality index in Port Harcourt Nigeria.J Environ Anal Chem 4:224. https://doi.org/10.4172/2380-2391.1000224 For Abuja, It was observed that the temperature and relative humidity data’s spread out more to the left of their mean value (negatively skewed) while PM1.0, PM2.5 and PM10.0 data’s spread out more to the right of their mean value (positively skewed) and PM1.0, PM2.5 and PM10.0 data’s have positive kurtosis which indicates a relatively peaked distribution and possibility of a leptokurtic distribution while temperature and relative humidity data have negative kurtosis which indicates a relatively flat distribution and possibility of a platykurtic distribution. COMPARATIVE ASSESSMENT OF PARTICUL… Meseke et al., For Kano, the skewness and kurtosis for Kano is quite lower than that of Abuja, the bottom part of the table show that all PMs, temperature and relative humidity spread out more to the right of their mean value (positively skewed) while for kurtosis only PM2.5 has a positive kurtosis indicating a peaked distribution and PM1.0, PM10.0, temperature and relative humidity has a negative kurtosis indicating a flat distribution. Akpan, I.O. and William, E.S. (2014). Assessment of Elemental Concentrations of Road DOI: 10.4236/gep.2020.811007 136 Journal of Geoscience and Environment Protection side Soils in Relation to Traffic Density in Calabar, Nigeria. International Journal of Scientific and Technology Research, 3, 3-10 Akpootu, D.O. and Iliyasu, M.I. (2017). A comparison of various Evapotranspiration models for estimating reference Evapotranspiration in Sokoto, North Western, Nigeria. Physical science International Journal.,14(2),1-14. DOI:10.9734/PSIJ/2017/32720 CONCLUSION Akpootu, D.O., Iliyasu, M.I., Mustapha, W., Aruna, S. and Yusuf, S.O. (2017). Developing Regression Models for estimating Atmospheric Visibility over Ikeja, Nigeria. Journal of Scientific Research & Reports.,15(6):1- 14.DOI:10.9734/JSRR/2017/36670. A comparative assessment of PM and the parameters of temperature and relative humidity for Abuja and Kano was investigated. The outcome shows that during the dry season in both locations, the monthly average of PM1.0, PM2.5 and PM10.0 surpasses the WHO 24-hour standard limit. Since both locations are the two major industrious locations in the northern part of Nigeria which causes the population to grow on a regular basis, this can be attributed to excessive pollution and an increase in car emissions. While during the wet season, PM1.0, PM2.5 and PM10.0 are below the WHO 24-hour guideline. When compared to AQI, PM2.5 was said to be unhealthy for sensitive individuals. People with respiratory or cardiovascular conditions are advised to be aware of their surroundings through the help of an air quality measures device that may determine whether the air they breathe is beneficial or hazardous for them. In order to achieve the goals of health is wealth and to lower the rate of morbidity caused by air pollution around the globe, air quality sensor manufacturers should also offer portable and less expensive sensors that are affordable for the general public. Akpootu, D.O., Iliyasu, M.I., Abubakar, M.B., Rabiu, A.M., Mustaspha, W., Okany, C.F. and Salifu, S.I. (2019a). Developing Empirical models for estimating photosynthetically active radiation over Akure, South Western, Nigeria. International Journals of Advances in Scientific research and engineering (ijasre).,5(10): 59-79. DOI:10.31695/IJASRE.2019.33546 Akpootu, D.O.,Tijjani, B.I. and Gana, U.M. (2019b). A Comparative study of Time Series, Empirical Orthogonal Transformation and Descriptive Statistical Analysis on Meteorological Parameters over Ogoja and Maiduguri. Journal of Energy Research and Reviews., 3(1): 1-14 DOI: 10.9734/JENRR/2019/v3i130088 Aliero, M.M., Ismail, M.H., Alias, A.M. and Sood, M.A. (2017). Evaluation of Land Cover Change and Vegetation Dynamics Using Remote Sensing and DPSIR Frame work in Kebbi State, Nigeria. COMPARATIVE ASSESSMENT OF PARTICUL… Meseke et al., Table 3 indicate that PM1.0 has strongly high positive correlation coefficient value with PM2.5 (r = 0.979), strong positively correlation was also found with PM10.0 (r = 0.965) while a positive correlation coefficient value with temperature (r = 0.607) and a strong negative correlation with relative Descriptive Statistical Analysis Table 4: Descriptive Summary Result PM1.0 (μg/m3) PM2.5 (μg/m3) PM10.0 (μg/m3) Temperature (℉) Relative Humidity (%) ABUJA Mean 35.4042 50.3403 56.7602 91.3346 41.1374 STD 16.9174 25.8333 30.1536 3.7818 17.7118 VAR 286.1993 667.3601 909.2363 14.3023 313.7060 MAX 75.5246 111.5265 124.6871 96.8348 60.3222 MIN 17.7918 25.2221 26.3632 85.4527 12.5083 Kurtosis 1.6436 1.6012 0.8004 -0.7534 -1.3662 Skewness 1.2405 1.3017 1.0909 -0.2150 -0.5542 KANO Mean 37.3479 49.0082 59.2021 -69.0990 26.9775 STD 16.2506 26.4256 32.5603 166.8355 11.8250 VAR 264.0827 698.3104 1060.1740 27834.0800 139.8302 MAX 69.3170 106.1048 121.2962 98.2482 43.1590 MIN 16.8333 19.6221 21.6803 -229.0000 12.8028 Kurtosis -0.4531 0.4238 -0.5438 -2.4369 -1.7877 Skewness 0.4323 0.9264 0.6913 0.0042 0.2150 Descriptive Statistical Analysis Table 4: Descriptive Summary Result Descriptive Statistical Analysis FUDMA Journal of Sciences (FJS) Vol. 6 No. 4, August, 2022, pp 203 - 211 208 FJS ACKNOWLEDGEMENTS ( ) g g Dynamics Using Remote Sensing and DPSIR Frame work in Kebbi State, Nigeria. http://dx.doi.org/10.20944/preprints201709.0090.v1 We acknowledge the management and staff of purple air for making the data available online. All data used in this study are publicly available. The particulate matter (PM1.0, PM2.5, PM10.0) and atmospheric parameters (temperature and humidity) were obtained from purpleair.com (https://www.purpleair.com/sensor) y g g Kebbi State, Nigeria. http://dx.doi.org/10.20944/preprints201709.0090.v1 http://dx.doi.org/10.20944/preprints201709.0090.v1 http://dx.doi.org/10.20944/preprints201709.0090.v1 Amato, F., Favez, O., Pandolfi, M., Alastuey, A., Querol, X., Moukhtar, S., Bruge, B., Verlhac, S., Orza, J.A.G., Bonnaire, N., Le Priolet, T., Petit, J.-F. and Sciare, J. (2016). Traffic Induced Particle Resuspension in Paris: Emission Factors and Source Contributions. Atmospheric Environment, 129, 114- REFERENCE Abulude, O.F. and Abulude, A.I. (2021). Monitoring Air Quality in Nigeria: The Case of Center for Atmospheric 124. https://doi.org/10.1016/j.atmosenv.2016.01.022 Feng, C., Li, J., Sun, W. et al. (2016). 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https://openalex.org/W3181621272	https://conflictandhealth.biomedcentral.com/counter/pdf/10.1186/s13031-021-00393-2	English	null	Built-environment attributes associated with refugee children’s physical activity: a narrative review and research agenda	Conflict and health	2,021	cc-by	7,498	© The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. REVIEW Open Access Abstract Research has identified built environmental attributes associated with children’s physical activity (PA); however, less is known for environmental correlates of refugee children’s PA. This narrative review summarised the current evidence of associations between built environment attributes and refugee children’s PA. Six databases were searched with three sets of terms related to exposure (built environment); outcome (PA); and target population (refugee children aged 6–12 years). Eight studies (one quantitative; seven qualitative) met the inclusion criteria. Key PA barriers were limited play space and lack of neighbourhood safety. Design of refugee facilities and surrounding environments should provide better access to formal, informal and safe spaces for children’s play. Keywords: Migrants, Outdoor play, Refugee facilities, Micro-environment, Meso-environment, Safety Participation in PA and sport can also help them to build social ties with peers, transcending national boundaries and language barriers [12]. Since refugee children have limited opportunities to engage in orga- nised sports and exercise [13, 14], taking part in informal PA such as active play is particularly important for them [15]. Given that the number of refugees and their chil- dren is increasing [16], and that lack of PA can have a long-term impact on children’s health and development [17], it is critical to develop policies and initiatives that can promote PA among refugee children. Built-environment attributes associated with refugee children’s physical activity: a narrative review and research agenda Siqi Chen1* , Alison Carver2, Takemi Sugiyama3 and Martin Knöll1 Introduction Physical activity (PA) is known to provide health benefits to children [1]. It helps children to build a robust body, stable mental health and healthy relationships with peers [2–4]. Despite the strong evidence supporting the health benefits of PA and public health efforts to promote chil- dren’s PA, over 80% children globally do not meet the recommendation of engaging in 60 min of moderateto- vigorous intensity PA per day [5]. Thus, increasing PA among children is a critical public health goal [6–8]. PA levels appear to be even lower among refugee chil- dren, who have recognised refugee status or are asylum seekers [9]. A UNICEF report showed that refugee chil- dren were rarely meeting the guidelines for daily PA [10]. Being physically active can be particularly beneficial for refugee children, who have to live in unfamiliar and uncertain situations, which can be stressful [11]. There are multiple factors that may be modified to fa- cilitate children to be physically active. One relevant do- main is the built environment, which refers to human- made space and structure in which people live, work/ study and engages in recreation on a day-to-day basis [18]. Built environmental attributes have been shown to be associated with non-refugee children’s PA. Several lit- erature reviews [19–23] have reported that built envir- onmental attributes such as access to physical activity facilities (playgrounds, greenspaces), availability of side- walks, neighbourhood perceived safety, and levels of * Correspondence: siqi.chen@stud.tu-darmstadt.de 1Urban Health Games Research Group (UHGs), Department of Architecture, Technische Universität Darmstadt, Darmstadt, Germany Full list of author information is available at the end of the article * Correspondence: siqi.chen@stud.tu-darmstadt.de 1Urban Health Games Research Group (UHGs), Department of Architecture, Technische Universität Darmstadt, Darmstadt, Germany Full list of author information is available at the end of the article Chen et al. Conflict and Health (2021) 15:55 https://doi.org/10.1186/s13031-021-00393-2 Chen et al. Conflict and Health (2021) 15:55 https://doi.org/10.1186/s13031-021-00393-2 Page 2 of 10 Chen et al. Conflict and Health (2021) 15:55 development (urban vs rural) are consistently associated with non-refugee children’s PA. designated playground [30]. The meso-environment is the intermediate layer beyond the immediate surround- ings but within the broader neighbourhood including local schools, communities, streets and open spaces. The macro-environment involves large-scale features of urban environments such as access to transport infrastructure and regional centres [31]. Figure 1 is a conceptual dia- gram illustrating these three layers. However, the existing findings of environmental attri- butes relevant to non-refugee children’s PA may not apply to refugee children. Non-refugee and refugee chil- dren live in very different settings. For example, refugee families and their children are typically assigned to refu- gee camps or other temporary accommodation once they arrive in a host country [24]. Such facilities are often built in isolated and inaccessible areas of cities [25]. Even those who were granted long-term/permanent visa tend to have limited options about where to live and are more likely to reside in disadvantaged areas [26]. Due to such living arrangements, it is possible to argue that refugee children are living in less favourable condi- tions than non-refugee children for engaging in PA [10]. An increasing number of studies begin to investigate en- vironmental attributes associated with refugee children’s PA. However, to build an evidence base that can inform relevant policies to promote refugee children’s PA, re- search findings on this topic need to be synthesised. The aim of this literature review is to summarise the evidence of associations of micro-, meso-, and macro- built environmental attributes with PA levels among refugee children. Study search and screening procedures A systematic search of peer-reviewed publications was conducted by one author (SC) in August 2020. Six elec- tronic databases (PubMed, Web of Science, SPORTDis- cus, ERIC, ScienceDirect, and SpringerLink) and one refugee-related journal (Journal of Refugee Studies) were individually searched using three sets of search terms on built environments, physical activity, and the target group. A full description of search queries is shown in Supplementary Material (Table S1). The study selection and screening process was managed using Zotero refer- ence manager software [32]. The articles identified in the search were screened based on their title and ab- stract first, then based on full text. The initial screening was performed by one author (SC), with randomly se- lected studies re-evaluated by another author (MK) for consistency. Screening based on full-text articles was Bronfenbrenner’s ecological systems theory [27] has been applied as a framework to understand refugee chil- dren’s day-to-day activities [28, 29]. The built environ- ment around refugee children includes three environmental layers of interest: micro-environment; meso-environment; and macro-environment. The micro- environment is the immediate vicinity of the child’s ac- commodation and contains the structures with which the children have direct contact in their daily lives [29]. Examples include the home/refugee camp and its Fig. 1 Diagram of environmental attributes on micro-, meso- and macro-level interacting with refugee children’s PA . 1 Diagram of environmental attributes on micro-, meso- and macro-level interacting with refugee children’s PA Chen et al. Conflict and Health (2021) 15:55 Page 3 of 10 carried out by SC, and the results were checked by AC. Any disagreements between them were resolved in con- sultation with TS. This review was preregistered in PROSPERO (CRD42020201186). meta-analysis. The final integrated synthesis consists of a narrative commentary for each of three built environ- ment levels and combines the results of quantitative and qualitative syntheses. The following inclusion criteria were applied: (1) peer- reviewed journal articles published in English between 2000 and 2020; (2) studies including healthy refugee children and unaccompanied refugee minors aged be- tween 6 and 12 years old; and (3) studies examining as- sociations of built environmental attributes with refugee children’s PA either quantitatively or qualitatively. Arti- cles with a broader age range were considered eligible if they included the 6–12 years age group, and distinct en- vironmental correlates may exist for PA among younger children (2–5 years) [33] and adolescents (13–18 years) [34, 35]. Data synthesis It was considered that assessing the quality of each study in a formal manner would not add useful information at this stage, due to the fact that research on refugee chil- dren’s PA and the built environment is still at an early stage, where most studies are cross-sectional, small scale, and exploratory. For quantitative studies, a relationship between an environmental attribute and a PA measure was considered as a distinct case. A positive relation be- tween them (e.g., more playgrounds related to more PA) was coded “+”, while non-significant relation was coded “0”. Qualitative studies were analysed thematically using NVivo software in three stages: (1) line-by-line coding of primary studies; (2) organising codes into themes and (3) development of analytical themes. Differences in opinion between the reviewers were discussed until con- sensus was reached. After a full-text evaluation of in- cluded studies, a narrative review was chosen due to a small number of eligible articles, most of which were qualitative in design. These reasons also precluded Data extraction h f ll The following information was extracted from each art- icle: author; publication year; study type (quantitative/ qualitative), study design (quantitative only); sample characteristics (size, age, country of origin); study set- tings (location/host country, length of stay); built envir- onmental attributes (categorised into micro, meso, and macro levels) and measurement methods; PA measures and measurement methods; analysis methods; and find- ings. Relevant data were extracted, double-checked and all studies were independently appraised by two authors (SC and AC). Any discrepancies were resolved through discussion between them. Results Characteristics of the studies reviewed Figure 2 shows the flowchart of the article search/ screening process according to the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta- Analyses) statement [36]. A total of 493 studies initially identified were reduced to 47 after screening based on title and abstract. Of these, eight studies (one added at the last stage from authors’ reference lists) remained after the full-text screening. Characteristics of the se- lected studies are presented in Table 1. Most (75%) of the studies were published in the past five years, and half of them were conducted in the USA. One of the in- cluded articles examined a local refugee camp in Palestine [41]. Most of the studies were qualitative, while there was one quantitative study, which observed the number of park users before and after park development for refugees [37]. PA was measured either as self-report or parent-report in 7 studies. One study used observa- tion by researchers [37], while two studies combined observation and self-report measures [39, 41]. Demo- graphics of participants in these studies were as follows: the majority (63%) of the studies investigated children from multi-ethnic backgrounds, and 37% of them came from Muslim countries. Half of the studies examined those with a transit period (in the host country), in which participants spent no more than six months. All of the studies investigated meso-environmental attri- butes (primarily neighbourhood-level factors), with four studies additionally examining attributes of micro- environments. A detailed description of each study is provided in Supplementary Material (Table S3–4). Study search and screening procedures Studies where parents reported children’s PA were also eligible. The review start date of 2000 was chosen, given that refugee children’s physical activity has been examined only recently. Meso-environment The meso-environment comprises refugee children’s school/community and broader neighbourhood. All studies reviewed (both quantitative and qualitative) ex- amined meso-environments in relation to refugee chil- dren’s PA (Table 2). Formal activity space Limited accessibility to formal space for PA was cited as a negative influence on refugee children’s PA. Quali- tative studies reported that limited or lack of access [14, 38] or lack of transportation to exercise facilities [39, 42] were barriers to refugee children’s PA. Moreover, one study indicated that access to outdoor facilities could in- crease refugee children’s PA [40]. It was found that there are two types of activity space relevant to refugee children’s PA. One is ‘formal’, while the other in ‘informal’ activity space (investigated in the next section). In this review, formal space is a play space/area built specifically for the purpose of physical activity, sports and exercise, including playgrounds, bas- ketball courts, and sports fields [14, 37, 38, 42]. A pre- and post-construction observational study [37] investigated refugee children’s physical activity before and after an undeveloped open space adjacent to transi- tional homes for refugees was transformed into a Micro-environment Available indoor space The micro-environment, which refers to refugee chil- dren’s home/refugee camp and its immediate vicinity, was examined in four qualitative studies [14, 38, 40, 41]. One factor found to be relevant to PA was the availabil- ity of sufficient indoor space for play at home. Two stud- ies [14, 38] reported that cramped living arrangements were a barrier to children playing actively indoors. For example, Somali mothers, who had migrated with their families to Bristol, UK and were residing in small apart- ments within residential tower blocks, described the lack of individual space and communal facilities within the housing schemes as barriers to their children’s physical activity [14]. Similarly, in a US study [38], Somali, Hmong, and Latino parents who had migrated to Chen et al. Conflict and Health (2021) 15:55 Page 4 of 10 Chen et al. Conflict and Health Fig. 2 Flow chart of database search and screening recreational park. Increased PA was observed in spaces designed for PA after renovation (e.g., play area, ball courts; garden) in children. Moreover, a higher propor- tion of female children observed within the park post- construction engaged in vigorous physical activity than those observed pre-construction. From the supplemen- tary material provided by the corresponding author, ob- served cases of girls inside the park boundaries rose from 13 to 79% after the construction. It rose from 35 to 75% for boys. Overall, 85% PA observed in the play area was of moderate to vigorous intensity. Purpose-built play spaces and sports facilities were associated with propor- tionally more moderate-to-vigorous physical activity and less sedentary behaviour compared with shaded sitting areas. The overall use of adjacent streets, alleys and sur- rounding parking lots has declined after a park redevelopment. Minnesota reported that lack of indoor space in their apartment blocks was a barrier to physical activity. Only one study conducted in a refugee camp setting included a reference to the design of refugee accommodation, and indicated that ‘dedicated spaces’ for play inside the camp (indoors and outdoors) helped children to engage in PA frequently by providing them with a safe environment [41]. There was no quantitative study on micro- environments and refugee children’s PA. Informal activity space The importance of ‘informal space for PA’ was also a prominent theme that emerged from the qualitative studies. Informal space for PA includes any urban spaces Page 5 of 10 Chen et al. Conflict and Health (2021) 15:55 Chen et al. Conflict and Health Table 1 Characteristics of eight studies included in the review No. Authors [ref] Publication year Study design Study settings Countries of origin Length of stay Environment-levels Sample size PA measurement 1 King et al. [37] 2015 quant. HIC, USA Ethnic minority 1–3 years meso park observation study observation 2 Allport et al. [14] 2019 qual. HIC, UK Somali > 3 years micro (home), meso N = 6 self- and parent-report 3 Arcan et al. [38] 2018 qual. HIC, USA Somali, Latino, Hmong > 3 years micro (home), meso N = 67 parent-report 4 Guest [39] 2013 qual. HIC, USA No specific, multi-ethnic < 6 months meso N = 239 of 380 observation and self-report 5 Hertting & Karlefors [15] 2013 qual. HIC, Sweden No specific, multi-ethnic < 6 months meso N = 20 self-report 6 MacMillan et al. [40] 2015 qual. HIC, Australia Iran, Indonesia,Pakistan, Malaysia, Kenya, Uganda < 6 months meso N = 19 self-report 7 Veronese et al. [41] 2020 qual. LMIC, Palestine Palestine < 6 months micro (refugee camp), meso (school, community) N = 29 observation and self-report 8 Wieland et al. [42] 2015 qual. HIC, USA Cambodia, Mexico, Somali, Sudan Not mentioned micro (home), meso N = 127 self-report : Muslim percentage (%) of total population > 70%; qual.: qualitative; quant.: quantitative; HIC: high income countries; LMIC: low- and middle-income countries; “meso” refers to neighbourhood environments unless otherwise specified Chen et al. Conflict and Health (2021) 15:55 Page 6 of 10 Table 2 Summary of built-environment attributes associated with refugee children’s PA Environmental level Built environmental attributes Quantitative Qualitative Relationships found Relationship identified Micro-environments Available indoor space 2, 3, 6, 7 Formal space for PA 7 Meso-environments Formal space for PA 1 (renovation of play area) Informal space for PA (public, outdoor, green, places for gathering) 2, 3, 5, 6, 7, 8 Neighbourhood safety (traffic-, sidewalk-organisation, violence) 2, 3, 4, 6, 7 Accessibility to formal space for PA 2, 3, 4, 6 1: King et al. [37], 2: Allport et al. [14], 3: Arcan et al. [38], 4: Guest [39], 5: Hertting & Karlefors [15], 6: MacMillan et al. Refugee children vs non-refugee children Another theme that emerged was neighbourhood safety. Four studies reported that neighbourhoods and school environments need to be safe for refugee children to play [14, 38, 40, 41]. Migrant mothers expressed their concerns about the existing traffic problems and danger from violence in the UK [14]. Since parents considered that adult supervision was required for children’s activ- ities outside, they preferred to keep their children at home [14]. Thus, parents’ safety concern can be a major factor restricting refugee children’s PA. The authors found that built environmental barriers and facilitators to physical activity for refugee children, i.e., access to physical activity facilities and neighbourhood safety, were similar to those identified for non-refugee children’s PA in earlier reviews. However, the findings do not necessarily mean that refugee and non-refugee children are equal in their access to physical activity fa- cilities. Future research needs to compare refugee and non-refugee children in terms of how active they are, where they engage in physical activity, and how access- ible activity spaces are. Such research would highlight the disparities in PA levels and opportunities between refugee and non-refugee children. With regard to safety concerns, they are often about road safety or local crime for non-refugee children [20]. However, refugee children need to adapt to new, unfamiliar environments when they come to their host country. Since they may have es- caped from war situations or have experienced military occupation [41], they may be more cautious and sensi- tive about safety issues than non-refugees [40]. Such concerns by their parents are particularly salient, as where children can play typically dictated by their par- ents [14]. Future research needs to pay particular atten- tion to how refugee children and parents perceive danger in surrounding environments and to what extent it is different from non-refugee children and parents. This review did not find studies that examined the role of macro-environment in refugee children’s PA, although it was found to be related to non-refugee children’s PA Macro-environment None of the studies included in this review investigated any attributes of macro-environment, such as transport systems or urban versus rural areas. Informal activity space [40], 7: Veronese et al. [41], 8: Wieland et al. [42] formal play area in undeveloped greenspace resulted in greater use of that area for PA by refugee children [37]. There was only one study reporting on a low- and middle-income country (LMIC) setting in which chil- dren stayed in a temporary refugee camp. All other stud- ies reported on refugee facilities (non-camps) within high-income countries (HICs). Our review shows that studies in LMICs are greatly under-represented, since the majority (68%) of refugees reside in low- and middle-income countries [43]. that are readily and freely available by refugee children. Such spaces enable children to engage in physically ac- tive, spontaneous play [14, 15, 40, 41]. Children men- tioned that there was a lack of space to gather and play as a group, and this appeared to discourage them from engaging in PA [42]. Another study of migrants in the USA reported that refugee children preferred being ac- tive in informal gathering spaces with friends rather than engaging in formal sport [42]. Camp and non-camp settings In meso-environments within HICs, one study argued that free access to outdoor space and parks are particu- larly important for refugee children since their financial situation would not allow them to participate in orga- nised sports and other fee-based activities [14]. However, local parks are not always a safe place to play in deprived areas [44], which are often chosen as a site for refugee accommodation [11]. Given that safety may be a particu- lar concern, research needs to identify what measures can be implemented to ensure parks are safe for refugee children to play. Natural surveillance, in which actions and behaviour in a park can be observed by “eyes on the street”, seems like an important principle [14]. Future studies from HICs can examine other park features (e.g., size, features, distance) that encourage refugee children’s active park use. Only one study was conducted in an LMIC setting [41]. It illustrated that refugee children without access to safe and suitable spaces for PA (e.g., parks) had to use space such as roads, streets and other open spaces despite dangers from military confrontation. Further studies should focus on settings in LMICs to identify PA barriers and facilitators in diverse contexts. The included studies were conducted in different refugee accommodation settings: a refugee camp in an LMIC [41], non-camp settings including designated refugee ac- commodations located in HICs [14, 15, 37, 39, 40] and community-accommodations specific to their culture in their host countries [38, 42]. It is difficult to compare these settings due to the small number of studies. How- ever, they are likely to differ in terms of the provision of spaces for children. Thus, it could be postulated that en- vironmental correlates of PA may be different for camp and non-camp settings. Further studies should identify environmental attributes related to children’s PA in these diverse contexts, and investigate whether similar environmental attributes may be relevant or there are unique environmental correlates in specific settings. Summary of research findings In this review, we identified eight studies examining as- sociations of micro- and meso-environments’ characteris- tics with refugee children’s PA. Firstly, all but one of the studies were qualitative, and most of them were con- ducted in the last five years (75%). The empirical re- search on associations between the built environment and refugee children’s physical activity is in its infancy. Secondly, qualitative studies suggest that both micro- and meso-environments are relevant to refugee children’s PA. These include available indoor spaces (micro) and accessible formal and informal spaces for PA and safety (meso). One quantitative study found that installing a Page 7 of 10 Page 7 of 10 Chen et al. Conflict and Health (2021) 15:55 Chen et al. Conflict and Health [19]. Considering that the location of refugee accommo- dation is a matter for the discretion of local authorities, future research on this topic is needed to inform where best to build refugee facilities to enhance refugee chil- dren’s activity, health and safety. subdivided functional activity zones. It suggests the im- portance of a high-quality park with suitable facilities and amenities rather than the mere presence of a park. Identifying design attributes of parks relevant to refugee children’s PA is informative for design and management of refugee-related facilities. Micro- and meso- environments Qualitative studies reviewed reported the importance of informal space for refugee children to engage in phys- ical activity [14, 42, 44]. However, this may be a reflec- tion of lack of opportunities for them to take part in sports and exercise. Given that it can be difficult to or- ganise sports in refugee settings, it is important that there is at least informal space such as open spaces where children can be active with friends during leisure time. It is thus conceivable that diverse opportunities (both formal and informal spaces) are important for refugee children’s PA. Considering that participation in sports activities involves not only physical activity but also social interactions, providing refugee children with such opportunities is likely to have multiple benefits [39]. Future studies can assess the effect and feasibility of sports and other activity programs targeting refugee children and investigate their benefits. It was reported that refugee children have limited access to neighbourhood places for their play [14]. In such a situation where meso environments are not conducive to children’s physical activity, micro-environments (refu- gee accommodation and its immediate vicinity) are likely to play an important role in refugee children’s PA in both camps and non-camp settings. However, existing studies on micro-environments do not seem to suggest that refugee facilities provide adequate opportunities for children’s PA. One study reported that being physically active indoors at home is not practical due to noise and space issues [38]. The other study found that passage- ways, stairwells and basement areas within apartment blocks were utilised as makeshift exercise spaces for oc- cupants [40]. However, they may not be totally safe for children to play. It is recommended that additional spaces suitable for children to be active should be pro- vided in/around their accommodations. Measurement issues for physical activity and built environment There was no objective measurement of PA in the stud- ies identified. It is evident that self-report measures con- tain errors and bias in capturing physical activity [45]. Future research needs to employ devices such as acceler- ometers to measure refugee children’s PA. Furthermore, there was little objective measurement of the built envir- onment in the studies reviewed. The quantitative study by King et al. (2015) provided the pre- and post- construction satellite images, which show the presence of some PA facilities after renovation [37]. The qualita- tive studies included in this review used self-report Formal vs informal spaces for refugee children’s PA The quantitative study reviewed highlights the import- ance of formal activity space quality [37]. It found that children’s energy expenditure in park areas increased from 2010 to 2012, after an undeveloped green space park had been transformed into a recreational park with Chen et al. Conflict and Health (2021) 15:55 Chen et al. Conflict and Health (2021) 15:55 Page 8 of 10 Gender and cultural differences Explore to what extent the quality of formal spaces (presence of physical activity facilities and amenities) and informal spaces (presence of green space and trees, seating, lighting, multiple things to do) are associated with refugee children’s PA; Previous studies have shown that refugee girls and boys are likely to play differently [47–49] and have different preferences for places where they would like to play [15, 39, 50]. There was only one study investigating gender differences in this review [37]. It found that more girls participating in vigorous physical activity were observed after park renovation. This seems to suggest that girls may require well-designed places for play, while the presence of open space (without facilities/amenities) may be sufficed for boy’s PA. There were studies examining refugee children from diverse cultural backgrounds [15, 37–40, 42], but they did not exam- ine whether there were between-culture differences in environmental correlates of PA. Further studies need to investigate gender-specific and culture-specific as- sociations between refugee children’s PA and environ- mental attributes. Understand the role of macro environments in refugee children’s PA, in particular, whether the location of a refugee accommodation within the city is relevant to their PA levels; Use objective measures (i.e., Geographic Information Systems) to identify environmental attributes; Identify children’s PA (duration and intensity) using objective measurement methods such as accelerometer; Compare environmental correlates of non-refugee and refugee children’s PA in a single study to further understand whether the previous findings on non- refugee children can apply to refugee children; Examine environmental correlates of refugee children’s PA in diverse contexts such as in camp and non-camp settings and in low- and middle- income countries; Limitations of the review There are a few limitations in this review. The inclusion of only peer-reviewed English-language articles may have excluded studies that were conducted in non-English speaking countries with relevant information. For ex- ample, much research on refugee children in Germany is reported in German [10, 51]. This review focused on the built environment of places where refugee children lived. However, there may be policies and regulations (e.g., organised PA program) [38, 42] within refugee accom- modations, which may be strong determinants of how active children can be. Future reviews may need to con- sider how policy and environmental factors may be re- lated (independently and jointly) to children’s PA. Finally, we conducted a narrative review, reflecting a small number of studies identified and an early stage of research on this topic. It is expected that more fruitful literature reviews will be conducted in future in light of an increasing interest in refugee’s health and well-being in international contexts. There are a few limitations in this review. The inclusion of only peer-reviewed English-language articles may have excluded studies that were conducted in non-English speaking countries with relevant information. For ex- ample, much research on refugee children in Germany is reported in German [10, 51]. This review focused on the built environment of places where refugee children lived. Conduct longitudinal studies that track refugee children’s PA patterns when they relocate from a temporary refugee facility to other accommodation; y g y Investigate environmental correlates of refugee boys’ and girls’ PA separately to produce gender-specific design recommendations; g Understand if environmental correlates of refugee children’s PA differ depending on their ethnic backgrounds. Research agenda: recommendations for future studies measures of the built environment, but these were, by their nature, descriptive and subjective. It is important that further studies employ objectively derived (GIS or audit) measures or validated self-report measures of rele- vant built environmental attributes. Future studies should learn from existing studies targeting non-refugee children, as they have developed a range of methods to assess the built environment [46]. Particular attention may be given to specific attributes of PA spaces (dis- tance, size, accessibility and features) and safety (per- ceived safety by parents and by children, objective measures such as crime statistics). This study identified gaps in the literature of environ- mental attributes associated with PA of school-aged (6– 12) refugee children. Overall, this research field requires more quantitative studies to better understand environ- mental features that are conducive to refugee children’s PA. Below are specific research topics that deserve de- tailed investigations: Examine specific features of environmental attributes (size, quality and accessibility of individual and communal spaces for PA) associated with refugee children’s PA; Funding This study has been conducted as a part of the corresponding author’s doctoral research project ‘Socio-spatial Interaction (SSI): Design strategies on promoting “Wartezustand” of School-aged refugees in Berlin’ funded by China Scholarship Council (CSC) [Grant No. 201708080019]. The funders had no role in undertaking this review. Open Access funding enabled and orga- nized by Projekt DEAL. 12. Block K, Gibbs L. Promoting Social Inclusion through Sport for Refugee- Background Youth in Australia: Analysing Different Participation Models. Soc Incl. 2017;5(2):91–100. https://doi.org/10.17645/si.v5i2.903. 12. Block K, Gibbs L. Promoting Social Inclusion through Sport for Refugee- Background Youth in Australia: Analysing Different Participation Models. Soc Incl. 2017;5(2):91–100. https://doi.org/10.17645/si.v5i2.903. 13. Montgomery C. The “Brown Paper Syndrome”: Unaccompanied Minors and Questions of Status. Refuge Can J Refug. 2002;20(2). https://refuge.journals. yorku.ca/index.php/refuge/article/view/21255. Accessed 9 Jan 2018. 14. Allport T, Mace J, Farah F, Yusuf F, Mandjoubi L, Redwood S. “Like a life in a cage”: understanding child play and social interaction in Somali refugee families in the UK. Health Place. 2019;56:191–201. https://doi.org/10.1016/j. healthplace.2019.01.019. Abbreviations PA h i l ti Abbreviations PA: physical activity; UNICEF: United Nations High Commissioner for Refugees; PRISMA: Preferred Reporting Items for Systematic Reviews and Meta-Analyses; HIC: high-income country; LMIC: low- and middle-income country 4. Ahn S, Fedewa AL. A meta-analysis of the relationship between Children’s physical activity and mental health. J Pediatr Psychol. 2011;36(4):385–97. https://doi.org/10.1093/jpepsy/jsq107. 5. Guthold R, Stevens GA, Riley LM, Bull FC. Global trends in insufficient physical activity among adolescents: a pooled analysis of 298 population- based surveys with 1·6 million participants. Lancet Child Adolesc Health. 2020;4(1):23–35. https://doi.org/10.1016/S2352-4642(19)30323-2. Acknowledgements 8. Okely A, Salmon J, Vella S, et al. A systematic review to update the Australian physical activity guidelines for children and young people. Fac Soc Seci - Pap Published online January 1, 2012. https://ro.uow.edu.au/sspa pers/1246 The authors would like to thank Dr. Diane K. King for providing additional information of the research. 9. Hek R. The experiences and needs of refugee and asylum seeking children in the UK : a literature review. Published online 2005. http://dera.ioe.ac.uk/53 98/1/RR635.pdf. Accessed 11 Jan 2018. 9. Hek R. The experiences and needs of refugee and asylum seeking children in the UK : a literature review. Published online 2005. http://dera.ioe.ac.uk/53 98/1/RR635.pdf. Accessed 11 Jan 2018. Author details 1 1Urban Health Games Research Group (UHGs), Department of Architecture, Technische Universität Darmstadt, Darmstadt, Germany. 2Mary Mackillop Institute for Health Research, Australian Catholic University, Melbourne, Australia. 3Centre for Urban Transitions, Swinburne University of Technology, Melbourne, Australia. Received: 23 February 2021 Accepted: 28 June 2021 Received: 23 February 2021 Accepted: 28 June 2021 Competing interests No potential conflict of interest was reported by the authors. Availability of data and materials The Search strategies and coding is listed in Supplemental material: Table S1; overview of included quantitative studies and qualitative studies are listed in Table S2 and Table S3 and datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. 15. Hertting K, Karlefors I. Sport as a context for integration : newly arrived immigrant children in Sweden drawing sporting experiences. Int J Humanit Soc Sci. 2013;3(18):35-44. http://urn.kb.se/resolve?urn=urn:nbn:se:ltu:diva-64 56. Accessed 9 Jan 2018. 15. Hertting K, Karlefors I. Sport as a context for integration : newly arrived immigrant children in Sweden drawing sporting experiences. Int J Humanit Soc Sci. 2013;3(18):35-44. http://urn.kb.se/resolve?urn=urn:nbn:se:ltu:diva-64 56. Accessed 9 Jan 2018. Authors’ contributions The initial screening was performed by SC, with randomly selected studies re-evaluated by MK for consistency. Screening based on full-text articles was carried out by SC, and the results were checked by AC. Any disagreements between them were resolved in consultation with TS. All authors read and approved the final manuscript. 10. Lewek M, Naber A. Kindheit im Wartezustand. UNICEF; 2017. https://www. unicef.de/informieren/aktuelles/presse/2017/studie-fluechtlingskinder-in- deutschland/137440. Accessed 9 Jan 2018. 10. Lewek M, Naber A. Kindheit im Wartezustand. UNICEF; 2017. https://www. unicef.de/informieren/aktuelles/presse/2017/studie-fluechtlingskinder-in- deutschland/137440. Accessed 9 Jan 2018. 11. Anderson P. ‘You Don’t belong Here in Germany … ’: on the social situation of refugee children in Germany. J Refug Stud. 2001;14(2):187–99. https://doi. org/10.1093/jrs/14.2.187. 11. Anderson P. ‘You Don’t belong Here in Germany … ’: on the social situation of refugee children in Germany. J Refug Stud. 2001;14(2):187–99. https://doi. org/10.1093/jrs/14.2.187. Declarations 16. 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Int J Pediatr Obes. 2009;4(4):338–42. https:// doi.org/10.3109/17477160902763317. Conclusion Children living in refugee accommodation rarely meet physical activity guidelines [10, 51]. This literature re- view suggests that the built environment where they live (micro and meso environments) is partly contributing to low levels of physical activity. In order to help refugee children to be more physically active, they need to have access to indoor/outdoor play areas in refugee facilities Page 9 of 10 Page 9 of 10 Page 9 of 10 Chen et al. Conflict and Health (2021) 15:55 Chen et al. Conflict and Health (2021) 15:55 and safe outdoor space for activity in their neighbour- hoods. To produce more specific evidence that can in- form designs of refugee facilities, more interdisciplinary research involving architecture, urban design, planning, sports science, public health, psychology, and education is necessary. Researchers also need to collaborate with policymakers in refugee-related programs, sports and re- creation, and planning to understand their concerns and to disseminate research findings. Given that the number of refugees continues to increase worldwide [52], it is important that host countries provide healthy living en- vironments, particularly for vulnerable groups such as children. Future studies need to build on research of non-refugee children’s physical activity, for which there is a wealth of evidence, to advance our understanding on this topic. and safe outdoor space for activity in their neighbour- hoods. To produce more specific evidence that can in- form designs of refugee facilities, more interdisciplinary research involving architecture, urban design, planning, sports science, public health, psychology, and education is necessary. Researchers also need to collaborate with policymakers in refugee-related programs, sports and re- creation, and planning to understand their concerns and to disseminate research findings. Given that the number of refugees continues to increase worldwide [52], it is important that host countries provide healthy living en- vironments, particularly for vulnerable groups such as children. Future studies need to build on research of non-refugee children’s physical activity, for which there is a wealth of evidence, to advance our understanding on this topic. Supplementary Information h l l The online version contains supplementary material available at https://doi. org/10.1186/s13031-021-00393-2. 6. Twisk JWR. Physical activity guidelines for children and adolescents. Sports Med. 2001;31(8):617–27. https://doi.org/10.2165/00007256-200131080-00006. 7. Tremblay MS, Warburton DER, Janssen I, Paterson DH, Latimer AE, Rhodes RE, et al. New Canadian physical activity guidelines. 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https://openalex.org/W3082743851	https://research-repository.st-andrews.ac.uk/bitstream/10023/21146/1/ElMkami_2020_MR_High_senstivity_CC.pdf	English	null	High-sensitivity Gd&lt;sup&gt;3+&lt;/sup&gt;–Gd&lt;sup&gt;3+&lt;/sup&gt; EPR distance measurements that eliminate artefacts seen at short distances	Magnetic resonance	2,020	cc-by	11,262	High-sensitivity Gd3+–Gd3+ EPR distance measurements that eliminate artefacts seen at short distances Hassane EL Mkami1, Robert I. Hunter1, Paul A. S. Cruickshank1, Michael J. Taylor1, Janet E. Lovett1, Akiva Feintuch2, Mian Qi3, Adelheid Godt3, and Graham M. Smith1 1SUPA, School of Physics and Astronomy, University of St Andrews, St Andrews, KY16 9SS, UK 2Department of Chemical Physics, Weizmann Institute of Science, Rehovot, Israel 3Faculty of Chemistry and Center of Molecular Materials (CM2), Bielefeld University, Universitätsstraße 25, 33615 Bielefeld, Germany Correspondence: Hassane EL Mkami (hem2@st-andrews.ac.uk) and Graham M. Smith (gms@st-andrews.ac.uk) Received: 4 August 2020 – Discussion started: 13 August 2020 Revised: 13 November 2020 Accepted: 18 November 2020 Published: 9 December 2020 Hassane EL Mkami1, Robert I. Hunter1, Paul A. S. Cruickshank1, Michael J. Taylor1, Janet E. Lovett1, Akiva Feintuch2, Mian Qi3, Adelheid Godt3, and Graham M. Smith1 1SUPA, School of Physics and Astronomy, University of St Andrews, St Andrews, KY16 9SS, UK 2Department of Chemical Physics, Weizmann Institute of Science, Rehovot, Israel 3Faculty of Chemistry and Center of Molecular Materials (CM2), Bielefeld University, Universitätsstraße 25, 33615 Bielefeld, Germany Correspondence: Hassane EL Mkami (hem2@st-andrews.ac.uk) and Graham M. Smith (gms@st-andrews.ac.uk) Received: 4 August 2020 – Discussion started: 13 August 2020 Revised: 13 November 2020 – Accepted: 18 November 2020 – Published: 9 December 2020 Received: 4 August 2020 – Discussion started: 13 August 2020 Revised: 13 November 2020 – Accepted: 18 November 2020 – Published: 9 December 2020 Abstract. Gadolinium complexes are attracting increasing attention as spin labels for EPR dipolar distance measurements in biomolecules and particularly for in-cell measurements. It has been shown that ﬂip-ﬂop tran- sitions within the central transition of the high-spin Gd3+ ion can introduce artefacts in dipolar distance mea- surements, particularly when measuring distances less than 3 nm. Previous work has shown some reduction of these artefacts through increasing the frequency separation between the two frequencies required for the double electron–electron resonance (DEER) experiment. Here we use a high-power (1 kW), wideband, non-resonant system operating at 94 GHz to evaluate DEER measurement protocols using two stiff Gd(III) rulers, consisting of two bis-Gd3+–PyMTA complexes, with separations of 2.1 nm and 6.0 nm, respectively. We show that by avoiding the −1 2 E → 1 2 E central transition completely, and placing both the pump and the observer pulses on either side of the central transition, we can now observe apparently artefact-free spectra and narrow distance distributions, even for a Gd–Gd distance of 2.1 nm. Importantly we still maintain excellent signal-to-noise ratio and relatively high modulation depths. These results have implications for in-cell EPR measurements at naturally occurring biomolecule concentrations. agn. Reson., 1, 301–313, 2020 tps://doi.org/10.5194/mr-1-301-2020 Author(s) 2020. This work is distributed under e Creative Commons Attribution 4.0 License. Open Access High-sensitivity Gd3+–Gd3+ EPR distance measurements that eliminate artefacts seen at short distances Hassane EL Mkami1, Robert I. Hunter1, Paul A. S. Cruickshank1, Michael J. Taylor1, Janet E. Lovett1, Akiva Feintuch2, Mian Qi3, Adelheid Godt3, and Graham M. Smith1 1SUPA, School of Physics and Astronomy, University of St Andrews, St Andrews, KY16 9SS, UK 2Department of Chemical Physics, Weizmann Institute of Science, Rehovot, Israel 3Faculty of Chemistry and Center of Molecular Materials (CM2), Bielefeld University, Universitätsstraße 25, 33615 Bielefeld, Germany Correspondence: Hassane EL Mkami (hem2@st-andrews.ac.uk) and Graham M. Smith (gms@st-andrews.ac.uk) Received: 4 August 2020 – Discussion started: 13 August 2020 Revised: 13 November 2020 – Accepted: 18 November 2020 – Published: 9 December 2020 Magn. Reson., 1, 301–313, 2020 https://doi.org/10.5194/mr-1-301-2020 © Author(s) 2020. This work is distributed under the Creative Commons Attribution 4.0 License. 1 Introduction based SLs have been of particular interest as they already ex- ist as a major class of contrast agents used in MRI and show a strong stability against the oxidation or reduction conditions found in cells, making them an ideal candidate for in-cell distance measurements. Double electron–electron resonance (DEER) spectroscopy combined with site-directed spin labelling (SDSL) is a pow- erful technique to probe structural and dynamic properties in a wide range of biological systems. Over the past decades, distance measurements have been mainly associated with ni- troxide spin labels (SLs). This has led to the development of new experimental protocols and reliable data analysis pro- grams for a routine extraction of distances and investiga- tion of conformational changes. Amongst other reasons, the increasing interest in characterising proteins in their native environment has extended the spin labelling family to new labels based on paramagnetic metal ion complexes. Gd3+- Gadolinium is a half integer high-spin S = 7/2 metal ion, characterised by a broad distribution of zero-ﬁeld-splitting (ZFS) parameters and an isotropic g value at high ﬁeld (Rait- simring et al., 2005). At lower temperatures its EPR spectrum is dominated by the central transition −1 2 E → 1 2 E superim- posed on a broad featureless background coming from all the other transitions. To ﬁrst order, perturbation theory shows the central transition is independent of the ZFS interaction, while Published by Copernicus Publications on behalf of the Groupement AMPERE. H. EL Mkami et al.: Gd3+–Gd3+ EPR distance measurements 302 al., 2016; Dalaloyan et al., 2015). The Gd–Gd distances were calculated for a temperature at 160.4 K, the glass transition temperature of the mixture of glycerol-d8 and D2O, 50/50 (v/v), applying the wormlike chain model as described pre- viously (Dalaloyan et al., 2015). We explore two different experimental protocols. The standard approach is where the pump pulse is positioned at the central transition, but with variable offset between pump and observer pulses of up to 900 MHz. In general, we observe narrower distance distri- butions and improved Pake patterns as frequency separation is increased. In the second approach we place the pump and observer pulses on either side of the −1 2 E → 1 2 E central transition, avoiding excitation of the central transition alto- gether. 1 Introduction In this case, we observe almost perfect Pake patterns, consistent with elimination of the artiﬁcial broadening of the distance distribution, even for the 2.1 nm Gd ruler. the other transitions scale linearly with the axial ZFS param- eter D. However, to second order the central line narrows as the operational frequency increases and its width scales pro- portionally with D2 gB0 . Therefore, high frequencies have been favoured for distance measurements using Gd3+-based spin labels due to an expected improved concentration sensitivity associated with placing the pump or observer frequency at the central line. Since their introduction in 2007, several Gd3+-based spin labels have been developed and a wide range of molecules have been successfully investigated, from simple model com- pounds to proteins, DNA, peptides and other biological sys- tems (Gordon-Grossman et al., 2011; Potapov et al., 2010; Raitsimring et al., 2007; Yagi et al., 2011; Shah et al., 2019; Wojciechowski et al., 2015). The good agreement between distance distributions derived from Gd–Gd DEER data and those resulting from other techniques has motivated re- searchers to attempt investigation of in-cell proteins and peptides (Qi et al., 2014; Yang et al., 2019; Dalaloyan et al., 2019; Martorana et al., 2014). In most of these stud- ies, Gd3+ was treated like an S = 1/2 system and standard data analysis software packages, developed initially for ni- troxides, have generally been applied. This approach has proven successful for Gd–Gd distances above 3–4 nm, but below 3–4 nm strongly damped dipolar distortions and arti- ﬁcially broadened distance distributions were obtained (Co- hen et al., 2016; Dalaloyan et al., 2015; Feintuch et al., 2015; Manukovsky et al., 2017). This is caused by unwanted ﬂip- ﬂop transitions, whose effects are enhanced by strong dipolar coupling (Cohen et al., 2016; Manukovsky et al., 2017). This effect can be ameliorated by increasing the frequency off- set between the pump and observer pulses (PO offset) (Co- hen et al., 2016). This has usually been achieved by having the pump pulse positioned at the central transition and posi- tioning the observer pulse with as large a frequency offset as possible. This is usually difﬁcult to achieve with standard res- onator bandwidths on commercial instruments. Nevertheless, a high-frequency dual-mode cavity with an ingenious design has been demonstrated, which can accommodate pump and observer pulses with separations of more than 1 GHz (Co- hen et al., 2016). 1 Introduction Unfortunately such cavities, particularly at high ﬁelds and low temperatures, can be challenging to set up precisely. Relaxation-induced dipolar modulation enhance- ment (RIDME) is another experimental alternative where no such restrictions apply, since it is a single-frequency tech- nique. However, it suffers from overtones of dipolar frequen- cies and requires a more complicated data analysis (Collauto et al., 2016; Keller et al., 2017; Meyer and Schiemann, 2016; Razzaghi et al., 2014). For this short 2.1 nm distance we show that any loss of sen- sitivity from not exciting the central transition is offset by the shorter time window now required to make the measurement. 2.1 Sample preparation The synthesis of the Gd rulers has been described else- where (Qi et al., 2016). Solutions of 40 µM concentration (molecules) of the Gd ruler (2.1 nm) and Gd ruler (6.0 nm) were prepared in 50/50 (v/v) deuterated glycerol and D2O (for chemical structure see Fig. 1). The use of the glycerol- d8/ D2O (1 : 1, v/v) was dictated by the desire to obtain a good glass, to reduce scattering losses and to extend the phase memory time. For the Q-band measurements the sam- ples were transferred to standard 3 mm quartz EPR tubes and ﬂash-frozen in liquid nitrogen prior to loading into the spectrometer. For the W-band experiments, the samples were transferred into 27 mm long ﬂuorinated ethylene propylene (FEP) tubes with 3 mm outer diameter and 2 mm inner di- ameter and ﬂash-frozen in liquid nitrogen prior loading into sample-holder cartridges that were separately pre-cooled in liquid nitrogen. These sample-holder cartridges were then loaded into the W-band spectrometer which had been pre- cooled to 150 K. 2.2.1 Non-resonant induction mode sample holder The sample is placed in a FEP tube within a sample- holder cartridge and mounted into a spring-loaded mount (see Fig. S1a in the Supplement). The latter co-locates to a smooth cylindrical waveguide transmission line of diameter 3 mm, which supports two orthogonal TE11 modes. Radia- tion is fed to the shorted waveguide via an adapted corrugated feed horn that feeds to a wider bore corrugated pipe which in turn feeds to an optical system. An adjustable backshort, con- sisting of a roof mirror with a shallow roof angle, is placed in the waveguide below the sample. Adjustment of this back- short allows optimisation of the cross-polarised signal com- ponent, which is important both for initial experimental set- up and for measurements. This adjustment is achieved using piezo motors (Attocube Systems AG) that separately control the roof angle and the relative distance of the backshort to the sample. A non-resonant induction mode sample holder is essentially a shorted symmetrical waveguide where two dominant orthog- onal linearly polarised modes can propagate. The incident linearly polarised beam can be decomposed into two orthog- onal circular polarisation components. At resonance, one cir- cular component is absorbed (or emitted) by the sample, re- sulting in a reﬂected beam containing a cross-polarised com- ponent perpendicular to the linear polarisation of the incident beam. In the system described here these reﬂected signals are diplexed via a wire grid polariser to separate the high-power incident beam from the very much smaller cross-polarised component which is passed to the detection system. The di- mensions of the empty waveguide sample holder (3 mm di- ameter) are chosen to be single-mode at the operating fre- quency of the spectrometer. The advantage of these shorted waveguide sample holders is that they are inherently wide- band since they are only weakly resonant. It might be thought that sensitivity would be strongly diminished as there is no resonant enhancement of either the excitation pulse or sig- nal. However, the critical parameter that determines the reso- nant enhancement is the microwave conversion efﬁciency of the sample holder, c, with units of gauss per root watt (Smith et al., 2008). A single-mode shorted waveguide at 94 GHz can have a comparable or better conversion efﬁciency (typ- ically about 0.5 G/W1/2 at the sample) than a commercial X-band pulsed resonator. H. EL Mkami et al.: Gd3+–Gd3+ EPR distance measurements 303 Figure 1. Chemical structures of Gd-ruler systems used in the current study. Figure 1. Chemical structures of Gd-ruler systems used in the current study. Figure 1. Chemical structures of Gd-ruler systems used in the current study. 2.2 Spectrometers The spectrometers used for these measurements were a Bruker ELEXSYS E580 high-power (150 W) Q-band pulsed spectrometer with ER 5106QT-2W resonator and a home- built 1 kW W-band spectrometer (93.5–94.5 GHz). This W- band spectrometer, widely known as HiPER, has been de- scribed previously (Cruickshank et al., 2009; Motion et al., 2017). It operates with a wideband non-resonant (or weakly-resonant) induction mode sample holder, which is now described in more detail. In the present work, we demonstrate a simpler approach that uses a wideband non-resonant or weakly resonant sam- ple holder to show the beneﬁt of wideband measurements. We use two Gd rulers with calculated distances between two Gd(III)–PyMTA complexes of 2.1 and 6.0 nm (Qi et Magn. Reson., 1, 301–313, 2020 https://doi.org/10.5194/mr-1-301-2020 H. EL Mkami et al.: Gd3+–Gd3+ EPR distance measurements 3.1 EPR spectra and relaxation times The simulation of the sub-spectra was performed using EasySpin by considering a distribution of the ZFS parameters D and E (Stoll and Schweiger, 2006). The magnitude and distribution of the ZFS depend primar- ily on the nature of the interactions between the Gd3+ ion and the ligand and/or solvent molecules coordinating to the Gd3+ ion. These are taken into account by the D and E strain parameters used by EasySpin, and they are deﬁned as monomodal Gaussian distributions. Furthermore, it was shown in some cases that a bimodal Gaussian distribution centred on D and −D considerably improved the simula- tion (Raitsimring et al., 2005; Clayton et al., 2018). The D parameters used for the simulations are reported with those obtained for other Gd3+ tags in Table S1 in the Supplement. The DEER experiments were carried out using the standard dead-time free four-pulse sequence (Pannier et al., 2000). π 2 (obs) →τ1 →π (obs) →t →π (pump) →τ1 + τ2 −t →π(obs) →τ2 →echo g pp The phase memory time, Tm, and the spin lattice relaxation time, T1, were measured for both samples at 10 K with the magnetic ﬁeld set on the central maximum of the ED-FS, and results were similar to other reported studies on Gd3+ com- plexes measured at low temperatures (Collauto et al., 2016; Raitsimring et al., 2014). The Tm time traces were ﬁtted ini- tially with a sum of two exponential functions with free expo- nent values, and excellent ﬁts were achieved with ﬁxed expo- nent values of 1 and 2, and the results are shown in Fig. S2a. The ﬁt to two exponentials (R2 = 0.9999) was rather better than could be achieved by ﬁtting to a stretched exponential. Tm values estimated from these ﬁts are shown in Table S2 and provide evidence for fast and slow relaxation contribu- tions to the echo decay. It is emphasised that these relaxation data were taken at the central maximum, but Tm relaxation The echo intensity was monitored as a function of t. For the Gd ruler (6.0 nm), the pump pulse was applied, for all ex- periments, at the maximum of the ED-FS spectrum, whereas the observer frequency was set at 94 GHz with different off- sets from the pump frequency as reported in Table 1. 3.1 EPR spectra and relaxation times The ED-FS spectra for both samples are similar to those reported for other Gd3+ complexes with a characteristic sharp line corresponding to the central transition and a broad featureless background resulting from contributions of all other transitions. The spectra recorded at Q- and W-bands are shown in Fig. 2. The simulation of the sub-spectra was performed using EasySpin by considering a distribution of the ZFS parameters D and E (Stoll and Schweiger, 2006). The magnitude and distribution of the ZFS depend primar- ily on the nature of the interactions between the Gd3+ ion and the ligand and/or solvent molecules coordinating to the Gd3+ ion. These are taken into account by the D and E strain parameters used by EasySpin, and they are deﬁned as monomodal Gaussian distributions. Furthermore, it was shown in some cases that a bimodal Gaussian distribution centred on D and −D considerably improved the simula- tion (Raitsimring et al., 2005; Clayton et al., 2018). The D parameters used for the simulations are reported with those obtained for other Gd3+ tags in Table S1 in the Supplement. The phase memory time, Tm, and the spin lattice relaxation time, T1, were measured for both samples at 10 K with the magnetic ﬁeld set on the central maximum of the ED-FS, and results were similar to other reported studies on Gd3+ com- plexes measured at low temperatures (Collauto et al., 2016; Raitsimring et al., 2014). The Tm time traces were ﬁtted ini- tially with a sum of two exponential functions with free expo- nent values, and excellent ﬁts were achieved with ﬁxed expo- nent values of 1 and 2, and the results are shown in Fig. S2a. The ﬁt to two exponentials (R2 = 0.9999) was rather better than could be achieved by ﬁtting to a stretched exponential. Tm values estimated from these ﬁts are shown in Table S2 and provide evidence for fast and slow relaxation contribu- tions to the echo decay. It is emphasised that these relaxation data were taken at the central maximum, but Tm relaxation The ED-FS spectra for both samples are similar to those reported for other Gd3+ complexes with a characteristic sharp line corresponding to the central transition and a broad featureless background resulting from contributions of all other transitions. The spectra recorded at Q- and W-bands are shown in Fig. 2. 2.2.2 Frequency-dependent power variation in HiPER It is necessary to re-optimise the pulse lengths for each fre- quency offset, due to power variation from the transmitter chain. For technical reasons, these measurements were made without phase cycling, but instead offsets were removed by separate automatic measurements of the baseline on either side of the echo (at a slight cost in SNR). It should be noted that the transmitted power level (from the ampliﬁer–isolator–switch combination) is not constant over the whole range of the frequency offsets used in this study. This is illustrated in Fig. S1b where we show the power level versus frequency monitored at different points along the transmitter chain of HiPER. As a consequence of this, absolute modulation depths should not be compared quanti- tatively. DEER data were processed using the DeerAnalysis (2019) program that allows extraction of distance distributions (Jeschke et al., 2006). Fits to the data were based on standard Tikhonov regularisation analysis using the bending point in the L-curve. The excitation proﬁles of the pump and observer pulses were simulated using a home-written MATLAB- based program using simple spin mechanics (Jeschke and Polyhach, 2007). The simulated ED-FS spectra and the asso- ciated sub-spectra for each transition were performed using the EasySpin program (Stoll and Schweiger, 2006). 2.2.1 Non-resonant induction mode sample holder Compared to a dual-mode W-band resonator, a shorted waveguide can also offer a hugely in- creased sample volume (up to 75 µL in the current system), provided that sample dielectric losses are low, which is usu- ally the case for measurements made at cryogenic tempera- tures. The non-resonant cavity also offers considerable ﬂexi- bility in optimising excitation frequencies and bandwidths at both pump and observer frequencies. Therefore these types of systems can have extremely high concentration sensitivity whilst offering very large instantaneous bandwidths. This po- tentially makes them ideal for Gd SL distance measurements, especially when large separations between the observer and pump pulses are required. p To facilitate the loading of pre-frozen samples, the samples and sample-holder cartridges are pre-cooled externally to the spectrometer in liquid nitrogen. The spring-loaded mount, feed horn and corrugated pipe are housed inside a vacuum vessel which forms an extension to the sample ﬂow cryostat and includes a vacuum window at the top to allow access for the microwave beams. The sample cryostat is cooled until the temperature of the spring-loaded mount reaches 150 K, which has been found to be a reliable temperature to use for loading of pre-frozen samples. In order to load the sample, the ﬂow cryostat must be stopped and returned to ambient pressure using helium gas. The vacuum vessel is hoisted up along with the corrugated pipe, feed horn and spring-loaded mount whilst a continuous ﬂow of helium gas is maintained to minimise icing of the cryostat and microwave feed sys- tem. The sample-holder cartridge is removed from the liquid nitrogen and inserted into the spring-loaded mount along a guide channel where it becomes located into sockets, ensur- ing accurate alignment of the waveguide. The vacuum vessel is then lowered back down and sealed to the cryostat and is then evacuated. Cryogen ﬂow is reinstated in the cryostat and the system is cooled to the measurement temperature. https://doi.org/10.5194/mr-1-301-2020 Magn. Reson., 1, 301–313, 2020 304 H. EL Mkami et al.: Gd3+–Gd3+ EPR distance measurements 2.3 Pulse EPR experiments For the Q-band experiments, echo detected ﬁeld sweep (ED- FS) measurements were carried out at 10 K. The π/2 and π pulse lengths were set at 16 and 32 ns respectively, with an inter-pulse delay of τ = 200 ns. For the W-band experiments all measurements were per- formed at 10 K, which corresponds to the optimum tempera- ture for these experiments when the central transition is ex- cited (Feintuch et al., 2015; Goldfarb, 2014; Raitsimring et al., 2013). The ED-FS spectra were recorded using a Hahn echo sequence with pulse lengths 6 and 12 ns as π/2 and π respectively and a delay of 300 ns. These pulse lengths were optimised by setting the magnetic ﬁeld to the peak of the spectrum. The echo decay (Tm) and the inversion recovery (Tl) experiments were recorded at the central maximum of the ED-FS spectrum by stepping the associated sequences with steps of 100 ns and 1 µs respectively. It should be noted that differences are expected particularly for the phase relax- ation when measuring away from the central transition (Rait- simring et al., 2014). The repetition rate for all W-band ex- periments was set at 3 kHz, unless otherwise stated, and this was again optimised at the maximum of the ED-FS spectrum. H. EL Mkami et al.: Gd3+–Gd3+ EPR distance measurements 305 Table 1. Settings for W-band DEER measurements on both rulers and the associated modulation depths obtained by ﬁtting the DEER data with DeerAnalysis (2019) (Jeschke et al., 2006). The interpulse delay τ1 was set to 300 ns for all experiments. To allow different DEER measurements to be compared more easily we take our sensitivity measure as the echo SNR multiplied by the modulation depth divided by the square root of the total number of measurements. It should be noted that this does not take into account differences in excitation bandwidth of pump and observer pulses. Table 1. Settings for W-band DEER measurements on both rulers and the associated modulation depths obtained by ﬁtting the DEER data with DeerAnalysis (2019) (Jeschke et al., 2006). The interpulse delay τ1 was set to 300 ns for all experiments. To allow different DEER measurements to be compared more easily we take our sensitivity measure as the echo SNR multiplied by the modulation depth divided by the square root of the total number of measurements. It should be noted that this does not take into account differences in excitation bandwidth of pump and observer pulses. H. EL Mkami et al.: Gd3+–Gd3+ EPR distance measurements e Number of averages calculated as number of scans × number of shots per point. f The sensitivity measure is calculated as = λ·SNR(Echo) (√total number of points measured) , where SNR (echo) is the ratio of the maximum echo height to the standard deviation of the noise. This is obtained by subtracting a smoothed ﬁt from the data and then calculating the standard deviation from the resulting noise trace. The total number of points measured is the total number of averages per point multiplied by the number of points in a scan. Figure 2. Simulated and experimental ED-FS spectra of the Gd ruler (6.0 nm) with the associated sub-spectra of the individual transitions, (a) at Q-band, (b) at W-band with wide magnetic ﬁeld ranges and (c) at W-band with narrow magnetic ﬁeld range respectively. The parameters used in the simulation are D = 1060 MHz, Dstrain = 850 MHz, E = 320 MHz and Estrain = 200 MHz. Figure 2. Simulated and experimental ED-FS spectra of the Gd ruler (6.0 nm) with the associated sub-spectra of the individual transitions, (a) at Q-band, (b) at W-band with wide magnetic ﬁeld ranges and (c) at W-band with narrow magnetic ﬁeld range respectively. The parameters used in the simulation are D = 1060 MHz, Dstrain = 850 MHz, E = 320 MHz and Estrain = 200 MHz. is expected to be faster away from the central maximum due to transition-dependent ﬂuctuations in the zero ﬁeld splitting (Raitsimring et al., 2014). data are shown in Fig. S3a. The pump and observer posi- tions with their associated excitation proﬁles are reported in Fig. 3b, c and d. In addition to the excitation proﬁles, the pump and observer pulse positions, with respect to the cen- tral transitions −1 2 E → 1 2 E , are shown in Fig. S4. The mod- ulation depths derived from the ﬁts are summarised in Ta- ble 1. The data and ﬁts at low-frequency offsets are very sim- ilar to those measured before at W-band, allowing for differ- ences in SNR and modulation depth (Dalaloyan et al., 2015). The modulation depth λ of 6 % obtained from the DEER data recorded with 120 MHz PO offset (see Table 1) is also in a good agreement with that derived from the concentra- T1 time traces were also well ﬁtted to a bi-exponential function as shown in Fig. S2b. H. EL Mkami et al.: Gd3+–Gd3+ EPR distance measurements Offseta Obsb Pumpc τ2 Data SRRd λ Echo Time Number of Sensitivity (MHz) π(ns) π(ns) (µs) points (kHz) (%) SNR averaging averagese measuref Pumping on the central line and observing on the side Gd ruler 120 (P1O1) 11 10 10.3 251 3 6.0 1111 1 h 30 min (14 scans) 42 000 2.06 (6.0 nm) 120 (P2O2) 16 16 10.3 251 3 5.0 769 44 min (7 scans) 21 000 1.68 420 (P3O3) 11 10 10.3 251 3 4.5 2000 10 h 20 min (99 scans) 297 000 1.02 Gd ruler 120 (P1O1) 24 24 2.2 251 3 2.5 1923 1 h (10 scans) 30 000 1.75 (2.1 nm) 420 (P2O2) 12 12 2.8 251 3 4.0 3125 1 h (10 scans) 30 000 4.56 840 (PO5) 12 12 1.5 121 1 2.9 3279 1 h 40 min (33 scans) 33 000 4.68 900 (PO6) 12 12 1.5 121 1 3.2 3846 0 h 45 min (15 scans) 15 000 8.99 Pumping and observing on the sides of the central line Gd ruler 800 (P3O) 8 8 1.5 121 1 2.1 8333 1 h 26 min (28 scans) 28 000 9.35 (2.1 nm) 900 (P4O) 12 12 1.5 121 1 1.1 10 000 4 h 27 min (71 scans) 71 000 3.69 a1 Frequency separation between pump pulse set at position i (Pi) and observer pulse at position j (Oj ). b, c Observer and pump π pulse lengths. The observer π/2 pulse was always half the observer π pulse. d SRR is shot repetition rate. e Number of averages calculated as number of scans × number of shots per point. f The sensitivity measure is calculated as = λ·SNR(Echo) (√total number of points measured) , where SNR (echo) is the ratio of the maximum echo height to the standard deviation of the noise. This is obtained by subtracting a smoothed ﬁt from the data and then calculating the standard deviation from the resulting noise trace. The total number of points measured is the total number of averages per point multiplied by the number of points in a scan. a1 Frequency separation between pump pulse set at position i (Pi) and observer pulse at position j (Oj ). b, c Observer and pump π pulse lengths. The observer π/2 pulse was always half the observer π pulse. d SRR is shot repetition rate. H. EL Mkami et al.: Gd3+–Gd3+ EPR distance measurements Fast and slow time constants were derived from these ﬁts and are reported in Table S3. 3.1 EPR spectra and relaxation times For the Gd ruler (2.1 nm), different settings were investigated, with either the pump frequency being set at the maximum of the ED-FS spectrum and the observer frequency placed on the side of the central line or with both the pump and observer frequencies being set on either side of the central line. The experimental parameters used in both cases are summarised in Table 1. Optimisation of the observer and pump pulse lengths was carried out systematically for each experiment given the wide range of frequency offsets used in this study. https://doi.org/10.5194/mr-1-301-2020 Magn. Reson., 1, 301–313, 2020 H. EL Mkami et al.: Gd3+–Gd3+ EPR distance measurements 3.2.1 Gd ruler (6.0 nm) The Pake patterns, for all experimental settings, show normal and typical shapes with clear dipolar singularities correspond- ing to parallel and perpendicular orientations. These DEER measurements were recorded, as mentioned, with the pump position set at the peak of the FS-ED spectrum, which pri- marily excites the −1 2 E → 1 2 E transition, whereas the ob- server frequency for both offsets was positioned where the −3 2 E → −1 2 E transition contributes most to the detected signal (see Fig. S4a, b). The Gd3+ spectrum is the result of a superposition of several transitions with different weights, and their contributions, either to the pumped and observed spins, are expected to be magnetic ﬁeld dependent. By in- creasing the PO offset, while keeping the pump position at the maximum of the FS-ED spectrum, the contribution of the −3 2 E → −1 2 E transition to the detected signal gradu- ally decreases whilst the contributions of the other transi- tions, −7 2 E → −5 2 E , −5 2 E → −3 2 E , increase. A further set of DEER experiments were performed by set- ting the pump and observer pulses on either side of the central transition with large PO offsets. With this we aimed to ex- clude completely the contribution of the −1 2 E → 1 2 E tran- sition of both the pumped and observed spins (see Fig. S6). Figure 7a shows the DEER data corresponding to 800 and 900 MHz frequency PO offsets. The pulse proﬁles associated with the pump and observer pulses are presented in Fig. 7b. For both PO offsets the obtained dipolar modulations show more than four clear oscillations and smooth damping, highly reminiscent of spectra of structurally related nitroxide rulers with similar distances (Godt et al., 2006). The Pake patterns reported in Fig. 6a show the expected shape with well-resolved perpendicular and parallel dipo- lar singularities. The corresponding distance distributions, shown in Fig. 6b, show a very narrow major peak centred at 2.1 nm with FWHH of 0.17 and 0.11 nm respectively. We also checked for signs of asymmetry in the distance distribu- tion associated with ﬂexibility of the ruler backbone, as pre- viously found with nitroxide rulers (Godt et al., 2006). 3.2.1 Gd ruler (6.0 nm) Background-corrected DEER data obtained with Gd ruler (6.0 nm) are shown in Fig. 3a, and the corresponding primary https://doi.org/10.5194/mr-1-301-2020 Magn. Reson., 1, 301–313, 2020 H. EL Mkami et al.: Gd3+–Gd3+ EPR distance measurements 306 tion dependence of a similar parent Gd3+ tag (Dalaloyan et al., 2015). By being slightly more selective with the pump pulse but keeping the same PO offset of 120 MHz, the modu- lation depth decreases to 5 % as expected due to fewer spins being excited. When the PO offset is increased to 420 MHz the modulation depth drops to 4.5 % mainly due to the output power drop off towards the band edges in our system. For this latter measurement, it should be noted the ﬁeld was different than in the former two experiments, and the pump pulse is a different frequency (see Fig. 3b, c and d). might expect for a stiff model system. The obtained modula- tion depths λ are reported in Table 1. The Pake pattern spectra reported in Fig. 6a show strong distortions and poorly resolved dipolar singularity points for the 120 and 420 MHz PO offsets. In contrast we observe sub- stantially improved Pake pattern spectra for the larger offsets of 840 and 900 MHz, particularly concerning the perpendic- ular dipolar singularities. In Fig. 6b, the distance distribu- tions are considerably broadened for the 120 and 420 MHz PO offsets, with FWHH, determined only for the major peak centred at 2.1 nm, of 0.83 and 0.45 nm. However, at 840 MHz PO offset, the peak in the distance distribution is centred at the expected 2.1 nm distance with a FWHH of 0.24 nm. The best results were obtained with the 900 MHz PO offset giving a distance distribution with a FWHH of only 0.17 nm. Note that results for 840 and 900 MHz are given as this is close to the edge of the extended interaction klystron (EIK) ampliﬁer bandwidth. The derived Pake pattern spectra and the associated dis- tance distributions are shown in Fig. 4. The distance distri- bution derived from DEER data measured with 120 MHz PO offset appears to be deviating slightly from 6.0 nm, the ex- pected distance for this Gd ruler, with a full width at half height (FWHH) of 0.56 nm, whereas with 420 MHz offset it is well centred on 6.0 nm with a FWHH of 0.48 nm. 3.2.1 Gd ruler (6.0 nm) This asymmetry is not clearly evident with Gd ruler (2.1 nm), pos- sibly because the ruler is too short, as mentioned by one of the referees. However, there are signs of asymmetry for Gd ruler (6.0 nm) in the measurement corresponding to P3O3 at large pump observer offsets. This asymmetry becomes less clear at smaller pump observer offsets as can be seen from Fig. 4. This is further evidence that even at long distances it can be beneﬁcial to have large offsets between pump and observer. 3.2.2 Gd ruler (2.1 nm) The DEER measurements with Gd ruler (2.1 nm) were conducted with a combination of different pump and ob- server positions and several PO offsets. Figure 5a shows background-corrected DEER data obtained with measure- ments performed with the pump pulse set at the position of the central transition with PO offsets of 120, 420, 840 and 900 MHz. The corresponding primary DEER data are shown in Fig. S3b. The excitation proﬁles of the pump and the ob- server at these positions are reported in Fig. 5b, c and d. The pump and observer positions with respect to the central tran- sition are shown in Fig. S5. At 120 and 420 MHz PO offsets, the time domain DEER data show severely damped dipolar modulations (see Fig. 5a), whereas in the cases of 840 and 900 MHz offsets, the dipolar modulations are signiﬁcantly recovered. However, they are still not as well deﬁned as one 4 Discussion High-quality DEER spectra from 40 µM concentration sam- ples were obtained with averaging times of an hour or two. Modulation depth, SNR of the echo and experimental param- eters are given in Table 1. As different traces were measured Magn. Reson., 1, 301–313, 2020 https://doi.org/10.5194/mr-1-301-2020 H. EL Mkami et al.: Gd3+–Gd3+ EPR distance measurements 307 Figure 3. (a) Background-corrected DEER data (black curves) of Gd ruler (6.0 nm) recorded with different PO offsets with the ﬁts (red) obtained by DeerAnalysis (2019) (Jeschke et al., 2006). Excitation proﬁles of the pump (P) and observer (O) at different frequency offsets, (b, c) P1O1 = P2O2 = 120 MHz with softer pulses for P2O2 and (d) P3O3 = 420 MHz. Figure 3. (a) Background-corrected DEER data (black curves) of Gd ruler (6.0 nm) recorded with different PO offsets with the ﬁts (red) obtained by DeerAnalysis (2019) (Jeschke et al., 2006). Excitation proﬁles of the pump (P) and observer (O) at different frequency offsets, (b, c) P1O1 = P2O2 = 120 MHz with softer pulses for P2O2 and (d) P3O3 = 420 MHz. Figure 4. (a) Pake pattern spectra obtained from ﬁtting of the DEER data of the Gd-ruler (6.0 nm) sample measured with different off- sets between pump and observer pulses and (b) corresponding distance distributions derived from the DEER data. The PO offsets are P1O1 = P2O2 = 120 MHz and P3O3 = 420 MHz. The red vertical dashed lines show in (a) the positions of the parallel and perpendicular singularities of the Pake pattern and in (b) the expected distance. Figure 4. (a) Pake pattern spectra obtained from ﬁtting of the DEER data of the Gd-ruler (6.0 nm) sample measured with different off- sets between pump and observer pulses and (b) corresponding distance distributions derived from the DEER data. The PO offsets are P1O1 = P2O2 = 120 MHz and P3O3 = 420 MHz. The red vertical dashed lines show in (a) the positions of the parallel and perpendicular singularities of the Pake pattern and in (b) the expected distance. https://doi.org/10.5194/mr-1-301-2020 https://doi.org/10.5194/mr-1-301-2020 Magn. Reson., 1, 301–313, 2020 H. EL Mkami et al.: Gd3+–Gd3+ EPR distance measurements 308 Figure 5. (a) Background-corrected DEER data (black curves) of Gd ruler (2.1 nm) recorded with different offsets between pump and observer pulses together with ﬁts (red curves) obtained by DeerAnalysis (2019) (Jeschke et al., 2006). (b, c, d) Excitation proﬁles of the pump (P) and observer (O) pulses at different frequency offsets. Please note the different frequency scales. The corresponding frequency offsets are P1O1 = 120 MHz, P2O2 = 420 MHz, PO5 = 840 MHz and PO6 = 900 MHz. The black arrow indicates the position of 94 GHz, the nominal centre frequency of our W-band EIK ampliﬁer, which has a bandwidth of just less than 1 GHz. Figure 5. (a) Background-corrected DEER data (black curves) of Gd ruler (2.1 nm) recorded with different offsets between pump and observer pulses together with ﬁts (red curves) obtained by DeerAnalysis (2019) (Jeschke et al., 2006). (b, c, d) Excitation proﬁles of the pump (P) and observer (O) pulses at different frequency offsets. Please note the different frequency scales. The corresponding frequency offsets are P1O1 = 120 MHz, P2O2 = 420 MHz, PO5 = 840 MHz and PO6 = 900 MHz. The black arrow indicates the position of 94 GHz, the nominal centre frequency of our W-band EIK ampliﬁer, which has a bandwidth of just less than 1 GHz. Figure 6. (a) Pake pattern spectra obtained from the ﬁtting of the DEER data of the Gd-ruler (2.1 nm) sample measured with different offsets between pump and observer pulses and (b) associated distance distributions derived from the DEER data. The corresponding frequency offsets are P1O1 = 120 MHz, P2O2 = 420 MHz, PO5 = 840 MHz, PO6 = 900 MHz, P3O = 800 MHz and P4O = 900 MHz. The red vertical dashed lines show in (a) the positions of the parallel and perpendicular singularities of the Pake pattern and in (b) the expected distance. Figure 6. (a) Pake pattern spectra obtained from the ﬁtting of the DEER data of the Gd-ruler (2.1 nm) sample measured with different offsets between pump and observer pulses and (b) associated distance distributions derived from the DEER data. The corresponding frequency offsets are P1O1 = 120 MHz, P2O2 = 420 MHz, PO5 = 840 MHz, PO6 = 900 MHz, P3O = 800 MHz and P4O = 900 MHz. https://doi.org/10.5194/mr-1-301-2020 The red vertical dashed lines show in (a) the positions of the parallel and perpendicular singularities of the Pake pattern and in (b) the expected distance. https://doi.org/10.5194/mr-1-301-2020 Magn. Reson., 1, 301–313, 2020 309 Figure 7. (a) Background-corrected DEER data (black curves) of Gd ruler (2.1 nm) recorded with different offsets between pump and observer pulses together with ﬁts (red curves) obtained by DeerAnalysis (2019) (Jeschke et al., 2006). (b) Excitation proﬁles of the pump (P) and observer (O) pulses at different frequency offsets. The corresponding frequency offsets are P3O = 800 MHz and P4O = 900 MHz. The black arrow indicates the position of 94 GHz, the nominal centre frequency of our W-band EIK ampliﬁer, which has a bandwidth of just less than 1 GHz. Figure 7. (a) Background-corrected DEER data (black curves) of Gd ruler (2.1 nm) recorded with different offsets between pump and observer pulses together with ﬁts (red curves) obtained by DeerAnalysis (2019) (Jeschke et al., 2006). (b) Excitation proﬁles of the pump (P) and observer (O) pulses at different frequency offsets. The corresponding frequency offsets are P3O = 800 MHz and P4O = 900 MHz. The black arrow indicates the position of 94 GHz, the nominal centre frequency of our W-band EIK ampliﬁer, which has a bandwidth of just less than 1 GHz. (Raitsimring et al., 2014). The need to ﬁt with two stretched exponentials suggests an additional fast dephasing process is contributing to the transverse relaxation. We tentatively spec- ulate that this additional dephasing process is driven by intra- molecular instantaneous diffusion due to the electron spin ﬂip-ﬂop processes resulting from simultaneous excitation of −1 2 E → 1 2 E transitions belonging to both Gd3+ ions of one Gd ruler. This seems to be consistent with the Tm values de- rived from the ﬁts (see Table S2) which show identical slow parts for both samples as one would expect for the same ma- trix and different fast parts as a result of two Gd3+ systems with different dipolar couplings, due to different Gd–Gd dis- tances, and therefore different spin ﬂip-ﬂop rates. The inter- molecular instantaneous diffusion process is less effective at concentrations as low as those used in this study and is there- fore considered to not contribute. However, this hypothesis needs further investigation at different concentrations and on a similar compound with a single Gd3+ label. https://doi.org/10.5194/mr-1-301-2020 with a different number of scans and different shot repeti- tion times, or have different number of points in the scan, we also provide a sensitivity measure for DEER measure- ments that normalises for these quantities. Results can be compared to W-band measurements on the same Gd rulers (Dalaloyan et al., 2015). The high concentration sensitivity, relative to conventional W-band resonator-based spectrom- eters, is attributed to much larger effective sample volumes (∼50–80 µL) and larger excitation bandwidths facilitated by the high available power that is only partially offset by the lower conversion factor. Large effective volumes are possi- ble with biological systems in non-resonant sample holders at W-band, as dielectric losses are expected to be small at cryogenic temperatures (tanδ < 0.001) if a high-quality glass is formed. The echo decays (measured at the central transition), shown in Fig. S2a, were well ﬁtted with a sum of two expo- nential functions with exponents found to be extremely close to 1 and 2 (R2 = 0.9999). Little difference in phase memory time was observed between the two rulers, with a slightly lower decay for Gd ruler (2.1 nm). However, no correlation had been found between the echo decay rate and the Gd– Gd distance of the same type of ruler as used in our study (Dalaloyan et al., 2015). This type of decay appears to be a characteristic of the Gd3+ complexes, and very similar re- sults have been obtained before (Collauto et al., 2016; Rait- simring et al., 2014; Dalaloyan et al., 2015). Nuclear spin diffusion is often the dominant process in phase relaxation of the central transition (Garbuio et al., 2015), when one would expect the data to be well ﬁtted with a single stretched exponential with an exponent of close to 2 (Kathirvelu et al., 2009). However nuclear spin diffusion is expected to be signiﬁcantly reduced by matrix deuteration, and contri- butions resulting from thermally assisted ﬂuctuations in the zero-ﬁeld splitting are then expected to become signiﬁcant Inversion recovery data shown in Fig. S2b have been well ﬁtted with the sum of two mono-exponential functions with fast and slow components (see Table S3). In addition, we pro- vided a single T1 value that was determined from a mono- exponential function ﬁt to the inversion recovery data. It is interesting to note that the longer Tm component is compara- ble to the shorter T1 component. https://doi.org/10.5194/mr-1-301-2020 This has been conﬁrmed theoretically and investigation has shown that the artefacts are only signiﬁcant for short distances, where the dipolar coupling is large, and when either the pump or ob- server pulse is set on the −3 2 E → −1 2 E transition adja- cent to the −1 2 E → 1 2 E or vice versa (Cohen et al., 2016; Manukovsky et al., 2017). However, when other transitions are selected by the observer pulse, it was shown that these artefacts are strongly reduced (Manukovsky et al., 2017). This was originally experimentally conﬁrmed in experiments with a dual-mode cavity (Cohen et al., 2016) and is also clearly seen in the experiments described here. This is par- ticularly demonstrated in Fig. 5 where the pump pulse is set on the −1 2 E → 1 2 E transition and the observer pulse is moved progressively further away from the central transi- tion, which gradually reduces the contribution of the adjacent −3 2 E → −1 2 E transition (see Fig. S5). For the short Gd ruler (2.1 nm) clearer modulations and narrower distance distributions are observed as the frequency offset is increased. Clearly visible modulations in the time domain are observed at the largest PO offset of 900 MHz Interestingly, small artefacts are even observed for the longer Gd ruler (6.0 nm) in Fig. 3 where better ﬁts to the expected Pake pattern are obtained at the larger 420 MHz frequency offset, and the related distance distribution has its peak at the expected 6.0 nm distance (see Fig. 4b). We note that in all the DEER studies reported so far, the central −1 2 E → 1 2 E transition has always been selected, ei- ther for the pump or the observer pulse. It had generally been assumed that there would otherwise be too big a sensitivity penalty, and the advantage of operating at high ﬁelds was achieving a higher degree of excitation of the central transi- tion by either pump or observer pulses. This led to the view that it is necessary to choose a Gd spin label with a large ZFS when measuring short distances to reduce the effect of unwanted ﬂip ﬂops (Dalaloyan et al., 2015). https://doi.org/10.5194/mr-1-301-2020 In this present work, we also investigated the DEER set- up where the pump and observer pulses are placed on either side of the central transition, thus avoiding any excitation of the central transition completely (see Fig. S6a). These DEER experiments, P3O and P4O, shown in Fig. 7a, now show time domain data with clear oscillations smoothly damped to the limit value (modulation depth), giving clear Pake patterns and narrow distance distributions that are strikingly simi- lar to those obtained for structurally related nitroxide rulers with comparable spin–spin distances (Godt et al., 2006). This is evidence that when the central transition does not play a role in the Gd3+–Gd3+ DEER measurements, the mixing of states has no major effect, as they do not share energy levels with those involved in the pump and observer transitions. The ability to measure shorter distances with Gd-based spin labels accurately has implications for sensitivity. Gd ruler (2.1 nm) would be expected to have at least 10 times higher echo SNR, compared to Gd ruler (6.0 nm) just from the shorter time window required for the DEER measure- ment, based on relaxation measurements at the central tran- sition. This increase in sensitivity is likely to be even larger for biological samples that are usually highly protonated and thus have signiﬁcantly shorter phase memory times. The rel- ative loss of sensitivity is therefore much less when shorter time windows become feasible, as shown with Gd ruler (2.1 nm) where an echo SNR of 8330, as well as a modu- lation depth of 2.1 %, was obtained after only 1 h and a half, even with a reduced 1 kHz SRR. For observer measurements made away from the cen- tral transition the relative gain, at short distances, also be- comes larger as relaxation times are expected to shorten due to transition-dependent phase relaxation (Raitsimring et al., 2014). In the measurements presented here, this is par- tially offset by the increased bandwidth required and the re- duced power available from the EIK–isolator–switch combi- nation at the band edges of the EIK ampliﬁer. The available power as a function of frequency is shown in Fig. S1b. How- ever, interestingly, and perhaps counter-intuitively, we see lit- tle degradation in SNR, when neither pump nor observer is E E For the short Gd ruler (2.1 nm) clearer modulations and narrower distance distributions are observed as the frequency offset is increased. https://doi.org/10.5194/mr-1-301-2020 For DEER experiments, the weak coupling approximation is generally expected to hold when the PO offset between the coupled spins is signiﬁcantly larger than the dipolar cou- pling between the coupled spins. This is usually fulﬁlled for Gd3+–Gd3+ distance measurements where a frequency off- set of at least 100 MHz is often used. In both Gd rulers, Gd ruler (2.1 nm) and Gd ruler (6.0 nm), the expected dipolar couplings are 5.6 and 0.2 MHz respectively and are far be- low the smallest frequency offset of 120 MHz used in our https://doi.org/10.5194/mr-1-301-2020 Magn. Reson., 1, 301–313, 2020 310 H. EL Mkami et al.: Gd3+–Gd3+ EPR distance measurements measurements. However, in the present work, as well as in the literature, artefacts in the spectra are observed for dis- tances below 3–4 nm (Raitsimring et al., 2007; Dalaloyan et al., 2015; Cohen et al., 2016; Manukovsky et al., 2017). Such artefacts mainly manifest themselves as a damping of the dipolar modulations in the time domain, which in turn results in an artiﬁcial broadening of the distance distribu- tions. This has previously been explained in terms of un- wanted excitation of ﬂip-ﬂop transitions within the central line. For the highest sensitivity in Gd3+–Gd3+ DEER mea- surements, the pump pulse is usually set at the maximum of the ED-FS spectrum to ensure the deepest modulation depth (and the observer is often set just outside the central line). Under such conditions, the central transition, −1 2 E → 1 2 E , contributes most to the pumped spins, whereas, just away from the central transition, the −3 2 E → −1 2 E transition becomes the more dominant contribution to the observer spins (see Fig. S4a, b). The DEER signal is thus the re- sult of the difference between the energy levels associated with the two transitions −3 2 (A), 1 2(B) E → −1 2 (A), 1 2(B) E and −3 2 (A),−1 2(B) E → −1 2 (A),−1 2(B) E . The associated energies of these two states are degenerate to ﬁrst order of the ZFS, and only a fairly small splitting is induced by the second-order ZFS contribution; this falls within the range of the dipolar couplings corresponding to short distances be- tween Gd3+ ions. Therefore, the weak coupling approxima- tion is no longer satisﬁed and the secular pseudo-terms de- scribing the ﬂip-ﬂop effects cannot be ignored. 5 Conclusion placed at the central transition. Modulation depth, although reduced, is still reasonable, and thus we still obtain excellent overall sensitivity under this condition. In the present work, we have investigated two Gd rulers, with Gd–Gd distances of 2.1 and 6.0 nm, using Gd3+ complexes with a moderate ZFS of 1060 MHz. We have performed a va- riety of Gd3+–Gd3+ DEER measurements with different off- sets between pump and observer pulses, using a non-resonant induction mode cavity. This is a ﬂexible wideband measure- ment set-up with relatively easy sample handling, where an excellent signal-to-noise ratio is observed. We therefore predict that Gd systems with smaller ZFS than used here (see Table S1) are to be preferred because not only is it easier to avoid the central transition, but we would also expect the amplitude of other transitions to increase (per unit bandwidth), which will further increase sensitivity. We would also predict that relaxation effects due to thermally assisted ﬂuctuations in the ZFS will reduce (Raitsimring et al., 2014). This is the subject of further investigation. We have shown, in agreement with previous experimental results, that larger PO offsets signiﬁcantly reduce the arte- facts observed for Gd–Gd distances below 3–4 nm, but they appear also to be of beneﬁt in the case of larger distances, such as 6.0 nm. It should also be noted, when pumping away from the central transition, at 40 µM molecular concentration, the re- quired background correction to DEER traces is small rel- ative to the experimental modulation depth. Indeed, the re- quirement for background correction is almost eliminated at short distances, as can be seen from the raw traces provided in Fig. S3b. This is particularly important for Gd3+–Gd3+ DEER measurements where modulation depths are low, as small errors in background correction can make a signiﬁcant contribution to uncertainties in the calculated distance distri- bution. The results can be compared to the larger corrections required at slightly higher spin label concentrations when pumping on the central transition (Dalaloyan et al., 2015). More importantly we have shown signiﬁcantly improved distance distributions at short distances by completely avoid- ing excitation of the central transition in the DEER exper- iment, −1 2 E → 1 2 E , and mostly selecting −7 2 E → −5 2 E , −5 2 E → −3 2 E and −3 2 E → −1 2 E transitions. https://doi.org/10.5194/mr-1-301-2020 Clearly visible modulations in the time domain are observed, at the largest PO offset of 900 MHz (see Fig. 5a), although simulations have indicated that some residual effects from the pseudo-secular term can be ob- served even at such PO offsets (Manukovsky et al., 2017). https://doi.org/10.5194/mr-1-301-2020 Magn. Reson., 1, 301–313, 2020 311 H. EL Mkami et al.: Gd3+–Gd3+ EPR distance measurements 5 Conclusion This still gives very high signal-to-noise ratio (per unit measurement time) while obtaining much improved ﬁtting to expected Pake patterns. This is a strong motivation to select and/or de- velop Gd-based SLs with as small a ZFS as possible and mea- sure using wideband spectrometers at moderately high mag- netic ﬁelds, where the central transition narrows. The sensi- tivity is already high, but we envisage considerable scope for improvement. There is also scope to further improve sensitivity, at W- band, by increasing both the shot repetition rate and averag- ing times and operating at lower temperatures if the central transition is not excited. Backshort positions in the sample holder (see Fig. S1a) were also optimised for cross-polar iso- lation rather than matching out the echo signal, which can make a difference of a factor of 2 in sensitivity. Other groups have demonstrated a signiﬁcant sensitivity beneﬁt from the use of broadband chirped pulses in DEER measurements on Gd3+ systems (Bahrenberg et al., 2017; Doll et al., 2015). These methodologies particularly lend themselves to high- power, wideband spectrometers like HiPER and promise sig- niﬁcant further gains. In contrast, we found both sensitivity and signal ﬁdelity were signiﬁcantly reduced at Q-band rel- ative to W-band, for the case where both pump and observer are placed on one side of the central transition (due to cavity bandwidth limitations). Example data sets, measured using a high-power (150 W) commercial Q-band spectrometer with comparable sample volumes, are shown in Fig. S7 and Ta- ble S4 for Gd ruler (2.1 nm). For the case where both pump and observer are offset to one side of the central transition, resolution was only partially improved and sensitivity was re- duced by a factor of 24 relative to W-band, using our deﬁned sensitivity measure. Data availability. The research data underpinning this publication can be accessed at https://doi.org/10.17630/96ab76ee-38f4-468f- 9ea8-e947f638261f (EL Mkami et al., 2020). Supplement. The supplement related to this article is available online at: https://doi.org/10.5194/mr-1-301-2020-supplement. Author contributions. MQ and AG designed and synthesised the Gd rulers. HEM, RIH, PASC and GMS designed and built HiPER. HEM, RIH, JEL, AF and GMS devised the experiments which were performed by HEM, RIH and MJT. HEM and GMS chieﬂy wrote the manuscript, with further input from all authors. Competing interests. The authors declare that they have no con- ﬂict of interest. Competing interests. The authors declare that they have no con- ﬂict of interest. The sensitivity achieved at W-band suggests that it will be feasible to obtain high-quality spectra for Gd3+ DEER measurements at sub-µM concentrations, even allowing for the shorter relaxation times commonly observed with spin- labelled biological samples. We have also observed promis- ing results with Gd3+ spin-labelled biological samples, which we will report in a future publication. Acknowledgements. It is a pleasure to acknowledge both Dun- can Robertson (University of St Andrews) for many useful dis- cussions on hardware and Daniella Goldfarb (Weizmann Institute of Science) for many useful discussions on Gd-based spin labels. Hassane EL Mkami, Graham M. Smith, Janet E. Lovett, Robert https://doi.org/10.5194/mr-1-301-2020 Magn. Reson., 1, 301–313, 2020 H. EL Mkami et al.: Gd3+–Gd3+ EPR distance measurements 312 I. Hunter, Paul A. S. Cruickshank and Michael J. Taylor are part of StAnD, which is a major collaboration between EPR groups at St Andrews and Dundee universities. We thank the Royal Society International Exchanges Scheme and the Weizmann–UK Making Connections Programme for allowing bilateral travel and research between the University of St Andrews and the Weizmann Institute of Science. Janet E. Lovett thanks the Royal Society for a Univer- sity Research Fellowship (URF). Michael J. Taylor thanks EPSRC for a CM-CDT studentship. Mian Qi and Adelheid Godt thank the Deutsche Forschungsgemeinschaft (DFG) Priority Program. I. Hunter, Paul A. S. Cruickshank and Michael J. Taylor are part of StAnD, which is a major collaboration between EPR groups at St Andrews and Dundee universities. We thank the Royal Society International Exchanges Scheme and the Weizmann–UK Making Connections Programme for allowing bilateral travel and research between the University of St Andrews and the Weizmann Institute of Science. Janet E. Lovett thanks the Royal Society for a Univer- sity Research Fellowship (URF). Michael J. Taylor thanks EPSRC for a CM-CDT studentship. Mian Qi and Adelheid Godt thank the Deutsche Forschungsgemeinschaft (DFG) Priority Program. Dalaloyan, A., Qi, M., Ruthstein, S., Vega, S., Godt, A., Fein- tuch, A., and Goldfarb, D.: Gd(III)-Gd(III) EPR distance mea- surements – the range of accessible distances and the impact of zero ﬁeld splitting, Phys. Chem. Chem. 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https://openalex.org/W4327910565	https://link.springer.com/content/pdf/10.1007/s10010-023-00653-y.pdf	English	null	Erweiterung der Systemgrenze des Digitalen Zwillings auf die Sensorik des Physischen Zwillings durch die Verwendung redundanter Softsensoren	Forschung im Ingenieurwesen	2,023	cc-by	6,690	Michel Fett michel.fett@tu-darmstadt.de Abstract The beneﬁt of Digital Twins depends to a large extent on the quality of the sensor data provided. In many cases, sensor failures are only detected late in operation which can lead to serious consequences. For this reason, one approach to reduce the resulting safety issues is to use redundant sensor systems that monitor the same measureand. However, due to the additional sensors required, this is associated with additional ﬁnancial and design effort. In this publication an alternative strategy is presented, which provides a redundant sensor system with the help of soft sensors. Soft sensors use already installed physical sensors to anticipate a new measured variable via algorithms. They are often used to avoid placing sensors in inaccessible locations, but are used here to perform redundant computation of already existing metrics. The sensor data of physical and soft sensors are used as input variables for a Digital Twin. Here, these are compared with each other and can be critically questioned by the twin itself. This makes it possible to extend the system boundary of the Digital Twin to the sensors themselves and provided input variables can be checked for their validity. This allows sensor failures to be detected at an early stage and consequential damage to be averted. 1 Institute for Product Development and Machine Elements, TU Darmstadt, Otto-Berndt-Straße 2, 64287 Darmstadt, Germany Extension of the system boundary of the Digital Twin onto the sensors of the Physical Twin through the introduction of redundant soft sensors Michel Fett1 · Eleanor Turner1 · Richard Breimann1 · Eckhard Kirchner1 Received: 1 November 2022 / Accepted: 27 January 2023 / Published online: 20 March 2023 © The Author(s) 2023 Received: 1 November 2022 / Accepted: 27 January 2023 / Published online: 20 March 2023 © The Author(s) 2023 Zusammenfassung Der Nutzen von Digitalen Zwillingen hängt in hohem Maße von der Qualität der bereitgestellten Sensordaten ab. Dabei werden in vielen Fällen Sensorausfälle erst spät im Betrieb erkannt, was zu schwerwiegenden Folgen führen kann. Ein möglicher Ansatz, um die daraus resultierenden Sicherheitsrisiken zu reduzieren, ist daher die Verwendung redundanter Sensorsysteme, welche die gleiche Messgröße erfassen. Aufgrund der größeren Anzahl benötigter physischer Sensoren ist dies allerdings mit zusätzlichen ﬁnanziellen und konstruktiven Herausforderungen verbunden. In dieser Publikation wird ein alternativer Ansatz vorgestellt, welcher Softsensoren nutzt um das redundante Sensorsystem zu erstellen. Softsensoren verwenden bereits integrierte physischen Sensoren, um über Algorithmen eine neue Messgröße zu antizipieren. Sie werden häuﬁg eingesetzt, wenn aufgrund der Unzugänglichkeit von Messstellen keine physischen Sensoren verbaut werden können. Im Rahmen dieser Publikation werden sie jedoch verwendet, um eine redundante Berechnung bereits vorhandener Messgrößen durchzuführen. Die Sensordaten von physischen Sensoren und Softsensoren dienen als Eingangsgrößen für einen Digitalen Zwilling. Dieser vergleicht die Werte miteinander und ist so imstande diese kritisch zu hinterfragen. Damit ist es möglich, die Systemgrenze des Digitalen Zwillings auf die Sensoren selbst zu erweitern, Sensorausfälle frühzeitig zu erkennen und Folgeschäden abzuwenden. Der Nutzen von Digitalen Zwillingen hängt in hohem Maße von der Qualität der bereitgestellten Sensordaten ab. Dabei werden in vielen Fällen Sensorausfälle erst spät im Betrieb erkannt, was zu schwerwiegenden Folgen führen kann. Ein möglicher Ansatz, um die daraus resultierenden Sicherheitsrisiken zu reduzieren, ist daher die Verwendung redundanter Sensorsysteme, welche die gleiche Messgröße erfassen. Aufgrund der größeren Anzahl benötigter physischer Sensoren ist dies allerdings mit zusätzlichen ﬁnanziellen und konstruktiven Herausforderungen verbunden. In dieser Publikation wird ein alternativer Ansatz vorgestellt, welcher Softsensoren nutzt um das redundante Sensorsystem zu erstellen. Softsensoren verwenden bereits integrierte physischen Sensoren, um über Algorithmen eine neue Messgröße zu antizipieren. Sie werden häuﬁg eingesetzt, wenn aufgrund der Unzugänglichkeit von Messstellen keine physischen Sensoren verbaut werden können. Im Rahmen dieser Publikation werden sie jedoch verwendet, um eine redundante Berechnung bereits vorhandener Messgrößen durchzuführen. Die Sensordaten von physischen Sensoren und Softsensoren dienen als Eingangsgrößen für einen Digitalen Zwilling. Dieser vergleicht die Werte miteinander und ist so imstande diese kritisch zu hinterfragen. Damit ist es möglich, die Systemgrenze des Digitalen Zwillings auf die Sensoren selbst zu erweitern, Sensorausfälle frühzeitig zu erkennen und Folgeschäden abzuwenden. https://doi.org/10.1007/s10010-023-00653-y Forsch Ingenieurwes (2023) 87:479–488 https://doi.org/10.1007/s10010-023-00653-y Forsch Ingenieurwes (2023) 87:479–488 ORIGINALARBEITEN/ORIGINALS 2.1 The Digital Twin concept Due to the high degree of novelty of the concept of Digital Twins, a multitude of partly contradictory understandings and deﬁnitions exist. In order to create a uniform under- standing the following deﬁnition of a Digital Twin applies in the context of this publication: In this way, data can be simulated for locations where placing physical sensors is difﬁcult due to design restric- tions or operating conditions. In the literature, soft sensors are also called virtual sensors [14–17], extended Kalman ﬁlters [18] or, in the context of control engineering, state observers, estimators or predictors [19]. Some authors de- scribe the soft sensor as a Digital Twin of a physical sensor [13, 14, 19]. Others use Digital Twins as comprehensive soft sensors of complex systems [20, 21]. In order to do justice to the previously mentioned deﬁnition of the term Digital Twin, the terms Digital Twin and soft sensor are not used synonymously in a general way. However, assuming that a physical counterpart exists in the product, a soft sen- sor will be regarded as a Digital Twin of the corresponding physical sensor in the scope of this publication. Soft sensors can, just like physical sensors, be used as input variables for a Digital Twin [22–24]. A digital twin is a digital representation of a real prod- uct instance (physical twin). The representation uses models that are fed with real-time data, e.g. by sensors installed on the physical twin, to simulate its behavior. The simulation results are then fed back into the physical world via a bidi- rectional connection and made use of there [3–6]. In order to distinguish the digital twin from its envi- ronment, it is deﬁned by a system boundary [7, 8]. The system boundary of the digital twin is based on the system boundary of the physical twin [9] and is drawn according to the intended use case [10]. The system boundary is pri- marily used to describe whether the respective digital twin is a single unit or an aggregated system of subsystems [8, 9, 11]. A more detailed consideration from a system theo- retical view to identify an optimum of the system boundary between twin and environment does not take place. It is therefore not deﬁned where the system boundary should be drawn and whether the sensors should be included as an in- formation interface between the physical and Digital Twin. 2 State of the art Ssoftsensor = f S1;physical ; :::; Sn;physical (1) (1) 1 Introduction The usability of Digital Twins is highly dependent on the quality of the sensor data provided [1, 2]. Malfunctions and failures of sensors can not only disturb the monitoring of the physical twin through the Digital Twin, but also lead K K 480 Forsch Ingenieurwes (2023) 87:479–488 2.2 Soft sensors to serious malfunctions of the physical twin, due to being controlled by the feedback of the Digital Twin. This prob- lem can be mitigated by the integration of redundant sen- sors, however, the installation of several physical sensors leads to additional ﬁnancial and constructive expenditure. For this reason, this publication examines the question of how redundancy can be built up by introducing soft sensors, which calculate the quantity of interest instead of measur- ing them. This is to be used to detect sensor failures with the resulting extended system boundary of the Digital Twin of a rolling bearing test bench. In the long term the results can be applied to different types of Digital Twins, such as test benches, production machines or customer products. In contrast to physical sensors, soft sensors describe an al- gorithm which calculates the quantity of interest in a prod- uct instead of measuring it with a physical entity [12]. The algorithms can be based on physical, empirical or data- driven models. The latter include machine learning ap- proaches [13]. These algorithms use the measurement data of various physical sensors integrated in the product as in- put variables to calculate the quantity of interest. The thus calculated variable of the soft sensor neither needs a local proximity or the same measured variable as the installed physical sensors. However, it must be ensured that a cor- relation is given and the soft sensor Ssoftsensor can be rep- resented in the form of a function of the physical sensors Si,physical shown in Eq. 1 [14]. 2.1 The Digital Twin concept However, the strong dependence of Digital Twins on correct sensor data [1, 2] leads to the conclusion that, in general, the behavior of sensors is not described by Digital Twins. For this reason, malfunctions and failures of sensors can disturb the monitoring of the physical twin by the Digital Twin. The calculations and simulations of the Digital Twin are based on sensor data and can be used to regulate or control the physical twin. With incorrect input values this can lead to serious malfunctions of the latter. 2.3 Redundancy through soft sensors One possible approach to mitigate the effects of sensor fail- ures is the redundant implementation of physical sensors. Redundancy can be differentiated into hot and cold redun- dancy. In the case of hot redundancy, two or more systems work in parallel at any given time, but can also fulﬁl the task on their own. In cold redundancy only one system is operated at a time. The replacement system is kept on standby and activated only in case of failure of the ﬁrst sys- tem [25]. Since a redundant sensor system records the same measured variables and compares the measured values, it is classiﬁed as hot redundancy. Discrepancies between the measured values allow sensor failures or misbehavior to be identiﬁed and appropriate measures to be initiated. A redun- dant design of physical sensors is associated with a num- K K 481 Forsch Ingenieurwes (2023) 87:479–488 ber of disadvantages. One drawback is increased cost, due to the usage of more sensors. Furthermore, the integration of sensors is accompanied by an increased design effort in order to meet requirements for functional fulﬁlment or construction space. this context, soft sensors are used to build analytical redun- dancy and are used within a holistic framework to identify conﬂicts. However, an isolated consideration of the practi- cal generation and use is not the focus of this publication. He et al. [26] use a multi-block PLS approach to model the system with the aim of monitoring the process through a digital twin. In addition to the (soft) sensors, the actuators and possible process faults are modelled in order to identify the cause of a process fault when it is detected. However, the identiﬁcation of the process error itself does not take place through the soft sensors. One alternative strategy is a data driven approach to val- idate sensor data and detect sensor failures like the imple- mentation of redundant soft sensors. Approaches in recent literature utilize the idea that soft sensors can not only be used to determine unknown measured variables, but also to perform a calculation of variables already measured from existing physical sensors. There are multiple ways to cal- culate the required soft sensor values. The Input can be either the last few values of the respective physical sensor [17] or the values all sensors except the respective physical sensor [16]. Another alternative is to identify reliable and unreliable sets of sensors in advance. 2.3 Redundancy through soft sensors Soft sensors are then created for the unreliable ones from the data of the reliable ones [17]. p g Darvishi et al. [17, 27] in contrast, use soft sensors speciﬁcally to identify sensor faults. For this purpose, soft sensors are used to calculate sensor values from unreliable physical sensors and the occurring deviations are compared. In a ﬁrst approach, the soft sensors consist of a predictor that takes into account the temporal development and an estimator that uses the remaining reliable sensors [27]. In a follow-up publication, an algorithm is used which takes both perspectives into account at the same time [17]. In each case, multilayer perceptron (MLP) neural networks are used to generate the soft sensors, using 70% [17] and 85% [27] of the available data to train each. The need for large amounts of data is explicitly mentioned as a limiting factor of neural networks. Darvishi et al. further state that the inclusion of correlations between the sensors can in- crease the performance compared to the use of all sensors simultaneously [17]. Using these methods, a redundant sensor system is built without the need to integrate additional physical sensors. This is referred to in the literature as analytical [2, 17] or virtual redundancy [13, 14, 16, 19]. In the context of redundancy alignment, the measured values of the physical sensors and the predicted values of the soft sensors can be compared and deviations can be determined. The deviations are utilized to verify the measured data from the physical sensors [2]. This way the condition of the physical sensor can be monitored and failures can be detected [16, 19, 26]. Soft sensors can also be used as backups to replace the physical sensors in case of a deviation [13, 17]. In this way, process stability can be improved [14]. In this contribution, the concept of soft sensors is consid- ered in isolated fashion in order to avoid implications that arise from interactions with related systems. For this pur- pose, the effects of the system boundary of Digital Twins are ﬁrst examined and the soft sensors are delimited. Redundant soft sensors cannot be used on their own, but must be processed and interpreted by a suitable data dissemination system. One possible approach is processing by a Digital Twin, for which some initial applications can be found in the literature. Fig. 1 Schematic presentation of the Digital Twin concept with the sensor outside (a) or inside (b) the system boundary 2.3 Redundancy through soft sensors 2 Schematic presentation of the information ﬂow from the physical twin to the Digital Twin including the processing by soft sensors Physical Twin PT Digital Twin of PT System boundary System component Informaon ﬂow Physical Sensor So sensor tions of the individual physical sensors are included. The entire procedure for creating the soft sensors is replicable and is presented in a methodical procedure model. 2.3 Redundancy through soft sensors In the practical side, redundant soft sensors are used to identify sensor errors. Neural networks are deliberately avoided, as they are not always suitable due to the lack of transparency, as well as the high demand for data. Instead, various alternative machine learning methods are used. To increase the performance of the soft sensors, the correla- Staudter et al. [2] consider soft sensors in a holistic in- vestigation of data-induced conﬂicts in Digital Twins. In Fig. 1 Schematic presentation of the Digital Twin concept with the sensor outside (a) or inside (b) the system boundary Physical Twin PT Digital Twin of PT Sensors Sensor data Feedback History data Physical Twin PT Digital Twin of PT Sensors Sensor data Feedback History data Digital Twin of Sensors (So Sensors) System boundary System component Informaon ﬂow a b K Physical Twin PT Digital Twin of PT Sensors Sensor data Feedback History data Digital Twin of Sensors (So Sensors) b Physical Twin PT Digital Twin of PT Sensors Sensor data Feedback History data System boundary System component Informaon ﬂow a b a Digital Twin of PT System boundary System component Informaon ﬂow 482 Forsch Ingenieurwes (2023) 87:479–488 Fig. 2 Schematic presentation of the information ﬂow from the physical twin to the Digital Twin including the processing by soft sensors Digital Twin of PT Digital Twin of sensors (so sensors system) Machine learning model Comparison A B C Physical Twin PT Sensor data A Sensor data B Sensor data C C* System boundary System component Informaon ﬂow Physical Sensor So sensor X X* tions of the individual physical sensors are included. The entire procedure for creating the soft sensors is replicable and is presented in a methodical procedure model. 3 Extension of the system boundary of a Digital Twin As described in Sect. 2.1, a Digital Twin is a virtual rep- Fig. 2 Schematic presentation of the information ﬂow from the physical twin to the Digital Twin including the processing by soft sensors Digital Twin of PT Digital Twin of sensors (so sensors system) Machine learning model Comparison A B C Physical Twin PT Sensor data A Sensor data B Sensor data C C* System boundary System component Fig. Fig. 3 Flowchart of the algo- rithm for the creation (a) and usage (b) the soft sensors 3 Extension of the system boundary of a Digital Twin As described in Sect. 2.1, a Digital Twin is a virtual rep- resentation of a physical counterpart (physical twin). Real- time data from the physical twin is captured by sensors and transferred to the Digital Twin. The behavior of the sensors is often not represented by Digital Twins. Instead, sensor data is taken as given. This corresponds to a system bound- Fig. 3 Flowchart of the algo- rithm for the creation (a) and usage (b) the soft sensors K K 483 Forsch Ingenieurwes (2023) 87:479–488 ary that is drawn around the physical twin, but excludes the sensors. This can be seen in Fig. 1a. All physical sensors are taken as input for the model, with the exception of the particular physical sensor i for which the respective redundancy is to be generated. The latter data are used as labels to train the model. Equation 2 shows the creation of the models using the example of linear regression with weights αi,j and intercept variables βi. ary that is drawn around the physical twin, but excludes the sensors. This can be seen in Fig. 1a. The ability to simulate and anticipate the behavior of physical sensors through a redundant soft sensor system extends this system boundary to include the physical sen- sors as shown in Fig. 1b. In this way, the Digital Twin can verify sensor data and detect sensor failures and malfunc- tions at an early stage. This helps prevent erroneous inputs to the Digital Twin and potentially harmful behavior. 2 66664 S1;softsensor ::: ::: Sn;softsensor 3 77775 = 2 66664 0 ˛1,2 ::: ˛1;n ˛2,1 0 ::: ::: ::: ˛n−1;n ˛n;1 ::: ˛n;n−1 0 3 77775 2 66664 S1;physical ::: ::: Sn;physical 3 77775 + 2 66664 ˇ1 ::: ::: ˇn 3 77775 (2) The information ﬂow from the physical twin to the Dig- ital Twin is of particular interest for the consideration and expansion of the system boundary through soft sensors. The soft sensor system is located in this information ﬂow. Fig- ure 2 schematically shows the information ﬂow in detail. (2) The measured values collected by the physical sensors A, B and C are fed into the soft sensor system. In this system, the soft sensor C* is initially created from the measured values of the physical sensors A and B and later supplied by them. 3 Extension of the system boundary of a Digital Twin The values of the physical sensor C and the soft sensor C* are compared before the information is fed into the Digital Twin. For reasons of clarity, only the creation and comparison of sensor C and soft sensor C* is shown in the illustration. In the same way, this must also be done for sensors A and B. Then, using data collected up to this point, the model scores of the different models are calculated. For this pur- pose, the respective score functions of the different model types of the Scikit-learn environment are used [29–32]. The best possible model for each individual sensor is selected and used as the core of the corresponding soft sensor i. De- pending on the correlations between the measured values, the individual soft sensors can only predict the measured values with a certain accuracy. For this reason, an individ- ual failure criterion must be determined for each soft sensor. To do this, each soft sensor predicts a value for the corre- sponding physical sensor. These values are compared and the normalized deviation over the value range of the data is calculated. This is shown in Eq. 3. In Fig. 2, the soft sensor system, which is the focus of this publication, is highlighted in red. It is not relevant for the scope of this publication how the information on the physical twin is collected in the form of physical sensors or how the information obtained is utilized in the Digital Twin. To create the soft sensors, different machine learning models are investigated. Neural networks are deliberately excluded due to their lack of transparency. The algorithms for the creation and usage of the soft sensors were realized in Python. To clarify the procedure a ﬂow chart according to DIN 66001 [28] is created, which contains both the creation and use of the soft sensors. The ﬂow chart is shown in Fig. 3, the two algorithms are described in the following sections. ˇˇSi;physical −Si;softsensor ˇˇ Si;physical;max −Si;phyiscal;min = Sn (3) (3) The failure criterion is then determined using the average deviation over the set of measured values. After a soft sen- sor is created for each physical sensor, the algorithm ends. The resulting models for each sensor, as well as the failure criteria in the form of the limit, exported for the later usage. 3.1 Creation of soft sensors New soft sensors are created for each individual use case, this is shown in in Fig. 3a. For this, data is collected at the beginning of the runtime, to create a soft sensor Si, softsensor for each physical sensor Si, physical. The soft sensors are based on correlations between the measured values of the individual physical sensors. In order to utilized these correlations in the best possible way, different machine learning models are investigated. In the context of this work, the models linear regression, polynominal regression, random forrest regressor and decision trees were used. For each sensor i, the k different model types are created. Fig. 5 Correlation matrix of the sensors installed on the test bench Fig. 4 Rolling bearing test bench “Athene” of the TU Darmstadt 4.2 Creation of the soft sensors Data from a long-term test was used to create/train the soft sensors. As described in Sect. 3.2, the ﬁrst hour of the ex- periment was used to create the models of the soft sensors. The inconsistent run-in phase was deliberately excluded. Figure 5 shows a correlation matrix of the physical sen- sor data during the training period. The brighter a single cell of the matrix is, the higher the correlation of the two corresponding physical sensors. It is emphasized that the correlations are differently pronounced. Table 1 Model score and deviations of selected sensors on the test bench Sensor Best ML model type Model qual- ity score Average devia- tion [%] Axial force Lr 0.00006964 24.22 Radial force Rfr 0.6024 7.502 Temperature L1 Rfr 0.7832 44.84 Temperature L2 Rfr 0.6910 44.54 Temperature L3 Dt 0.9999 46.96 Temperature L4 Rfr 0.8054 26.87 Vibration radial RMS Rfr 0.9964 0.9726 Shock level radial Pr 0.9774 1.444 Vibration axial RMS Rfr 0.9992 1.870 Shock level axial Pr 0.9934 1.188 Torque Pr 0.0110 23.89 Temperature oil input Rfr 0.9563 13.56 Temperature oil output Rfr 0.9279 13.39 Voltage RMS Pr 0.9999 <0.001 Current RMS Pr 0.9999 1.719 Resistance Pr 0.9999 3.274 Table 1 Model score and deviations of selected sensors on the test bench Table 1 Model score and deviations of selected sensors on the test b h The data of the training period is ﬁrst split using a train test split (0.75; 0.25). The training data is used to create four different machine learning models (linear regression (lr), polynomial regression (pr), random forrest regressor (rfr) and decision trees (dt)) for each of the physical sensors and to determine the model scores. The model with the highest model score is then automatically selected and used as the basis for the corresponding soft sensors. The test data is then used to determine the deviations be- tween data of the physical sensors and the predicted values of the soft sensors. For each sensor 10,000 random data points are used and the average deviation is determined. The deviations are then normalized to the range of values of the data. Table 1 shows the most suitable ML models for each sen- sor, their model score and the average deviation of the pre- dictions that can be obtained with them. 4.1 Introduction of the test bench In the following, a soft sensor system is created and applied for the sensors of this test bench. As illustrated in Fig. 2, the digital twin of the test bench itself is not the focus of this work and will therefore not be discussed further below. At the Department of Product Development and Machine Elements at TU Darmstadt, rolling bearings can be exam- ined on the rolling bearing test bench “Athene” (see Fig. 4; [33–35]). This test bench can apply loads to rolling bearings at up to 8000rpm in four test chambers. For this purpose, two hydraulic cylinders apply up to 40kN axially and radi- ally. The reactions of the bearing in the form of radial and axial vibration and impact levels are measured via sensors. Furthermore, sensors for speed, torque, axial and radial force are installed and the voltage and current is recorded. A listing of all sensors can be seen in Fig. 5 or Table 1. Since the test bench is already equipped with extensive sen- sor systems, the corresponding sensor data can be used to feed a Digital Twin. In contrast to consumer products, no integration of further sensor technology is necessary. 3.2 Usage of soft sensors The created soft sensors are applied during the rest of the runtime of the physical twin operation. For this purpose the saved soft sensors in the form of models of each physical sensor as well as the individual failure criteria are loaded in for the utilization. This is shown in Fig. 3b. For each time step, the data of the physical sensors to be examined is imported. The model of each soft sensor is fed with the sensor data of the other sensors. Depending on K K 484 Forsch Ingenieurwes (2023) 87:479–488 Radial hydraulic cylinder Axial hydraulic cylinder Temperature sensors Internal bearing seat Vibraon sensors Electric motor Fig. 4 Rolling bearing test bench “Athene” of the TU Darmstadt Radial hydraulic cylinder Vibraon sensors Electric motor are calculated. If these deviations exceed the previously deﬁned limits, a warning is issued and appropriate measures are initiated. If the deviation of all measured variables is within their individual limits, the data of the entire time the respective trained weights, these are used to predict the value of the corresponding soft sensor. The predicted values of the soft sensor are then compared with the values of the physical sensors and the deviations K K 485 Forsch Ingenieurwes (2023) 87:479–488 step is released for use and passed on to the actual Digital Twin of the superordinate system or product. step is released for use and passed on to the actual Digital Twin of the superordinate system or product. Since the necessary IT infrastructure for real-time pro- cessing of the data is currently still under development, Since the necessary IT infrastructure for real-time pro- cessing of the data is currently still under development, stored data sets are used. The data sets are not loaded as a whole but row wise in discrete time steps of one second, so a real time data input is simulated and the transferability of the results is increased. This applies to both the Digital Twin and the soft sensors implemented later. 4.3 Usage of the soft sensors To evaluate the function of the soft sensors developed, a physical sensor is artiﬁcially damaged. For this purpose, an offset is applied to the physical sensor of the axial vi- bration from second 60 of the observed time range. This leads to the violation of the failure criterion of the maxi- mum permissible deviation in form of the yellow line. The sensor of the axial vibration is indicated as defective. This is shown in Fig. 7. For each physical sensor, the corresponding previously cre- ated model is loaded. This is fed with the corresponding physical sensor data and thus the values of the respec- tive soft sensors are calculated based on the weights of the model. The calculated values of the soft sensor are then com- pared with the real measured values of the physic sensor and the relative deviation is calculated. This is then com- pared with the limit values derived from the mean deviation. If this limit value is exceeded for a certain time, an error message is displayed. K 4.2 Creation of the soft sensors The normalization to the value range of the data leads to sensors with quasi- static measured values with a small value range showing high average deviations. The temperatures ﬂuctuate with about ±1°C around a constant value range, so that small de- viations of 0.5°C already lead to the high deviations shown here. The model scores and average deviations of the soft sensors for axial force and torque are also striking. This may be due to defective sensors during data collection, but requires closer investigation. For the purposes of this example, the failure criteria for each sensor are set as ten times the average deviation. K K K Forsch Ingenieurwes (2023) 87:479–488 486 Fig. 6 Dashboard of calculated values of the soft sensors (nor- mal operation) Temperature L2 [°C] Temperature oil input L2 [°C] Vibraon axial RMS [m/s^2] Vibraon axial RMS - Oﬀset [%] Fig. 7 Dashboard of calculated values of the soft sensors (sensor failure) Vibraon axial RMS [m/s^2] Vibraon axial RMS - Oﬀset [%] 4.3 Usage of the soft sensors For each physical sensor, the corresponding previously cre- ated model is loaded. This is fed with the corresponding physical sensor data and thus the values of the respec- tive soft sensors are calculated based on the weights of the model. The calculated values of the soft sensor are then com- pared with the real measured values of the physic sensor and the relative deviation is calculated. This is then com- 4.4 Detection of sensor failures To evaluate the function of the soft sensors developed, a physical sensor is artiﬁcially damaged. For this purpose, an offset is applied to the physical sensor of the axial vi- bration from second 60 of the observed time range. This leads to the violation of the failure criterion of the maxi- mum permissible deviation in form of the yellow line. The sensor of the axial vibration is indicated as defective. This is shown in Fig. 7. shboard of calculated he soft sensors (nor- ion) Temperature L2 [°C] Temperature oil input L2 [°C] Vibraon axial RMS [m/s^2] Vibraon axial RMS - Oﬀset [%] Temperature L2 [°C] Temperature oil input L2 [°C] Fig. 6 Dashboard of calculated values of the soft sensors (nor- mal operation) Fig. 4.2 Creation of the soft sensors 6 Dashboard of calculated values of the soft sensors (nor- mal operation) Temperature L2 [°C] Temperature oil input L2 [°C] Temperature oil input L2 [ C] Vibraon axial RMS [m/s^2] p p [ ] Vibraon axial RMS [m/s^2] Vibraon axial RMS [m/s^2] Vibraon axial RMS - Oﬀset [%] Vibraon axial RMS - Oﬀset [%] Vibraon axial RMS [m/s^2] Vibraon axial RMS - Oﬀset [%] 4.4 Detection of sensor failures Vibraon axial RMS [m/s^2] Vibraon axial RMS - Oﬀset [%] Fig. 7 Dashboard of calculated values of the soft sensors (sensor failure) Vibraon axial RMS [m/s^2] Vibraon axial RMS - Oﬀset [%] Fig. 7 Dashboard of calculated values of the soft sensors (sensor failure) Fig. 7 Dashboard of calculated values of the soft sensors (sensor failure) Vibraon axial RMS [m/s^2] 4.4 Detection of sensor failures References At the current time, the IT infrastructure required for real-time processing is still under development, which is why the exemplary implementation was only possible with existing data sets. Care was taken to process these data sets row wise in one-second increments, which increases the transferability of the results, but this still needs to be ﬁnally evaluated. Furthermore, the models are trained for individ- ual use cases. In the future, the use of measurement data from four-quadrant experiments can be used, to create soft sensors, which can be used with various use cases at dif- ferent operating points. Furthermore, the choice of failure criteria should be reconsidered. Although the derivation of the selection criteria from the mean deviations during the training leads to satisfying results, the interdependencies of the different soft sensors cannot be covered sufﬁciently. Even if the evaluation an the rolling bearing test bench shows that the soft sensors fulﬁll their purpose, the deter- mination of the failure criteria from the average deviations must be critically questioned and extensively veriﬁed. 1. 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https://openalex.org/W4321375547	https://essd.copernicus.org/preprints/essd-2022-392/essd-2022-392.pdf	English	null	Reply on RC2	null	2,023	cc-by	22,110	20 m Annual Paddy Rice Map for Mainland Southeast Asia Using Sentinel-1 SAR Data Good agreement was obtained when comparing our rice map with statistical data and other rice maps at the national d i i l l l Th i ffi i t f d t i ti R2 0 93 t th ti l l l d 0 97 t th i i l 20 backscattering characteristics in mainland Southeast Asia, the combination of spatio-temporal statistical features with the 15 generalization ability to complex rice growth patterns was selected, then input into the U-Net semantic segmentation model and combined with WorldCover data to eliminate false alarms, and finally the 20-meter resolution rice map of five countries in mainland Southeast Asia in 2019 was obtained. On the validation sample set, the proposed method achieved an accuracy of 92.20%. Good agreement was obtained when comparing our rice map with statistical data and other rice maps at the national backscattering characteristics in mainland Southeast Asia, the combination of spatio-temporal statistical features with the 15 generalization ability to complex rice growth patterns was selected, then input into the U-Net semantic segmentation model and combined with WorldCover data to eliminate false alarms, and finally the 20-meter resolution rice map of five countries in mainland Southeast Asia in 2019 was obtained. On the validation sample set, the proposed method achieved an accuracy of 92.20%. Good agreement was obtained when comparing our rice map with statistical data and other rice maps at the national and provincial levels. The maximum coefficient of determination R2 was 0.93 at the national level and 0.97 at the provincial 20 level. These results demonstrate the advantages of the proposed method in rice extraction with complex cropping patterns and the reliability of the generated rice maps. The 20 m annual paddy rice map for mainland Southeast Asia is available at https://doi.org/10.5281/zenodo.7315076(Sun, 2022). and provincial levels. The maximum coefficient of determination R2 was 0.93 at the national level and 0.97 at the provincial 20 level. These results demonstrate the advantages of the proposed method in rice extraction with complex cropping patterns and the reliability of the generated rice maps. The 20 m annual paddy rice map for mainland Southeast Asia is available at https://doi.org/10.5281/zenodo.7315076(Sun, 2022). https://doi.org/10.5194/essd-2022-392 Preprint. Discussion started: 6 December 2022 c⃝Author(s) 2022. CC BY 4.0 License. 20 m Annual Paddy Rice Map for Mainland Southeast Asia Using Sentinel-1 SAR Data Chunling Sun1,2,3, Hong Zhang1,2,3, Lu Xu1,2, Ji Ge1,2,3, Jingling Jiang1,2,3, Lijun Zuo1,2, Chao Wang1,2,3 1Key Laboratory of Digital Earth Science, Aerospace Information Research Institute, Chinese Academy of Sciences, Beijing 100094, China 5 2International Research Center of Big Data for Sustainable Development Goals, Beijing 100094, China 3College of Resources and Environment, University of Chinese Academy of Sciences, Beijing 100049, China Correspondence to: Hong Zhang (zhanghong@radi.ac.cn) Chunling Sun1,2,3, Hong Zhang1,2,3, Lu Xu1,2, Ji Ge1,2,3, Jingling Jiang1,2,3, Lijun Zuo1,2, Chao Wang1,2,3 1Key Laboratory of Digital Earth Science, Aerospace Information Research Institute, Chinese Academy of Sciences, Beijing 100094, China 5 2International Research Center of Big Data for Sustainable Development Goals, Beijing 100094, China 3College of Resources and Environment, University of Chinese Academy of Sciences, Beijing 100049, China Correspondence to: Hong Zhang (zhanghong@radi.ac.cn) Abstract. Over 90% of the world’s rice is produced in the Asia-Pacific Region. Synthetic aperture radar (SAR) enables all- 10 day and all-weather observations of rice distribution in tropical and subtropical regions. Rice growth patterns in tropical and subtropical regions are complex, and it is difficult to construct representative rice growth patterns, which makes it much more difficult to extract rice distribution based on SAR data. To address this problem, a rice mapping method based on time-series Sentinel-1 SAR data is proposed in this study for large regional tropical or subtropical areas. Based on the analysis of rice backscattering characteristics in mainland Southeast Asia, the combination of spatio-temporal statistical features with the 15 generalization ability to complex rice growth patterns was selected, then input into the U-Net semantic segmentation model and combined with WorldCover data to eliminate false alarms, and finally the 20-meter resolution rice map of five countries in mainland Southeast Asia in 2019 was obtained. On the validation sample set, the proposed method achieved an accuracy of 92.20%. Among the 17 sustainable development goals (SDGs) set by the United Nations in 2015, eradicating hunger, achieving food 25 security and improving nutrition, and promoting sustainable agriculture are key components of Goal 2 "Zero Hunger"(Desa, 2016). Rice feeds more than half of the world's population as a staple food and is a major crop for world food security (Kuenzer and Knauer, 2012). Asia is the largest rice-producing region in the world (Chen et al., 2012). With 48 million hectares under rice cultivation, Southeast Asia accounts for 40% of global rice exports(Yuan et al., 2022). The dual pressure of population and environment threatens the sustainability of global food security(Faostat, 2010; Godfray et al., 2010). Accurate information 30 1 Introduction on planted area and spatial distribution is the basis for monitoring rice growth and predicting yield(Mosleh et al., 2015; Laborte et al., 2017; Clauss et al., 2018; Jin et al., 2018; Yu et al., 2020; Hoang-Phi et al., 2021). Remote sensing technology plays a crucial role in rice growth monitoring and distribution mapping (Weiss et al., 2020; Zhao et al., 2021; Tsokas et al., 2022). Rice area mapping at the national scale usually uses medium- and low-resolution optical remote sensing data, such as MODIS and Landsat data. Some researchers used MODIS multitemporal data to produce rice 35 maps of China with resolutions of 500 m, 250 m and 500 m respectively (Sun et al., 2009; Clauss et al., 2016; Qiu et al., 2022). Guan et al. produced rice maps of Vietnam at 500 m resolution using MODIS time series data in 2010(Guan et al., 2016). The National Agricultural Statistics Service (NASS) released the state-based Crop Data Layer (CDL), a 30-m resolution crop distribution map product for the entire continental United States, using multisource medium resolution remote sensing data on planted area and spatial distribution is the basis for monitoring rice growth and predicting yield(Mosleh et al., 2015; Laborte et al., 2017; Clauss et al., 2018; Jin et al., 2018; Yu et al., 2020; Hoang-Phi et al., 2021). on planted area and spatial distribution is the basis for monitoring rice growth and predicting yield(Mosleh et al., 2015; Laborte et al., 2017; Clauss et al., 2018; Jin et al., 2018; Yu et al., 2020; Hoang-Phi et al., 2021). Remote sensing technology plays a crucial role in rice growth monitoring and distribution mapping (Weiss et al., 2020; Zhao et al., 2021; Tsokas et al., 2022). Rice area mapping at the national scale usually uses medium- and low-resolution optical remote sensing data, such as MODIS and Landsat data. Some researchers used MODIS multitemporal data to produce rice 35 maps of China with resolutions of 500 m, 250 m and 500 m respectively (Sun et al., 2009; Clauss et al., 2016; Qiu et al., 2022). Guan et al. produced rice maps of Vietnam at 500 m resolution using MODIS time series data in 2010(Guan et al., 2016). 1 Introduction The National Agricultural Statistics Service (NASS) released the state-based Crop Data Layer (CDL), a 30-m resolution crop distribution map product for the entire continental United States, using multisource medium resolution remote sensing data 35 (Landsat, IRS-p6, DEIMOS-1, etc.) (Johnson and Mueller, 2010).Luo et al. and Wei et al. used Landsat time-series data to 40 produce 1 km and 30 m resolution rice datasets for China, respectively (Luo et al., 2020a; Luo et al., 2020b; Wei et al., 2022). Recently，the Sentinel -2 satellite sensor opens up new possibilities for paddy rice monitoring. Liu et al. obtained medium- resolution rice maps of China using Sentinel-2 time series data in 2020 (Liu et al., 2022). At the continental scale, MODIS time-series data were frequently used to map the distribution of rice cultivation (Dong et al., 2016a; Dong et al., 2016b). Xiao et al. and Gumma et al. produced low- and medium-resolution rice area maps for several 45 South and Southeast Asian countries using MODIS data at the 500 m spatial resolution, respectively (Xiao et al., 2005; Xiao et al., 2006; Gumma et al., 2011a; Gumma et al., 2011b; Bridhikitti and Overcamp, 2012; Gumma et al., 2014; Nelson and Gumma, 2015). Using MODIS time-series data, Zhang et al. generated rice acreage maps for China and India from 2000 to 2015 (Zhang et al., 2017). Han et al. used MODIS data to complete 500 m annual rice maps for the Asian monsoon region from 2000 to 2020(Han et al., 2022). SPOT data were also used for continent-wide rice mapping. Manjunath et al. used 2009- 50 2010 multi-temporal SPOT VGT normalized difference vegetation index (NDVI) data to produce 1km resolution rice maps for South and Southeast Asia(Manjunath et al., 2015). Most of the rice in the world is distributed in hot and rainy areas. However, optical data are easily obscured by clouds, which also poses a challenge for rice extraction in humid and sub-humid climates with abundant water resources such as Southeast Asia(Zhu and Woodcock, 2012; Liu et al., 2019; Sun et al., 2021). Compared with traditional optical remote sensing, synthetic 55 aperture radar (SAR) is an active microwave radar with the advantages of all-day and all-weather, which is weather- independent and can penetrate clouds, and is very sensitive to the geometric structure and dielectric properties of crops(Huang et al., 2017; Orynbaikyzy et al., 2019; Sun et al., 2022). 1 Introduction Among the 17 sustainable development goals (SDGs) set by the United Nations in 2015, eradicating hunger, achieving food 25 security and improving nutrition, and promoting sustainable agriculture are key components of Goal 2 "Zero Hunger"(Desa, 2016). Rice feeds more than half of the world's population as a staple food and is a major crop for world food security (Kuenzer and Knauer, 2012). Asia is the largest rice-producing region in the world (Chen et al., 2012). With 48 million hectares under rice cultivation, Southeast Asia accounts for 40% of global rice exports(Yuan et al., 2022). The dual pressure of population and environment threatens the sustainability of global food security(Faostat, 2010; Godfray et al., 2010). Accurate information 30 Among the 17 sustainable development goals (SDGs) set by the United Nations in 2015, eradicating hunger, achieving food 25 security and improving nutrition, and promoting sustainable agriculture are key components of Goal 2 "Zero Hunger"(Desa, 2016). Rice feeds more than half of the world's population as a staple food and is a major crop for world food security (Kuenzer and Knauer, 2012). Asia is the largest rice-producing region in the world (Chen et al., 2012). With 48 million hectares under rice cultivation, Southeast Asia accounts for 40% of global rice exports(Yuan et al., 2022). The dual pressure of population Among the 17 sustainable development goals (SDGs) set by the United Nations in 2015, eradicating hunger, achieving food 25 security and improving nutrition, and promoting sustainable agriculture are key components of Goal 2 "Zero Hunger"(Desa, 2016). Rice feeds more than half of the world's population as a staple food and is a major crop for world food security (Kuenzer and Knauer, 2012). Asia is the largest rice-producing region in the world (Chen et al., 2012). With 48 million hectares under rice cultivation, Southeast Asia accounts for 40% of global rice exports(Yuan et al., 2022). The dual pressure of population d i h h i bili f l b l f d i (F 2010 G df l 2010) A i f i 30 and environment threatens the sustainability of global food security(Faostat, 2010; Godfray et al., 2010). Accurate information 30 1 1 https://doi.org/10.5194/essd-2022-392 Preprint. Discussion started: 6 December 2022 c⃝Author(s) 2022. CC BY 4.0 License. 1 Introduction The phenology-based approach refers to the extraction of rice by defining 70 phenological indicators or identifying phenological periods by combining the time-series data of the rice growth cycle and the analysis of the growth phenology calendar (Nelson et al., 2014; Chen et al., 2016; Nguyen and Wagner, 2017; Liu et al., 2018; Xin et al., 2020; Ni et al., 2021). The phenological periods such as transplanting, flooding, heading and maturity are most often used to extract rice. Shew et al. combined vegetation indices extracted from Landsat time-series data with a rule-based machine learning relying on image information. The phenology-based approach refers to the extraction of rice by defining 70 phenological indicators or identifying phenological periods by combining the time-series data of the rice growth cycle and the analysis of the growth phenology calendar (Nelson et al., 2014; Chen et al., 2016; Nguyen and Wagner, 2017; Liu et al., 2018; Xin et al., 2020; Ni et al., 2021). The phenological periods such as transplanting, flooding, heading and maturity are most often used to extract rice. Shew et al. combined vegetation indices extracted from Landsat time-series data with a rule-based algorithm for phenological stages to map a 30 m dry season rice map of Bangladesh from 2014 to 2018 (Shew and Ghosh, 75 2019). Li et al. extracted the minimum and maximum values of permanent water backscatter coefficients and three thresholds of phenological characteristics, namely, the date of the beginning of the season, date of maximum backscatter during the peak growing season, and length of the vegetative stage from 402 scenes of Sentinel-1 data in 2017 to map rice paddies in the Mun River basin ,Thailand (Li et al., 2020).Kang et al. completed a 10 m resolution rice map of Cambodia from Sentinel-1 (2015) and Sentinel-2 (2015-2017) time-series data using three key rice phenological periods in the dry and rainy seasons, respectively 80 (Kang et al., 2022). However, the phenology-based methods rely too much on human intervention and are not suitable for rice extraction with complex cropping cycles. The approaches based on the combination of time series data and machine learning method refer to the direct use of time series as the input features for machine learning (Ndikumana et al., 2018; Chang et al., 2020; Mansaray et al., 2021; Yang et al., 2021). 1 Introduction Machine learning methods are used to extract rice information by mining fixed relationships 85 across growth periods of rice (Yang et al., 2019; You et al., 2021). Torbick et al. used Sentinel-1, Landsat-8 and PALSAR-2 time series data and a random forest algorithm to map rice planting area and planting intensity of Myanmar with 20 m resolution in 2015 (Torbick et al., 2017). Inoue et al. developed a 30 m resolution map of paddy rice in Japan for 2018 using Sentinel-1 SAR data and Sentinel-2 data with the conventional decision tree methods (Inoue et al., 2020). Wei et al. completed rice area mapping for the Arkansas River Basin, USA, by entering dual-polarized Sentinel-1 data from 2017-2019 into a modified U- 90 Net model (Wei et al., 2021). Soh et al. used Sentinel-1 and Sentinel-2 time series data and a K-means clustering method to map rice in West Malaysia (Soh et al., 2022). The climate in tropical or subtropical regions such as Southeast Asia is suitable for rice growth throughout the year, increasing the difficulty of rice extraction. First, it is difficult to obtain accurate phenological information because the climate in Southeast Asia is hot and humid for rice growth, the timing of rice seedling and transplanting is more flexible (Xu et al., 2021). This 95 makes it impossible to use empirical methods to determine effective weathering indicators and suitable periods. Second, rice growth patterns in Southeast Asia are too complex to construct a representative rice growth model (Kang et al., 2022). This poses obstacles for rice extraction methods that utilize time-fixed relationships in time-series data. Asia is hot and humid for rice growth, the timing of rice seedling and transplanting is more flexible (Xu et al., 2021). This 95 makes it impossible to use empirical methods to determine effective weathering indicators and suitable periods. Second, rice growth patterns in Southeast Asia are too complex to construct a representative rice growth model (Kang et al., 2022). This poses obstacles for rice extraction methods that utilize time-fixed relationships in time-series data. Asia is hot and humid for rice growth, the timing of rice seedling and transplanting is more flexible (Xu et al., 2021). This 95 makes it impossible to use empirical methods to determine effective weathering indicators and suitable periods. Second, rice growth patterns in Southeast Asia are too complex to construct a representative rice growth model (Kang et al., 2022). 1 Introduction In recent years, free SAR data represented by Sentinel-1 data have been widely used in rice mapping over large regions. Singha et al. obtained seasonal rice maps at 10 m resolution for Bangladesh and northeast India using time-series Sentinel-1VH data for 2017 (Singha et al., 2019). Pan et al. used 2016- 60 2020Sentinel-1VH data to produce 10-m spatial resolution double-season rice maps for nine provinces in southern China (Pan et al., 2021). Xu et al. used time-series Sentinel-1VH data to obtain a 20 m rice map for Thailand in 2019 (Xu et al., 2021). To take full advantage of multi-source remote sensing data, some researchers combined optical and SAR time-series data in the large-scale rice mapping studies (Thenkabail et al., 2009; Zhang et al., 2018; You and Dong, 2020). Phan et al. used 2 https://doi.org/10.5194/essd-2022-392 Preprint. Discussion started: 6 December 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/essd-2022-392 Preprint. Discussion started: 6 December 2022 c⃝Author(s) 2022. CC BY 4.0 License. Sentinel 1/2 and Landsat data to produce the first Vietnam land use/land cover annual dataset with 30m resolution from 1990 65 to 2020 (Phan et al., 2021). Han et al. obtained 500m resolution rice maps from 2017 to 2019 in Northeast and Southeast Asia using Sentinel-1 and MODIS time-series data (Han et al., 2021). At present, large-scale rice mapping methods based on remote sensing data can be divided into two categories, one is the combination of phenological information and remote sensing images, and the other is the combination of time series data and Sentinel 1/2 and Landsat data to produce the first Vietnam land use/land cover annual dataset with 30m resolution from 1990 65 to 2020 (Phan et al., 2021). Han et al. obtained 500m resolution rice maps from 2017 to 2019 in Northeast and Southeast Asia using Sentinel-1 and MODIS time-series data (Han et al., 2021). At present, large-scale rice mapping methods based on remote sensing data can be divided into two categories, one is the combination of phenological information and remote sensing images, and the other is the combination of time series data and combination of phenological information and remote sensing images, and the other is the combination of time series data and machine learning relying on image information. 1 Introduction Section 2 describes the study area and the data information used; Section 3 presents the rice mapping scheme; Section 4 presents the temporal characteristics analysis of the data and the rice map results; Section 5 discusses the results; and finally, Section 6 draws conclusions. 1 Introduction (2) A deep combination of the above features and the U-Net model will be used to fully exploit the pixel-level semantic features to complete the annual rice mapping of five Southeast Asian countries in 2019, enriching the available Southeast Asian rice maps, and providing support information for the scientific community and scientific decision-making. 115 The rest of the paper is organized as follows. Section 2 describes the study area and the data information used; Section 3 presents the rice mapping scheme; Section 4 presents the temporal characteristics analysis of the data and the rice map results; Section 5 discusses the results; and finally, Section 6 draws conclusions. of rice maps. 105 Therefore, in this study, to meet the requirements of high-precision rice area mapping inSoutheast Asia, the objectives accomplished using Sentinel-1 time-series data are as follows. Therefore, in this study, to meet the requirements of high-precision rice area mapping inSoutheast Asia, the objectives accomplished using Sentinel-1 time-series data are as follows. (1) A new feature extraction method is proposed by analyzing the time-series backscattering variation of rice in mainland Southeast Asia. The method does not need to summarize the general evolutionary model from rice backscatter coefficients with diverse growth patterns Using three simple but effective temporal statistical features defined in this study it is 110 Southeast Asia. The method does not need to summarize the general evolutionary model from rice backscatter coefficients with diverse growth patterns. Using three simple but effective temporal statistical features defined in this study, it is possible to capture features that provide key information about the rice growth process. This study provided a new idea for rice mapping methods in tropical or subtropical regions. with diverse growth patterns. Using three simple but effective temporal statistical features defined in this study, it is 110 possible to capture features that provide key information about the rice growth process. This study provided a new idea for rice mapping methods in tropical or subtropical regions. (2) A deep combination of the above features and the U-Net model will be used to fully exploit the pixel-level semantic features to complete the annual rice mapping of five Southeast Asian countries in 2019, enriching the available Southeast Asian rice maps, and providing support information for the scientific community and scientific decision-making. 115 The rest of the paper is organized as follows. 1 Introduction This poses obstacles for rice extraction methods that utilize time-fixed relationships in time-series data. 3 https://doi.org/10.5194/essd-2022-392 Preprint. Discussion started: 6 December 2022 c⃝Author(s) 2022. CC BY 4.0 License. Current publicly downloadable rice products for Southeast Asia include the Asia rice map (IRRI Rice Data, 500 m) (Nelson and Gumma, 2015), Vietnam-wide annual land use/land cover datasets from 1990 to 2020 (VLUCD, 30 m) (Phan et al., 2021), 100 annual paddy rice maps for Northeast and Southeast Asia from 2017 to 2019 (NESEA-Rice10, 10 m) (Han et al., 2021), and annual rice in the Asian monsoon region from 2000 to 2020 (500 m) (Han et al., 2022). Except for Vietnam's VLUCD, the source data for the public rice maps in Southeast Asia were mainly MODIS. Rice maps using MODIS data contained a large number of mixed pixels due to low spatial resolution (Dong et al., 2015; Shew and Ghosh, 2019), which affected the accuracy Current publicly downloadable rice products for Southeast Asia include the Asia rice map (IRRI Rice Data, 500 m) (Nelson and Gumma, 2015), Vietnam-wide annual land use/land cover datasets from 1990 to 2020 (VLUCD, 30 m) (Phan et al., 2021), 100 annual paddy rice maps for Northeast and Southeast Asia from 2017 to 2019 (NESEA-Rice10, 10 m) (Han et al., 2021), and annual rice in the Asian monsoon region from 2000 to 2020 (500 m) (Han et al., 2022). Except for Vietnam's VLUCD, the source data for the public rice maps in Southeast Asia were mainly MODIS. Rice maps using MODIS data contained a large number of mixed pixels due to low spatial resolution (Dong et al., 2015; Shew and Ghosh, 2019), which affected the accuracy of rice maps. 105 Therefore, in this study, to meet the requirements of high-precision rice area mapping inSoutheast Asia, the objectives accomplished using Sentinel-1 time-series data are as follows. (1) A new feature extraction method is proposed by analyzing the time-series backscattering variation of rice in mainland Southeast Asia. The method does not need to summarize the general evolutionary model from rice backscatter coefficients with diverse growth patterns. Using three simple but effective temporal statistical features defined in this study, it is 110 possible to capture features that provide key information about the rice growth process. This study provided a new idea for rice mapping methods in tropical or subtropical regions. 2.1 Study area 120 Approximately 90% of the world's rice is grown on 140 million hectares of land in Asia. The rice production in mainland Southeast Asian accounts for about 15% of the world rice production(Fao, 2020).. The study area is five countries in mainland Southeast Asia, namely, Myanmar, Thailand, Laos, Cambodia, and Vietnam, as shown in Figure 1. These countries have more land under rice cultivation than any other crop. And Vietnam and Thailand are the two largest rice exporters in the world (Yuan et al 2022) Indeed changes in rice production in these countries could destabilize international rice markets and have a clear 125 Approximately 90% of the world's rice is grown on 140 million hectares of land in Asia. The rice production in mainland Southeast Asian accounts for about 15% of the world rice production(Fao, 2020).. The study area is five countries in mainland Southeast Asia, namely, Myanmar, Thailand, Laos, Cambodia, and Vietnam, as shown in Figure 1. These countries have more land under rice cultivation than any other crop. And Vietnam and Thailand are the two largest rice exporters in the world (Yuan et al., 2022). Indeed, changes in rice production in these countries could destabilize international rice markets and have a clear 125 impact on global food security. Southeast Asia has a tropical monsoon climate with an average annual temperature of 20-27 °C and abundant rainfall. Therefore, rice can be grown at any time of the year. Agricultural systems in Southeast Asia are dominated by rainfed lowland rice and irrigated lowland rice (Kuenzer and Knauer, 2012). Under suitable irrigation conditions, rice can be harvested two to three times per year. 130 4 4 https://doi.org/10.5194/essd-2022-392 Preprint. Discussion started: 6 December 2022 c⃝Author(s) 2022. CC BY 4.0 License. Figure 1: Location of the study area. Orbit-frame 99-16 images are used for the training samples, and the Rice and Non-rice flags show the distribution of the validation sample set. The base map is from Esri. 135 Figure 1: Location of the study area. Orbit-frame 99-16 images are used for the training samples, and the Rice and Non-rice flags show the distribution of the validation sample set. The base map is from Esri. 35 135 2.2.1 Satellite imagery and auxiliary data The European Space Agency (ESA) provides a free data source for large-scale land cover monitoring through Sentinel-1A, launched in 2014, and Sentinel-1B, launched in 2016 (Torres et al., 2012). The Sentinel-1 satellites carry a C-band (5.405 GHz) synthetic-aperture radar with a 12-day revisit period. In this study, the 2019 dual-polarized (VV/VH) GRD products in 140 Interferometric Wide Swath (IW) mode were downloaded from the ASF website. In total, 12 tracks, 90 frames and 2665 scenes of data were acquired. Details are shown in Table 1. The European Space Agency (ESA) provides a free data source for large-scale land cover monitoring through Sentinel-1A, launched in 2014, and Sentinel-1B, launched in 2016 (Torres et al., 2012). The Sentinel-1 satellites carry a C-band (5.405 GHz) synthetic-aperture radar with a 12-day revisit period. In this study, the 2019 dual-polarized (VV/VH) GRD products in 40 The European Space Agency (ESA) provides a free data source for large-scale land cover monitoring through Sentinel-1A, launched in 2014, and Sentinel-1B, launched in 2016 (Torres et al., 2012). The Sentinel-1 satellites carry a C-band (5.405 GHz) synthetic-aperture radar with a 12-day revisit period. In this study, the 2019 dual-polarized (VV/VH) GRD products in 140 Interferometric Wide Swath (IW) mode were downloaded from the ASF website. In total, 12 tracks, 90 frames and 2665 scenes of data were acquired. Details are shown in Table 1. And, the DEM and land use/land cover product were also collected. Shuttle Radar Topography Mission (SRTM) 3sec DEM synthetic-aperture radar with a 12-day revisit period. In this study, the 2019 dual-polarized (VV/VH) GRD products in 140 Interferometric Wide Swath (IW) mode were downloaded from the ASF website. In total, 12 tracks, 90 frames and 2665 scenes of data were acquired. Details are shown in Table 1. And, the DEM and land use/land cover product were also collected. Shuttle Radar Topography Mission (SRTM) 3sec DEM product was used for terrain correction of SAR data. WorldCover data were used to reduce false alarms caused by water and of data were acquired. Details are shown in Table 1. And, the DEM and land use/land cover product were also collected. Shuttle Radar Topography Mission (SRTM) 3sec DEM product was used for terrain correction of SAR data. WorldCover data were used to reduce false alarms caused by water and woodland. 2.2.2 Agricultural statistics The statistical yearbooks of each country were collected to compile annual census data of rice harvested area at different 150 administrative levels in these countries. The administrative levels include national and subnational levels (state, province, or regions, uniformly represented by province in this study). The unit of area in the statistical data is uniformly converted to hectares (ha). The statistical yearbooks of each country were collected to compile annual census data of rice harvested area at different 150 administrative levels in these countries. The administrative levels include national and subnational levels (state, province, or regions, uniformly represented by province in this study). The unit of area in the statistical data is uniformly converted to hectares (ha). 2.2.1 Satellite imagery and auxiliary data CC BY 4.0 License. 2.2.3 Available rice maps based on remote sensing data From the perspective of resolution and coverage area, 2 publicly downloadable rice maps were selected for comparison. 155 (1) Vietnam-wide annual land use/land cover datasets (VLUCD) Researchers from the Japan Aerospace Exploration Agency (JAXA) produced the first 30-m resolution Vietnam-wide annual land use/land cover datasets (VLUCD) using multiple sources of data (including Landsat and Sentinel-1/2) and a random forest algorithm (Phan et al., 2021). The VLUCD contains annual land cover products for 1990-2020, including a primary From the perspective of resolution and coverage area, 2 publicly downloadable rice maps were selected for comparison. 155 (1) Vietnam-wide annual land use/land cover datasets (VLUCD) Researchers from the Japan Aerospace Exploration Agency (JAXA) produced the first 30-m resolution Vietnam-wide annual land use/land cover datasets (VLUCD) using multiple sources of data (including Landsat and Sentinel-1/2) and a random forest algorithm (Phan et al., 2021). The VLUCD contains annual land cover products for 1990-2020, including a primary algorithm (Phan et al., 2021). The VLUCD contains annual land cover products for 1990-2020, including a primary classification (containing 10 different categories of primary land cover, including rice) and a secondary classification (18 160 different categories of secondary primary land cover, including rice). The rice layer was extracted from the 2019 annual land cover products for comparison. classification (containing 10 different categories of primary land cover, including rice) and a secondary classification (18 160 different categories of secondary primary land cover, including rice). The rice layer was extracted from the 2019 annual land cover products for comparison. classification (containing 10 different categories of primary land cover, including rice) and a secondary classification (18 160 different categories of secondary primary land cover, including rice). The rice layer was extracted from the 2019 annual land cover products for comparison. classification (containing 10 different categories of primary land cover, including rice) and a secondary classification (18 160 different categories of secondary primary land cover, including rice). The rice layer was extracted from the 2019 annual land cover products for comparison. 2.2.1 Satellite imagery and auxiliary data WorldCover is a global land cover product produced by ESA and several scientific institutions using Sentinel-1 145 and -2 data (Zanaga et al., 2021). It provides information on 11 land cover types for 2020 with a resolution of 10 m and an overall accuracy of 80.7% for the Asian region. 5 5 https://doi.org/10.5194/essd-2022-392 Preprint. Discussion started: 6 December 2022 c⃝Author(s) 2022. CC BY 4.0 License. Table 1 List of SAR data Country Orbit Frame Satellite Number of images Country Orbit Frame Satellite Number of images Experimental Data Thailand 172 17 S1A 31 Laos 99 1240 S1A 30 18 S1A 31 1245 S1A 30 135 16 S1A 23 1250 S1A 30 17 S1A 23 26 44 S1A 31 18 S1A 23 49 S1A 31 19 S1A 23 54 S1A 31 62 1 S1B 29 59 S1A 31 2 S1B 29 64 S1A 31 3 S1B 29 69 S1A 31 4 S1B 29 128 44 S1A 30 5 S1B 29 49 S1A 30 20 S1A 27 54 S1A 30 21 S1A 27 59 S1A 30 22 S1A 26 64 S1A 30 23 S1A 24 Myanmar 41 44 S1A 31 24 S1A 25 50 S1A 31 164 1 S1B 32 55 S1A 31 2 S1B 32 60 S1A 31 3 S1B 32 65 S1A 31 4 S1B 32 70 S1A 31 5 S1B 32 143 46 S1A 30 20 S1A 13 51 S1A 30 91 1 S1B 32 56 S1A 30 2 S1B 32 61 S1A 30 3 S1B 32 66 S1A 30 4 S1B 32 71 S1A 30 Cambodia 99 1220 S1A 30 76 S1A 30 1225 S1A 30 70 1217 S1A 31 31 S1B 31 1222 S1A 31 26 29 S1A 28 1227 S1A 31 32 S1B 30 1232 S1A 31 38 S1B 30 1237 S1A 31 43 S1B 30 1242 S1A 31 128 34 S1A 30 1247 S1A 31 39 S1A 30 1252 S1A 31 Vietnam 26 23 S1A 28 1257 S1A 31 34 S1A 31 1262 S1A 31 55 31 S1A 31 1267 S1A 31 37 S1A 31 172 1248 S1A 28 42 S1A 31 1253 S1A 28 47 S1A 31 1258 S1A 28 62 S1A 31 1263 S1A 28 67 S1A 31 1268 S1A 28 72 S1A 31 1273 S1A 28 128 29 S1A 30 69 S1A 30 Training data set Thailand 99 16 S1A 29 6 6 https://doi.org/10.5194/essd-2022-392 Preprint. Discussion started: 6 December 2022 c⃝Author(s) 2022. 2.2.3 Available rice maps based on remote sensing data (2) Rice data of Asia from International Rice Research Institute (IRRI Rice Data) (2) Rice data of Asia from International Rice Research Institute (IRRI Rice Data) The International Rice Research Institute (IRRI) is an international agricultural research and The International Rice Research Institute (IRRI) is an international agricultural research and training organization with its The International Rice Research Institute (IRRI) is an international agricultural research and training organization with its headquarters in Los Baños, Laguna, in the Philippines, and offices in seventeen countries. IRRI is one of 15 agricultural 165 research centers in the world that form the Consortium of International Agricultural Research Centers(CGIAR), a global partnership of organizations engaged in research on food security. IRRI is also the largest non-profit agricultural research center in Asia. headquarters in Los Baños, Laguna, in the Philippines, and offices in seventeen countries. IRRI is one of 15 agricultural 165 research centers in the world that form the Consortium of International Agricultural Research Centers(CGIAR), a global partnership of organizations engaged in research on food security. IRRI is also the largest non-profit agricultural research center in Asia. The IRRI produced a 500 m resolution map of the general distribution of rice in Asia from 2001 to 2012 using MODIS time- series data (Nelson and Gumma, 2015), which is freely available to the public. 170 Table 2 shows details of the SAR data, auxiliary data, available rice maps, land cover data and statistics used in the study. Table 2 Details of the data used in the study Data type Data product or country name Year Resolution Description Data access Last access (dd/mm/yyy y) SAR imagery Sentinel-1 2019 2022 m (rgaz) The backscatter characteristics extraction https://search.asf.alaska.e du/#/ 11/10/2022 DEM Shuttle Radar Topography 2000 90m Terrain correction https://search.earthdata.n asa.gov/search?q=SRTM 11/10/2022 The IRRI produced a 500 m resolution map of the general distribution of rice in Asia from 2001 to 2012 using MODIS time- series data (Nelson and Gumma, 2015), which is freely available to the public. 170 The IRRI produced a 500 m resolution map of the general distribution of rice in Asia from 2001 to 2012 using MODIS time- series data (Nelson and Gumma, 2015), which is freely available to the public. 170 Table 2 shows details of the SAR data, auxiliary data, available rice maps, land cover data and statistics used in the study. 2.2.3 Available rice maps based on remote sensing data 7 7 Mission (SRTM) 3sec Land cover data ESA WorldCover 2020 2020 10 m Extraction of water and forest mask https://esa- worldcover.org/en 11/10/2022 Available Rice Map Vietnam-wide annual land use/land cover datasets (VLUCD) 2019 30 m Spatial consistency comparison of rice extraction results https://www.eorc.jaxa.jp/ ALOS/en/dataset/lulc/lul c_vnm_v2109_e.htm 11/10/2022 Rice data of Asia from IRRI (IRRI Rice Data) 2000 to 2012 500 m Spatial consistency comparison of rice extraction results https://www.irri.org/mapp ing 11/10/2022 Statistical yearbook Vietnam 2019 Province scale verifying the classification accuracy https://www.gso.gov.vn/e n/homepage/ 11/10/2022 Cambodia 2019 Province scale verifying the classification accuracy http://nis.gov.kh/index.ph p/km/ 11/10/2022 Laos 2019 Province scale verifying the classification accuracy https://www.lsb.gov.la/en /home/ 11/10/2022 Thailand 2019 Province scale verifying the classification accuracy http://www.nso.go.th/site s/2014en 11/10/2022 Myanmar 2019 State scale verifying the classification accuracy https://www.mopf.gov.m m/en/page/planning/centr al-statistical- organization-cso/752 11/10/2022 3 Method The flowchart of this study is shown in Figure 2. First, the Sentinel-1 time series images were preprocessed. Then, key features 175 in the rice growth process are extracted from the time series SAR data. To make full use of the pixel-level semantics of the features, the extracted features were fed into the U-Net model to obtain rice extraction results with spatial details. Finally, to reduce false alarms from water bodies and vegetation, the results were postprocessed using masks generated based on high- precision land cover products to obtain the annual rice map of five Southeast Asian countries. The flowchart of this study is shown in Figure 2. First, the Sentinel-1 time series images were preprocessed. Then, key features 175 in the rice growth process are extracted from the time series SAR data. To make full use of the pixel-level semantics of the features, the extracted features were fed into the U-Net model to obtain rice extraction results with spatial details. Finally, to reduce false alarms from water bodies and vegetation, the results were postprocessed using masks generated based on high- precision land cover products to obtain the annual rice map of five Southeast Asian countries. 3.1 Preprocessing 180 3.1 Preprocessing 180 The Sentinle-1 time-series data were preprocessed using SNAP software (Filipponi, 2019). The steps were as follows: (1) orbit correction; (2) thermal noise removal; (3) radiometric calibration; (4) coregistration; (5) terrain correction; (6) multitemporal The Sentinle-1 time-series data were preprocessed using SNAP software (Filipponi, 2019). The steps were as follows: (1) orbit correction; (2) thermal noise removal; (3) radiometric calibration; (4) coregistration; (5) terrain correction; (6) multitemporal 8 https://doi.org/10.5194/essd-2022-392 Preprint. Discussion started: 6 December 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/essd-2022-392 Preprint. Discussion started: 6 December 2022 c⃝Author(s) 2022. CC BY 4.0 License. speckle noise filtering; and (7) conversion of the multitemporal intensity map to sigma 0 (𝜎0) on the decibel (dB) scale. The final 𝜎0 images with 20 m resolution in the WGS84 geographic coordinate system were obtained. speckle noise filtering; and (7) conversion of the multitemporal intensity map to sigma 0 (𝜎0) on the decibel (dB) scale. The final 𝜎0 images with 20 m resolution in the WGS84 geographic coordinate system were obtained. 185 Figure 2: Flowchart of the proposed rice mapping method using Sentinel-1 data. 185 Figure 2: Flowchart of the proposed rice mapping method using Sentinel-1 data. 3.2 Feature Extraction As described in many previous studies (Singha et al., 2019; Chang et al., 2020; Crisóstomo De Castro Filho et al., 2020; Sun et al., 2022), VH polarization was more sensitive to the flooding period of rice than VV polarization and has been more widely used for rice extraction. Therefore, VH data were selected in this study. To analyze the time-series characteristics of the 90 backscattering coefficients of rice and other land cover types in the study area, representative sample plots of four typical land cover types (rice, water bodies, buildings, and vegetation) were selected. Based on Google Earth data and other land cover data, four rice regions that belongs to different cropping systems were chosen. The average VH polarization time series data As described in many previous studies (Singha et al., 2019; Chang et al., 2020; Crisóstomo De Castro Filho et al., 2020; Sun et al., 2022), VH polarization was more sensitive to the flooding period of rice than VV polarization and has been more widely used for rice extraction. Therefore, VH data were selected in this study. To analyze the time-series characteristics of the 190 backscattering coefficients of rice and other land cover types in the study area, representative sample plots of four typical land cover types (rice, water bodies, buildings, and vegetation) were selected. Based on Google Earth data and other land cover data, four rice regions that belongs to different cropping systems were chosen. The average VH polarization time series data of these land cover types were calculated, as shown in Figure 3. used for rice extraction. Therefore, VH data were selected in this study. To analyze the time-series characteristics of the 190 backscattering coefficients of rice and other land cover types in the study area, representative sample plots of four typical land cover types (rice, water bodies, buildings, and vegetation) were selected. Based on Google Earth data and other land cover data, four rice regions that belongs to different cropping systems were chosen. The average VH polarization time series data of these land cover types were calculated, as shown in Figure 3. In Figure 3, the backscattering coefficients of water bodies were small, as they exhibited single specular scattering, and their 195 return power level was lower than other land covers. In contrast, buildings exhibited double bounce and their return powers were much stronger, leading to larger backscattering coefficients. 3.2 Feature Extraction The scattering process of radar waves of vegetation was more complicated, and the backscattering coefficients of vegetation were between buildings and water bodies. For different kinds of rice samples, the curve fluctuations were significant, due to the effects of flooding and the multi-season planting In Figure 3, the backscattering coefficients of water bodies were small, as they exhibited single specular scattering, and their 195 return power level was lower than other land covers. In contrast, buildings exhibited double bounce and their return powers were much stronger, leading to larger backscattering coefficients. The scattering process of radar waves of vegetation was more complicated, and the backscattering coefficients of vegetation were between buildings and water bodies. For different kinds of rice samples, the curve fluctuations were significant, due to the effects of flooding and the multi-season planting patterns. But generally, their backscattering intensities ranged between buildings and water bodies. More specifically, the land 200 patterns. But generally, their backscattering intensities ranged between buildings and water bodies. More specifically, the land 200 parcel of Rice 1 was planted with two seasons of rice during the observation period: the first season was from April to July, patterns. But generally, their backscattering intensities ranged between buildings and water bodies. More specifically, the land 200 parcel of Rice 1 was planted with two seasons of rice during the observation period: the first season was from April to July, 9 https://doi.org/10.5194/essd-2022-392 Preprint. Discussion started: 6 December 2022 c⃝Author(s) 2022. CC BY 4.0 License. and the second season was from August to October. The land parcel of Rice 2 was planted with only one season of rice, from April to September. The land parcel of Rice 3 was planted with two seasons of rice: the first season was from March to July and the second season was from July to October. The land parcel of Rice 4 was planted with three seasons of rice: the first season was from February to June, the second season was from June to October, and only part of the third season (October- 205 December) was observed. It can be seen that the time steps of each growing season for the selected Rice 1- Rice 4 were inconsistent. In fact, the high heterogeneity of rice backscattering coefficients in Southeast Asia is caused by the high heterogeneity in climate and topography. 3.2 Feature Extraction This makes the backscatter coefficient curves of the rice growth cycle more diverse and does not allow us to summarize a generalized model of rice evolution. Therefore, it will be difficult to accomplish the rice extraction task using a direct reliance on the fixed relationship between phenology and time. 210 and the second season was from August to October. The land parcel of Rice 2 was planted with only one season of rice, from April to September. The land parcel of Rice 3 was planted with two seasons of rice: the first season was from March to July and the second season was from July to October. The land parcel of Rice 4 was planted with three seasons of rice: the first season was from February to June, the second season was from June to October, and only part of the third season (October- 205 December) was observed. It can be seen that the time steps of each growing season for the selected Rice 1- Rice 4 were inconsistent. In fact, the high heterogeneity of rice backscattering coefficients in Southeast Asia is caused by the high heterogeneity in climate and topography. This makes the backscatter coefficient curves of the rice growth cycle more diverse and does not allow us to summarize a generalized model of rice evolution. Therefore, it will be difficult to accomplish the rice extraction task using a direct reliance on the fixed relationship between phenology and time. 210 Figure 3: The average VH polarization backscattering coefficient curve of typical landcovers Figure 3: The average VH polarization backscattering coefficient curve of typical landcovers Through a large number of comparative experimental analysis and combined with our previous research work (Xu et al., 2022), three time-series statistical features that can describe the most significant SAR characteristics during rice growth were selected for rice mapping in the study area, namely, the sharpness of the change in 𝜎0 (𝜎𝑣𝑎𝑟 0 ), the minimum value of the backscatter 215 coefficients in the time-series (𝜎𝑚𝑖𝑛 0 ), the maximum value of backscatter coefficients in the time-series (𝜎𝑚𝑎𝑥 0 ). for rice mapping in the study area, namely, the sharpness of the change in 𝜎0 (𝜎𝑣𝑎𝑟 0 ), the minimum value of the backscatter 215 coefficients in the time-series (𝜎𝑚𝑖𝑛 0 ), the maximum value of backscatter coefficients in the time-series (𝜎𝑚𝑎𝑥 0 ). 3.2 Feature Extraction Land covers with less variation in backscatter intensity, such as buildings and vegetation, generally have smaller 𝜎𝑣𝑎𝑟 0 and higher 𝜎𝑚𝑖𝑛 0 . Therefore, the colors of these land covers are usually yellow or green in the pseudo-color image 240 3.3 Training and validation sets The above analysis shows that, the rice and non-rice landcovers of these Southeast Asian countries have consistent features in the pseudo-color image, which means that the model trained by one scene was applicable for all other scenes. Therefore, a training dataset generated from the orbit-frame 99-16 images of Thailand from previous work (Xu et al., 2021) was used, as 245 shown in Figure 1. A sliding window with a pixel size of 224224 was used to slice the training images into image patches with 50%. The training dataset consisted of 15659 image patches with a pixel size of 224224. A validation sample set for accuracy evaluation was collected using auxiliary data such as Google Earth optical images and other rice maps. The validation samples were divided into two categories, rice category and non-rice category, and the distribution is shown in Figure 1. Specific information is shown in Table 3. 250 Table 3 Validation sample set information Class Number of plots Number of pixels Rice 1913 2,128,431 Non-rice 2032 2,188,477 The above analysis shows that, the rice and non-rice landcovers of these Southeast Asian countries have consistent features in the pseudo-color image, which means that the model trained by one scene was applicable for all other scenes. Therefore, a training dataset generated from the orbit-frame 99-16 images of Thailand from previous work (Xu et al., 2021) was used, as 245 shown in Figure 1. A sliding window with a pixel size of 224224 was used to slice the training images into image patches with 50%. The training dataset consisted of 15659 image patches with a pixel size of 224224. A validation sample set for accuracy evaluation was collected using auxiliary data such as Google Earth optical images and other rice maps. The validation samples were divided into two categories, rice category and non-rice category, and the distribution is shown in Figure 1. Specific information is shown in Table 3. 250 Specific information is shown in Table 3. 250 Table 3 Validation sample set information Class Number of plots Number of pixels Rice 1913 2,128,431 Non-rice 2032 2,188,477 3.2 Feature Extraction The interaction between the crop canopy and microwave radiation varies with time during plant growth. In contrast, the backscattering coefficients of non-crops, such as water bodies, buildings and forest, are more stable. Therefore, the sharpness of the change in 𝜎0 with time will be a key factor in distinguishing cropland from other land cover types. 𝜎𝑣𝑎𝑟 0 is given by the The interaction between the crop canopy and microwave radiation varies with time during plant growth. In contrast, the backscattering coefficients of non-crops, such as water bodies, buildings and forest, are more stable. Therefore, the sharpness of the change in 𝜎0 with time will be a key factor in distinguishing cropland from other land cover types. 𝜎𝑣𝑎𝑟 0 is given by the following equation. 220 following equation. 220 10 https://doi.org/10.5194/essd-2022-392 Preprint. Discussion started: 6 December 2022 c⃝Author(s) 2022. CC BY 4.0 License. 𝜎𝑣𝑎𝑟 0 = 1 𝑛∑\|𝜎𝑖 0 −𝜎𝑚𝑒𝑎𝑛 0 \| 2 𝑛 𝑖 (1) 0 1 ∑ 0 𝑛 𝜎𝑣𝑎𝑟 0 = 1 𝑛∑\|𝜎𝑖 0 −𝜎𝑚𝑒𝑎𝑛 0 \| 2 𝑛 𝑖 𝜎𝑣𝑎𝑟 0 = 1 𝑛∑\|𝜎𝑖 0 −𝜎𝑚𝑒𝑎𝑛 0 \| 2 𝑛 𝑖 where 𝜎𝑚𝑒𝑎𝑛 0 = 1 𝑛∑𝜎𝑖 0 𝑛 𝑖 , 𝑛 is the number of images. where 𝜎𝑚𝑒𝑎𝑛 0 = 1 𝑛∑𝜎𝑖 0 𝑛 𝑖 , 𝑛 is the number of images. The presence of a flooding period makes rice differ significantly from other crops in terms of backscattering characteristics. The backscattering coefficient of rice in the flooding period is close to that of the water body. Therefore, this study identified the flooding period by calculating the minimum value of the backscatter coefficient in the time-series images to distinguish 225 rice from other crops. 𝜎𝑚𝑖𝑛 0 is given by Equation (2). 225 𝜎𝑚𝑖𝑛 0 = 𝑚𝑖𝑛{𝜎1 0, 𝜎2, 0𝜎3 0, … , 𝜎𝑛0} (2) 𝜎𝑚𝑖𝑛 𝑚𝑖𝑛{𝜎1 , 𝜎2,𝜎3 , … , 𝜎𝑛} ( ) Figure 4: The Pseudo-color image of typical land cover types in different countries (R: 𝝈𝒎𝒂𝒙 𝟎 ; G: 𝝈𝒎𝒊𝒏 𝟎 ; B: 𝝈𝒗𝒂𝒓 𝟎 ;Optical images are from Google Earth ©Google Earth). Figure 4: The Pseudo-color image of typical land cover types in different countries (R: 𝝈𝒎𝒂𝒙 𝟎 ; G: 𝝈𝒎𝒊𝒏 𝟎 ; B: 𝝈𝒗𝒂𝒓 𝟎 ;Optical images are from Google Earth ©Google Earth). 11 11 https://doi.org/10.5194/essd-2022-392 Preprint. Discussion started: 6 December 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/essd-2022-392 Preprint. Discussion started: 6 December 2022 c⃝Author(s) 2022. CC BY 4.0 License. 3.2 Feature Extraction The seasonal backscattering variation exhibited by water bodies can interfere with the identification of rice. In contrast to the 230 seasonal variation of water bodies, the backscatter coefficient of rice shows a substantial increase during the growth process. Therefore, false alarms generated by water bodies can be reduced by identifying the maximum value of backscatter coefficients in the time-series images. 𝜎𝑚𝑎𝑥 0 is given by the following equation. The seasonal backscattering variation exhibited by water bodies can interfere with the identification of rice. In contrast to the 230 seasonal variation of water bodies, the backscatter coefficient of rice shows a substantial increase during the growth process. Therefore, false alarms generated by water bodies can be reduced by identifying the maximum value of backscatter coefficients in the time-series images. 𝜎𝑚𝑎𝑥 0 is given by the following equation. 𝜎𝑚𝑎𝑥 0 = 𝑚𝑎𝑥{𝜎1 0, 𝜎2, 0𝜎3 0, … , 𝜎𝑛0} (3) A pseudo-color image is synthesized in the order of R: 𝜎𝑚𝑎𝑥 0 , G: 𝜎𝑚𝑖𝑛 0 , and G: 𝜎𝑣𝑎𝑟 0 . False color images of typical land cover The seasonal backscattering variation exhibited by water bodies can interfere with the identification of rice. In contrast to the 230 seasonal variation of water bodies, the backscatter coefficient of rice shows a substantial increase during the growth process. Therefore, false alarms generated by water bodies can be reduced by identifying the maximum value of backscatter coefficients in the time-series images. 𝜎𝑚𝑎𝑥 0 is given by the following equation. 𝜎𝑚𝑎𝑥 0 = 𝑚𝑎𝑥{𝜎1 0, 𝜎2, 0𝜎3 0, … , 𝜎𝑛0} (3) A pseudo-color image is synthesized in the order of R: 𝜎𝑚𝑎𝑥 0 , G: 𝜎𝑚𝑖𝑛 0 , and G: 𝜎𝑣𝑎𝑟 0 . False color images of typical land cover (3) A pseudo-color image is synthesized in the order of R: 𝜎𝑚𝑎𝑥 0 , G: 𝜎𝑚𝑖𝑛 0 , and G: 𝜎𝑣𝑎𝑟 0 . False color i for several countries and the corresponding optical images in Google Earth are given in Figure 4. Due to the higher 𝜎𝑣𝑎𝑟 0 and 235 𝜎𝑚𝑎𝑥 0 and lower 𝜎𝑚𝑖𝑛 0 of rice, the color of rice in the pseudo-color composite image is mainly purplish red, sometimes red or dark blue. Compared to other land covers, water bodies have lower 𝜎𝑣𝑎𝑟 0 , 𝜎𝑚𝑎𝑥 0 and 𝜎𝑚𝑖𝑛 0 . Therefore, water bodies are black in the false color composite. 3.4 U-Net Model In the final stage of the decoder, the feature vector of the last upsampling unitis converted into a probability mapping by the 11 convolutional layer. The dimension of the probability mapping is 2 and the pixel value indicates 260 the probability that the pixel belongs to rice and non-rice. 265 To solve the problem of uneven data distribution, a batch normalization (BN) layer was added before each convolutional layer (Ioffe and Szegedy, 2015). The BN layer allows the input data to follow the same distribution to achieve regularization of the model. To solve the problem of uneven data distribution, a batch normalization (BN) layer was added before each convolutional layer (Ioffe and Szegedy, 2015). The BN layer allows the input data to follow the same distribution to achieve regularization of the model To solve the problem of uneven data distribution, a batch normalization (BN) layer was added before each convolutional layer (Ioffe and Szegedy, 2015). The BN layer allows the input data to follow the same distribution to achieve regularization of the model. Figure 5: Structure of the U-Net model. 270 3.5 Postprocessing In rice mapping, water bodies (e.g., rivers and lakes) can confuse the flooding period signal of rice. In addition, vegetation may cause some disturbances due to weather effects. Therefore, as drawn on many studies (Lavreniuk et al., 2018; Cué La Rosa et al., 2019; Sun et al., 2021), water body masks and woodland masks produced by WorldCover were used to reduce false alarms of rice extraction results to some extent. 275 Figure 5: Structure of the U-Net model. 270 Figure 5: Structure of the U-Net model. 270 3.4 U-Net Model In this paper，high-resolution rice production was accomplished using U-Net. U-Net is a classical semantic segmentation model widely used in biomedical image segmentation and remote sensing (Wei et al., 2019). It outputs semantically labeled 255 255 12 https://doi.org/10.5194/essd-2022-392 Preprint. Discussion started: 6 December 2022 c⃝Author(s) 2022. CC BY 4.0 License. pixel-by-pixel images corresponding to the input image while extracting high-level semantic features, so that the spatial details of the input image can be maintained (Ronneberger et al., 2015). The structure of U-Net model is shown in Figure 5. The model has 23 convolutional layers, including eighteen 33 convolutional layers, four 22 convolutional layers and one 11 convolutional layer. U-Net consists of encoder (contracting path) and decoder (expansive path). The encoder part consists of five downsampling units, where each unit consists of two 260 33 convolutional layers and a 22 max-pool layer. The output of the downsampling unit is input to the next downsampling unit by max-pooling. The decoder contains four upsampling units, each of which consists of two 33 convolutional layers and a 22 deconvolutional layer. In the final stage of the decoder, the feature vector of the last upsampling unitis converted into a probability mapping by the 11 convolutional layer. The dimension of the probability mapping is 2 and the pixel value indicates the probability that the pixel belongs to rice and non-rice. 265 path) and decoder (expansive path). The encoder part consists of five downsampling units, where each unit consists of two 260 33 convolutional layers and a 22 max-pool layer. The output of the downsampling unit is input to the next downsampling unit by max-pooling. The decoder contains four upsampling units, each of which consists of two 33 convolutional layers and a 22 deconvolutional layer. In the final stage of the decoder, the feature vector of the last upsampling unitis converted into a probability mapping by the 11 convolutional layer. The dimension of the probability mapping is 2 and the pixel value indicates path) and decoder (expansive path). The encoder part consists of five downsampling units, where each unit consists of two 260 33 convolutional layers and a 22 max-pool layer. The output of the downsampling unit is input to the next downsampling unit by max-pooling. The decoder contains four upsampling units, each of which consists of two 33 convolutional layers and a 22 deconvolutional layer. 3.5 Postprocessing In rice mapping, water bodies (e.g., rivers and lakes) can confuse the flooding period signal of rice. In addition, vegetation may cause some disturbances due to weather effects. In rice mapping, water bodies (e.g., rivers and lakes) can confuse the flooding period signal of rice. In addition, vegetation may cause some disturbances due to weather effects. Therefore, as drawn on many studies (Lavreniuk et al., 2018; Cué La Rosa et al., 2019; Sun et al., 2021), water body masks and woodland masks produced by WorldCover were used to reduce false alarms of rice extraction results to some extent. 275 Therefore, as drawn on many studies (Lavreniuk et al., 2018; Cué La Rosa et al., 2019; Sun et al., 2021), water body masks and woodland masks produced by WorldCover were used to reduce false alarms of rice extraction results to some extent. 275 13 https://doi.org/10.5194/essd-2022-392 Preprint. Discussion started: 6 December 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/essd-2022-392 Preprint. Discussion started: 6 December 2022 c⃝Author(s) 2022. CC BY 4.0 License. 3.6 Accuracy evaluation 285 𝑅2 = (∑ (𝑥𝑖−𝑥̅𝑖) 𝑛 𝑖=1 ×(𝑘𝑖−𝑘̅ 𝑖)) 2 ∑ (𝑥𝑖−𝑥̅𝑖)2 𝑛 𝑖=1 ×∑ (𝑘𝑖−𝑘̅𝑖)2 𝑛 𝑖=1 𝑅2 = (∑ (𝑥𝑖−𝑥̅𝑖) 𝑛 𝑖=1 ×(𝑘𝑖−𝑘̅ 𝑖)) 2 ∑ (𝑥𝑖−𝑥̅𝑖)2 𝑛 𝑖=1 ×∑ (𝑘𝑖−𝑘̅𝑖)2 𝑛 𝑖=1 (9) (9) where 𝑛 is the total number of administrative units, 𝑥𝑖 is the area of extracted rice, 𝑥̅𝑖 is its corresponding mean value, 𝑘𝑖 is the area of statistical data or other rice maps and 𝑘̅𝑖 is its corresponding mean value. where 𝑛 is the total number of administrative units, 𝑥𝑖 is the area of extracted rice, 𝑥̅𝑖 is its corresponding mean value, 𝑘𝑖 is the area of statistical data or other rice maps and 𝑘̅𝑖 is its corresponding mean value. 3.6 Accuracy evaluation In this study, several strategies were used to evaluate our rice map product, including accuracy assessments based on validation sets and comparisons with statistical data and other rice maps at the national and provincial levels. First, common accuracy metrics based on the validation set were calculated to measure the classification effectiveness of the model, including accuracy, precision, recall, and kappa (Congalton, 1991; Vapnik, 1999; Mchugh, 2012). 0 280 𝐴𝑐𝑐𝑢𝑟𝑎𝑐𝑦= 𝑇𝑃+ 𝑇𝑁 𝑇𝑃+ 𝑇𝑁+ 𝐹𝑁+ 𝐹𝑃 (4) 𝑃𝑟𝑒𝑐𝑖𝑠𝑖𝑜𝑛= 𝑇𝑃 𝑇𝑃+ 𝐹𝑃 (5) 𝑅𝑒𝑐𝑎𝑙𝑙= 𝑇𝑃 𝑇𝑃+ 𝐹𝑁 (6) 𝐾𝑎𝑝𝑝𝑎= 𝑎𝑐𝑐𝑢𝑟𝑎𝑐𝑦 − 𝑃𝑒 1 − 𝑃𝑒 (7) 𝐴𝑐𝑐𝑢𝑟𝑎𝑐𝑦= 𝑇𝑃+ 𝑇𝑁 𝑇𝑃+ 𝑇𝑁+ 𝐹𝑁+ 𝐹𝑃 𝑃𝑟𝑒𝑐𝑖𝑠𝑖𝑜𝑛= 𝑇𝑃 𝑇𝑃+ 𝐹𝑃 𝑅𝑒𝑐𝑎𝑙𝑙= 𝑇𝑃 𝑇𝑃+ 𝐹𝑁 𝐾𝑎𝑝𝑝𝑎= 𝑎𝑐𝑐𝑢𝑟𝑎𝑐𝑦 − 𝑃𝑒 1 − 𝑃𝑒 𝐴𝑐𝑐𝑢𝑟𝑎𝑐𝑦= 𝑇𝑃+ 𝑇𝑁 𝑇𝑃+ 𝑇𝑁+ 𝐹𝑁+ 𝐹𝑃 (4) 𝑃𝑟𝑒𝑐𝑖𝑠𝑖𝑜𝑛= 𝑇𝑃 𝑇𝑃+ 𝐹𝑃 (5) 𝑅𝑒𝑐𝑎𝑙𝑙= 𝑇𝑃 𝑇𝑃+ 𝐹𝑁 (6) 𝐾𝑎𝑝𝑝𝑎= 𝑎𝑐𝑐𝑢𝑟𝑎𝑐𝑦 − 𝑃𝑒 1 − 𝑃𝑒 (7) (𝑇𝑃+ 𝐹𝑃) × (𝑇𝑃+ 𝐹𝑁) + (𝐹𝑁+ 𝑇𝑁) × (𝐹𝑃+ 𝑇𝑁) (8) (4) 𝑃𝑒= (𝑇𝑃+ 𝐹𝑃) × (𝑇𝑃+ 𝐹𝑁) + (𝐹𝑁+ 𝑇𝑁) × (𝐹𝑃+ 𝑇𝑁) (𝑇𝑃+ 𝑇𝑁+ 𝐹𝑁+ 𝐹𝑃)2 (8) 𝑃𝑒= (𝑇𝑃+ 𝐹𝑃) × (𝑇𝑃+ 𝐹𝑁) + (𝐹𝑁+ 𝑇𝑁) × (𝐹𝑃+ 𝑇𝑁) (𝑇𝑃+ 𝑇𝑁+ 𝐹𝑁+ 𝐹𝑃)2 𝑃𝑒= (𝑇𝑃+ 𝐹𝑃) × (𝑇𝑃+ 𝐹𝑁) + (𝐹𝑁+ 𝑇𝑁) × (𝐹𝑃+ 𝑇𝑁) (𝑇𝑃+ 𝑇𝑁+ 𝐹𝑁+ 𝐹𝑃)2 (8) (8) where TP denotes the number of pixels correctly classified as rice, TN denotes the number of pixels correctly classified as non- rice, FP denotes the number of pixels misclassified as rice among non-rice pixels, FN denotes the number of pixels misclassified as non-rice among rice pixels, and Pe is the desired accuracy. where TP denotes the number of pixels correctly classified as rice, TN denotes the number of pixels correctly classified as non- Second, the spatial consistency of rice extraction results with statistical data and other rice maps was compared at the national and provincial levels. The coefficient of determination (R2) of the rice map with statistical data and other rice maps was 285 calculated using the following equation (Draper and Smith, 1998). Second, the spatial consistency of rice extraction results with statistical data and other rice maps was compared at the national and provincial levels. The coefficient of determination (R2) of the rice map with statistical data and other rice maps was 285 calculated using the following equation (Draper and Smith, 1998). 4 Results The 2019 rice map for mainland Southeast Asia using Sentinel-1 SAR data was shown in Figure 6. According to the extraction 290 result, the main rice production areas in Myanmar are located in the Ayeyarwady, Bago and Yangon delta regions, which are crossed by river systems. In addition, Mandalay, Sagaing and Magwayue in the northern arid mountainous region also play an important role in rice production. Thailand's rice fields are concentrated in the central plains, north and northeast. The main rice-producing areas in Laos are located in the central and southern lowland areas. Many of the major rice-producing provinces The 2019 rice map for mainland Southeast Asia using Sentinel-1 SAR data was shown in Figure 6. According to the extraction 290 result, the main rice production areas in Myanmar are located in the Ayeyarwady, Bago and Yangon delta regions, which are crossed by river systems. In addition, Mandalay, Sagaing and Magwayue in the northern arid mountainous region also play an important role in rice production. Thailand's rice fields are concentrated in the central plains, north and northeast. The main rice-producing areas in Laos are located in the central and southern lowland areas. Many of the major rice-producing provinces are located along the Mekong River, such as Bolikhamxai, Khammouan, Savannakhet, Salavan, and Champasak. Rice fields 295 are located along the Mekong River, such as Bolikhamxai, Khammouan, Savannakhet, Salavan, and Champasak. Rice fields 295 14 https://doi.org/10.5194/essd-2022-392 Preprint. Discussion started: 6 December 2022 c⃝Author(s) 2022. CC BY 4.0 License. in Cambodia are concentrated in the Tonle Sap Lake basin and the southern Mekong River basin. The representative rice growing areas in Vietnam are the Mekong Delta and the Red River Delta. in Cambodia are concentrated in the Tonle Sap Lake basin and the southern Mekong River basin. The representative rice growing areas in Vietnam are the Mekong Delta and the Red River Delta. Next, the rice map was evaluated as comprehensive as possible from three different scales. First, the validation sample set introduced in the previous section was used to evaluate the accuracy of rice mapping from the methodological level. Second, at the national level, the rice maps were compared with statistical data on rice harvested area and other available rice maps, respectively. 4 Results It could b seen that the statistics of rice harvested area were much higher than the area of rice extracted. The statistical data were the tot rice harvest areas in different growing seasons each year, but the extracted rice area was the land area where rice was plante In Vietnam, there are three seasons of rice, namely, spring rice, autumn rice and winter rice, while the harvested areas of sprin 325 rice and autumn rice are comparable, and the harvested area of winter rice is smaller. In this way, part of the statistical data rice harvest area is repeated and accounts for a large proportion of the area, resulting in a larger rice statistical area than th extracted rice area. Although other countries also have multiple rice seasons, the areas of rice in the main season are larg while that in other seasons is small, so the area proportion calculated repeatedly is small. The extracted rice area was closer the paddy land area in statistical yearbook of Vietnam and VLUCD, indicating that the extraction result was reliable. 330 Table 5 Statistics, other rice maps and the extracted rice area in Vietnam Country Statistics of rice cultivation area IRRI rice data Paddy Land area (10^6 ha) VLUCD (10^6ha) Extracted rice cultivation area Therefore, these precision metrics illustrated that the rice mapping results were in good agreement with the validation samples. It also further demonstrated the capability of the proposed method for rice mapping in large tropical regions. 310 Table 4 Accuracy of the rice map based on the validation set Class Accuracy Precision Recall Kappa Rice 92.20% 92.45% 90.26% 0.8425 Therefore, these precision metrics illustrated that the rice mapping results were in good agreement with the validation samples. It also further demonstrated the capability of the proposed method for rice mapping in large tropical regions. 10 Therefore, these precision metrics illustrated that the rice mapping results were in good agreement with the validation samples. It also further demonstrated the capability of the proposed method for rice mapping in large tropical regions. 0 4.2 Comparison with statistical data and other rice maps at the national scale Figure 7 showed the comparison of the extracted rice area with statistical data and the IRRI rice data at the national level scale for five Southeast Asian countries. As seen from the figure, the extraction results were consistent with both statistical data and 315 IRRI rice data. Most points were distributed in the vicinity of the 1:1 line. In contrast, the extraction result was better consistent with IRRI, R2 can reach 0.93, while R2 with statistical data was 0.78. C d ith IRRI i d t th t ti f C b di L d Th il d l t th t f IRRI hil th t f in good agreement with the statistical data. The extraction area of rice in Myanmar and Vietnam was lower, while that in 320 Thailand was slightly higher. Table 5 showed the statistical area of rice, the area of other rice maps and the area of rice extraction in Vietnam. It could be seen that the statistics of rice harvested area were much higher than the area of rice extracted. The statistical data were the total rice harvest areas in different growing seasons each year, but the extracted rice area was the land area where rice was planted. in good agreement with the statistical data. The extraction area of rice in Myanmar and Vietnam was lower, while that in 320 Thailand was slightly higher. Table 5 showed the statistical area of rice, the area of other rice maps and the area of rice extraction in Vietnam. It could be seen that the statistics of rice harvested area were much higher than the area of rice extracted. The statistical data were the total rice harvest areas in different growing seasons each year, but the extracted rice area was the land area where rice was planted. rice harvest areas in different growing seasons each year, but the extracted rice area was the land area where rice was planted. In Vietnam, there are three seasons of rice, namely, spring rice, autumn rice and winter rice, while the harvested areas of spring 325 rice and autumn rice are comparable, and the harvested area of winter rice is smaller. In this way, part of the statistical data of rice harvest area is repeated and accounts for a large proportion of the area, resulting in a larger rice statistical area than the extracted rice area. 4 Results Finally, at the provincial level, more detailed comparisons were made with statistical data and other provincial rice maps to measure the spatial consistency between the extracted rice distribution and these data. 300 Figure 6: 2019 20m resolution rice map of five countries in mainland Southeast Asia Figure 6: 2019 20m resolution rice map of five countries in mainland Southeast Asia 4.1 Accuracy based on the validation set 305 305 The accuracies of the rice map based on the validation sample set is shown in Table 4. Among them, the accuracy was as high as 92.20%, and the Kappa was 0.8425, which proved that the proposed method had good classification performance. The precision was 92.45%, which indicated that the method could effectively reduce the false alarms in the rice extraction results. 15 https://doi.org/10.5194/essd-2022-392 Preprint. Discussion started: 6 December 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/essd-2022-392 Preprint. Discussion started: 6 December 2022 c⃝Author(s) 2022. CC BY 4.0 License. Therefore, these precision metrics illustrated that the rice mapping results were in good agreement with the validation sample It also further demonstrated the capability of the proposed method for rice mapping in large tropical regions. 310 Table 4 Accuracy of the rice map based on the validation set Class Accuracy Precision Recall Kappa Rice 92.20% 92.45% 90.26% 0.8425 4.2 Comparison with statistical data and other rice maps at the national scale Figure 7 showed the comparison of the extracted rice area with statistical data and the IRRI rice data at the national level sca for five Southeast Asian countries. As seen from the figure, the extraction results were consistent with both statistical data an 315 IRRI rice data. Most points were distributed in the vicinity of the 1:1 line. In contrast, the extraction result was better consiste with IRRI, R2 can reach 0.93, while R2 with statistical data was 0.78. Compared with IRRI rice data, the extraction area of Cambodia, Laos and Thailand was close to that of IRRI, while that Myanmar and Vietnam was slight lower. Compared with the statistical data, the extraction areas of Cambodia and Laos we in good agreement with the statistical data. The extraction area of rice in Myanmar and Vietnam was lower, while that 320 Thailand was slightly higher. Table 5 showed the statistical area of rice, the area of other rice maps and the area of rice extraction in Vietnam. 4.2 Comparison with statistical data and other rice maps at the national scale Although other countries also have multiple rice seasons, the areas of rice in the main season are large, while that in other seasons is small, so the area proportion calculated repeatedly is small. The extracted rice area was closer to the paddy land area in statistical yearbook of Vietnam and VLUCD, indicating that the extraction result was reliable. 330 In Vietnam, there are three seasons of rice, namely, spring rice, autumn rice and winter rice, while the harvested areas of spring 325 rice and autumn rice are comparable, and the harvested area of winter rice is smaller. In this way, part of the statistical data of rice harvest area is repeated and accounts for a large proportion of the area, resulting in a larger rice statistical area than the extracted rice area. Although other countries also have multiple rice seasons, the areas of rice in the main season are large, while that in other seasons is small, so the area proportion calculated repeatedly is small. The extracted rice area was closer to Table 5 Statistics, other rice maps and the extracted rice area in Vietnam Country Statistics of rice cultivation area (10^6ha) IRRI rice data (10^6ha) Paddy Land area (10^6 ha) VLUCD (10^6ha) Extracted rice cultivation area (*10^6 ha) Vietnam 7.4695 6.1527 4.1205 3.8210 3.3270 16 Figure 7: Comparison of the extracted rice area with statistical rice harvested area and IRRI dataset at national level scale. M is 335 the number of countries. https://doi.org/10.5194/essd-2022-392 Preprint. Discussion started: 6 December 2022 c⃝Author(s) 2022. CC BY 4.0 License. Figure 7: Comparison of the extracted rice area with statistical rice harvested area and IRRI dataset at national level scale. M is 335 the number of countries. 4.3 Comparison with statistical data and other rice maps at the provincial scale Figure 8 shows the comparison of the extracted rice area with the statistical data of rice harvested area and IRRI rice data at the provincial scale for five Southeast Asian countries. The available rice maps contain a 500 m resolution rice map of mainland Southeast Asia (IRRI Rice Data) and a 30 m resolution rice map of Vietnam (VLUCD) (see Sect. 2.2.3 for details). In general, 340 the rice extraction results were in good agreement with the statistical area, IRRI data and VLUCD. Among them, the R2 ranged from 0.82 to 0.88 with statistical data and from 0.83 to 0.97 with IRRI, as shown in Figure 8. Figure 8 shows the comparison of the extracted rice area with the statistical data of rice harvested area and IRRI rice data at the provincial scale for five Southeast Asian countries. The available rice maps contain a 500 m resolution rice map of mainland Figure 8 shows the comparison of the extracted rice area with the statistical data of rice harvested area and IRRI rice data at the provincial scale for five Southeast Asian countries. The available rice maps contain a 500 m resolution rice map of mainland Southeast Asia (IRRI Rice Data) and a 30 m resolution rice map of Vietnam (VLUCD) (see Sect. 2.2.3 for details). In general, 340 the rice extraction results were in good agreement with the statistical area, IRRI data and VLUCD. Among them, the R2 ranged from 0.82 to 0.88 with statistical data and from 0.83 to 0.97 with IRRI, as shown in Figure 8. As shown in Figure 8 (a) and (b), the rice planting areas in Thailand and Cambodia extracted by our method had a good correlation with the statistical data and IRRI data at the provincial scale. The R2 was distributed in the range of 0.83-0.88. Southeast Asia (IRRI Rice Data) and a 30 m resolution rice map of Vietnam (VLUCD) (see Sect. 2.2.3 for details). In general, 340 the rice extraction results were in good agreement with the statistical area, IRRI data and VLUCD. Among them, the R2 ranged from 0.82 to 0.88 with statistical data and from 0.83 to 0.97 with IRRI, as shown in Figure 8. 4.3 Comparison with statistical data and other rice maps at the provincial scale As shown in Figure 8 (a) and (b), the rice planting areas in Thailand and Cambodia extracted by our method had a good l i i h h i i l d d IRRI d h i i l l Th R2 di ib d i h f 0 83 0 88 the rice extraction results were in good agreement with the statistical area, IRRI data and VLUCD. Among them, the R2 ranged from 0.82 to 0.88 with statistical data and from 0.83 to 0.97 with IRRI, as shown in Figure 8. As shown in Figure 8 (a) and (b), the rice planting areas in Thailand and Cambodia extracted by our method had a good correlation with the statistical data and IRRI data at the provincial scale. The R2 was distributed in the range of 0.83-0.88. As shown in Figure 8 (a) and (b), the rice planting areas in Thailand and Cambodia extracted by our method had a good correlation with the statistical data and IRRI data at the provincial scale. The R2 was distributed in the range of 0.83-0.88. There were no provinces with large deviations. 345 There were no provinces with large deviations. 345 In Figure 8(c), in Myanmar, the R2 between the extracted area of rice and the statistical data, IRRI rice data was 0.83 and 0.84, respectively. However, the extracted area of Ayeyarwady Province was significantly lower than that of the statistical data and IRRI data. The extraction results of Ayeyarwady were compared with the IRRI data, as shown in Figure 9. As reported by Han et al. (Han et al., 2021), due to the influence of mixed pixels, the IRRI data divides too many rivers and vegetation into rice. There were no provinces with large deviations. 345 In Figure 8(c), in Myanmar, the R2 between the extracted area of rice and the statistical data, IRRI rice data was 0.83 and 0.84, respectively. However, the extracted area of Ayeyarwady Province was significantly lower than that of the statistical data and IRRI data. The extraction results of Ayeyarwady were compared with the IRRI data, as shown in Figure 9. As reported by Han et al. (Han et al., 2021), due to the influence of mixed pixels, the IRRI data divides too many rivers and vegetation into rice. 4.3 Comparison with statistical data and other rice maps at the provincial scale A d th t t d i t i th d t il f i d d 350 And the extracted rice map retains the details of rivers and roads. 350 The R2 of the extracted rice area in Laos with statistical data was 0.82, and the highest agreement with IRRI data was 0.97, as shown in Figure 8(d). For the same reason as Ayeyarwady Province, the rice extraction area in Savannakhet Province was lower than the IRRI data because the details of rivers and roads were preserved in the extraction results. And the extracted rice map retains the details of rivers and roads. 350 The R2 of the extracted rice area in Laos with statistical data was 0.82, and the highest agreement with IRRI data was 0.97, as shown in Figure 8(d). For the same reason as Ayeyarwady Province, the rice extraction area in Savannakhet Province was lower than the IRRI data because the details of rivers and roads were preserved in the extraction results. 17 https://doi.org/10.5194/essd-2022-392 Preprint. Discussion started: 6 December 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/essd-2022-392 Preprint. Discussion started: 6 December 2022 c⃝Author(s) 2022. CC BY 4.0 License. Different from other sub figures, Vietnam added data comparison results with 30m VLUCD. The extraction results in Vietnam correlated well with the statistical data, VLUCD and IRRI data, with all R2 values greater than 0.80, as shown in Figure 8(e). 355 The area of rice extraction in Vietnam was in higher agreement with VLUCD (R2 of 0.87) than with statistics (R2 of 0.86) and IRRI Rice Data (R2 of 0.83). Most of the points of VLUCD were distributed on the 1:1 line. 355 Figure 8: Comparison of the extracted rice area with statistical rice harvested area and IRRI dataset at provincial scale. N is the 360 number of provinces in each country. Figure 8: Comparison of the extracted rice area with statistical rice harvested area and IRRI dataset at provincial scale. N is the 360 number of provinces in each country. 18 https://doi.org/10.5194/essd-2022-392 Preprint. Discussion started: 6 December 2022 c⃝Author(s) 2022. CC BY 4.0 License. Figure 9: Ayeyarwady's extracted rice area plotted against IRRI data. Our extraction results (a-b), False color image(c), IRRI data (d-e), Google Earth optical image (f) ©Google Earth. Figure 9: Ayeyarwady's extracted rice area plotted against IRRI data. 4.3 Comparison with statistical data and other rice maps at the provincial scale Our extraction results (a-b), False color image(c), IRRI data (d-e), Google Earth optical image (f) ©Google Earth. 5 Discussion 365 In this study, annual rice maps for five Southeast Asian countries in 2019 were generated using temporal features extracted based on Sentinel-1SAR time series and an improved U-Net. Accuracy, Precision, and Recall based on the validation set exceeded 90% with a Kappa of 0.8425. Accuracy evaluation of rice mapping showed that the proposed temporal features were able to portray the unique growth characteristics of rice, and the improved U-Net model was able to suppress the false alarms 5 Discussion 365 In this study, annual rice maps for five Southeast Asian countries in 2019 were generated using temporal features extracted based on Sentinel-1SAR time series and an improved U-Net. Accuracy, Precision, and Recall based on the validation set exceeded 90% with a Kappa of 0.8425. Accuracy evaluation of rice mapping showed that the proposed temporal features were able to portray the unique growth characteristics of rice, and the improved U-Net model was able to suppress the false alarms of sporadic distrib tion ca sed b comple topograph The proposed method has s perior capabilit in mapping rice 370 of sporadic distribution caused by complex topography. The proposed method has superior capability in mapping rice 370 distribution in large tropical regions. The rice extraction results were compared with statistical data from the national and provincial levels in Sections 4.2 and 4.3. The results of multiple comparisons show that our rice extraction results are in high agreement with the statistical data. However, there were also minor inconsistencies. A possible reason is that the statistical cycle is not strictly aligned with the of sporadic distribution caused by complex topography. The proposed method has superior capability in mapping rice 370 distribution in large tropical regions. The rice extraction results were compared with statistical data from the national and provincial levels in Sections 4.2 and 4.3. The results of multiple comparisons show that our rice extraction results are in high agreement with the statistical data. However, there were also minor inconsistencies. A possible reason is that the statistical cycle is not strictly aligned with the , p y y g data collection cycle. The rice area extracted in this study is the total area of all fields that have been planted with rice in a 375 year. Most agricultural statistics record the total area of rice planted in different growing seasons on an annual basis or even from one month of one year to the next. In addition, the statistical methods may cause errors in the statistics. The well-organized rice growing seasons were mainly considered in all statistics, and the random and irregular planting behavior of individual farmers was inevitably ignored. Considering the data collection conditions and statistical errors, it is understandable that the extracted rice maps differ from the official statistics. 380 data collection cycle. 5 Discussion 365 The rice area extracted in this study is the total area of all fields that have been planted with rice in a 375 year. Most agricultural statistics record the total area of rice planted in different growing seasons on an annual basis or even from one month of one year to the next. In addition, the statistical methods may cause errors in the statistics. The well-organized rice growing seasons were mainly considered in all statistics, and the random and irregular planting behavior of individual farmers was inevitably ignored. Considering the data collection conditions and statistical errors, it is understandable that the data collection cycle. The rice area extracted in this study is the total area of all fields that have been planted with rice in a 375 year. Most agricultural statistics record the total area of rice planted in different growing seasons on an annual basis or even from one month of one year to the next. In addition, the statistical methods may cause errors in the statistics. The well-organized rice growing seasons were mainly considered in all statistics, and the random and irregular planting behavior of individual farmers was inevitably ignored. Considering the data collection conditions and statistical errors, it is understandable that the extracted rice maps differ from the official statistics 380 extracted rice maps differ from the official statistics. 380 The comparison results between rice area products extracted based on different remote sensing data show that our rice extraction results are in good agreement with the available rice products at the national and provincial levels. To fully extracted rice maps differ from the official statistics. 380 The comparison results between rice area products extracted based on different remote sensing data show that our rice extraction results are in good agreement with the available rice products at the national and provincial levels. To fully The comparison results between rice area products extracted based on different remote sensing data show that our rice extraction results are in good agreement with the available rice products at the national and provincial levels. To fully 19 https://doi.org/10.5194/essd-2022-392 Preprint. Discussion started: 6 December 2022 c⃝Author(s) 2022. CC BY 4.0 License. demonstrate the reliability of the rice extraction results, three subregions from the rice map were selected for comparison in Thailand and Vietnam, as shown in Figure 10. 7 Conclusions Ending hunger and malnutrition in Southeast Asia is essential and rice plays a critical role. Rice is the single most important staple in the region as it provides 50% of calorie intake for its population. Satellite-based remote sensing offers the most practical means of monitoring changes on the vast area of land under rice cultivation in mainland Southeast Asia, given the synoptic coverage, repeated and regular observation, and archival nature of satellite imagery. Questions remain, however, as 405 to appropriate timing, number of satellite observations, spatial resolution of satellite imagery, and effective data processing methods for accurately capturing changes in factors such as rice production extent, growing seasons, and land management. For large-scale rice mapping in tropical and subtropical regions, a rice mapping method based on time series SAR features and deep learning models is proposed. Rice mapping was completed for mainland Southeast Asia using the 2019 Sentinel-1 time series data and the proposed rice extraction method. The accuracy of the proposed method on the validation sample set was 410 92.20%. Our rice maps correlated significantly with statistical data and were consistent with other rice maps. These results demonstrate the advantages of the proposed method for rice extraction with complex cropping patterns. The rice maps we produced will provide data support for agricultural resource studies, such as yield prediction and agricultural management. series data and the proposed rice extraction method. The accuracy of the proposed method on the validation sample set was 410 92.20%. Our rice maps correlated significantly with statistical data and were consistent with other rice maps. These results demonstrate the advantages of the proposed method for rice extraction with complex cropping patterns. The rice maps we produced will provide data support for agricultural resource studies, such as yield prediction and agricultural management. Author Contributions: Conceptualization, methodology, software, C.S. and H.Z.; validation, formal analysis, H.Z.; 415 investigation, C.S. and L.X.; resources, data curation, J.J. and J.G.; writing—original draft preparation, C.S. and H.Z.; writing—review and editing, H.Z., L.X., J.G. and L.Z.; visualization, L.X. and J.G.; supervision, project administration, H.Z. and C.W. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the National Natural Science Foundation of China under Grants 41971395, 41930110 Author Contributions: Conceptualization, methodology, software, C.S. and H.Z.; validation, formal analysis, H.Z.; 415 investigation, C.S. and L.X.; resources, data curation, J.J. and J.G.; writing—original draft preparation, C.S. 7 Conclusions and H.Z.; writing—review and editing, H.Z., L.X., J.G. and L.Z.; visualization, L.X. and J.G.; supervision, project administration, H.Z. and C.W. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the National Natural Science Foundation of China under Grants 41971395, 41930110 Funding: This research was funded by the National Natural Science Foundation of China under Grants 41971395, 41930110 and 42001278 and the Strategic Priority Research Program of Chinese Academy of Sciences (XDA19090119). 420 and 42001278 and the Strategic Priority Research Program of Chinese Academy of Sciences (XDA19090119). 420 Acknowledgments: The authors would like to thank ESA and EU Copernicus Program for providing the Sentinel-1 SAR data. Conflicts of Interest: The authors declare no conflict of interest. 5 Discussion 365 As mentioned in other literature (Dong et al., 2015; Han et al., 2021), the MODIS-based IRRI rice map with 500 m resolution contains a large number of mixed image elements, and thus 385 misclassification exists in the rice results. The spatial distribution characteristics of our rice map were generally consistent with those of the IRRI data, and our rice map retained more details with fewer mixed pixels. In addition, our rice map also had better agreement with the spatial distribution and detailed information of rice from VLUCD. Overall, comparisons based on the validation set, statistical data, and other rice products confirmed the reliability of our generated rice maps. 390 385 0 In the study, we found that the temporal features along rivers and wetlands are more similar to rice and have similar colors in the feature map, which can be easily misclassified as rice. The backscattered information of scattered rice fields is subject to interference from topography and surrounding non-rice land cover, resulting in missed detection. 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https://openalex.org/W1545687875	https://europepmc.org/articles/pmc4506590?pdf=render	English	null	Generational differences in the physical activity of UK South Asians: a systematic review	The international journal of behavioural nutrition and physical activity	2,015	cc-by	13,828	Bhatnagar et al. International Journal of Behavioral Nutrition and Physical Activity (2015) 12:96 DOI 10.1186/s12966-015-0255-8 Bhatnagar et al. International Journal of Behavioral Nutrition and Physical Activity (2015) 12:96 DOI 10.1186/s12966-015-0255-8 Abstract Background: South Asians are some of the least active people in the UK, but we know very little about how physical activity varies within and between different UK South Asian groups. There is much socio-economic and cultural heterogeneity among UK Indians, Pakistanis and Bangladeshis, and the same approaches to increasing physical activity may not be appropriate for all people of these ethnic groups. We report on the variation in physical activity behaviour prevalence in quantitative studies and the variations in attitudes, motivations and barriers to physical activity among South Asians in qualitative papers. Methods: We performed systematic searches in MEDLINE, Embase and Psychinfo for papers written in English and published between 1990 and 2014. We also attempted to search literature not published in peer-review journals (the ‘grey’ literature). We reported data for the quantitative observational studies and synthesised themes from the qualitative literature according to age-group. We assessed the quality of studies using a National Institute of Health and Clinical Excellence tool. Results: We included 29 quantitative papers and 17 qualitative papers. Thirteen papers reported on physical activity prevalence in South Asian children, with the majority comparing them to White British children. Four papers reported on adult second-generation South Asians and the rest reported on South Asian adults in general. Second-generation South Asians were more active than the first-generation but were still less active than the White British. There were no high quality qualitative studies on second-generation South Asian adults, but there were some studies on South Asian children. The adult studies indicated that the second-generation might have a more favourable attitude towards physical activity than the first-generation. Conclusions: There is clear variation in physical activity levels among UK South Asians. Second-generation South Asians appear to be more physically active than the first-generation, but still less active than the White British. More qualitative research is needed to understand why, but there are indications that second-generation South Asians have a more positive attitude towards physical activity than the first-generation. Different strategies to increase physical activity may be needed for different generations of UK South Asians. Keywords: South Asian, Physical activity, Review * Correspondence: prachi.bhatnagar@dph.ox.ac.uk 1British Heart Foundation Centre on Population Approaches for Non-Communicable Disease Prevention, Nuffield Department of Population Health, University of Oxford, Old Road Campus, OX3 7LF Oxford, UK Full list of author information is available at the end of the article © 2015 Bhatnagar et al. 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. Generational differences in the physical activity of UK South Asians: a systematic review Prachi Bhatnagar1*, Alison Shaw2 and Charlie Foster1 © 2015 Bhatnagar et al. 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. RESEARCH Open Access Methods We performed systematic searches in MEDLINE, Embase and Psychinfo for papers written in English and published between 1990 and 2014. We also attempted to search literature not published in peer-review journals (the ‘grey’ literature). The website www.better-health.org.uk has a comprehensive list of resources and organisations that work on ethnicity and health; we searched through the website of each organisation that worked with a relevant ethnic group or on physical activity to find additional papers. Within the UK, the Health Survey for England 2004 was the latest health survey to boost the ethnic minority sample, therefore providing the most reliable estimates for physical activity levels in English ethnic minorities. According to the old physical activity guidelines in place in 2004, 37 % of men and 25 % of women in the general population were doing the recommended levels of physical activity. Compar- isons between ethnic minorities show that Bangladeshi men and women had the lowest proportion of people meet- ing the guidelines (26 % and 11 % respectively) [3]. The Health Survey for England also reported that differences between younger and older people were greater for Indian women and for Bangladeshi respondents than for the gen- eral population (mostly the White British group). 18 % of Indian women aged 16 to 34 compared to 2 % of Indian women aged over 55 were highly active [3]. We combined three groups of search terms using the ‘AND’ command. The search terms related to South Asian ethnicity were, ‘Asian’, ‘Indian’, ‘Pakistani’ and ‘Bangladeshi’. The terms on physical activity included ‘physical activity’, ‘exercise’, ‘walking’, ‘leisure’, ‘sports’ and ‘transport’. The third group limited the search to the United Kingdom, and the MeSH term ‘United Kingdom’ was used with all its subheadings. Both MeSH terms and keywords were used; the full search strategy used to search the databases is available on request. We assessed the quality of the papers after deter- mining whether they met the inclusion/exclusion criteria. The inclusion and exclusion criteria for the reviews were as follows: Inclusion/exclusion criteria for all: Inclusion/exclusion criteria for all: Papers must report on South Asian populations residing within the UK (Indian, Pakistani or Bangladeshi). There are no ethnic-specific cohorts in the UK, although there are studies with a high-proportion of ethnic groups in their sample. Background current measurement methods, such as those used in the national census. Consequently, when we aim to understand physical activity in ethnic groups, we may miss the influence of other factors related to having an ethnic minority background, such as country of birth or the country where most schooling took place. In this paper we review the literature on physical activity preva- lence and attitudes within the UK Indian, Pakistani and Bangladeshi populations to explore how physical activity varies between and within these UK South Asian groups. Ethnicity is poorly measured in epidemiological studies, failing to reflect both its social nature and socio- economic and cultural heterogeneity within ethnic groups. Ethnicity is a multifaceted concept encompass- ing factors such as language, marriage patterns and com- mon ancestry, but this is not accurately reflected by the Bhatnagar et al. International Journal of Behavioral Nutrition and Physical Activity (2015) 12:96 Bhatnagar et al. International Journal of Behavioral Nutrition and Physical Activity (2015) 12:96 Page 2 of 19 activity patterns that differ from those of the first- generation. There have been two reviews on physical activity among UK South Asians during the past decade. Fischbacher et al. 2004 [1] reviewed physical activity in the UK South Asian population, and Babakus & Thompson 2012 [2] reviewed physical activity in South Asian women inter- nationally. Both reviews reported low levels of physical activity among South Asians, with women in particular having a low level of physical activity. Babakus & Thompson 2012 also reported on the barriers and motiva- tions for physical activity in South Asian women found in the literature. While providing valuable information, nei- ther of these reviews discussed how physical activity prevalence varies within different South Asian groups. Methods However, these cohorts are all set up to explore the health and behaviour of children, and infor- mation on the health behaviours of UK-born ethnic mi- nority adults will not be available for some years. Since children’s behaviour is heavily influenced by parental be- haviour, it is necessary to study adults in order to obtain an accurate picture of how the second-generation differs from their parents. Papers must have been published between 1990 and 2014. Review articles will be excluded. Patient groups will be excluded. For the quantitative literature: For the quantitative literature: For the quantitative literature: Papers must report on the prevalence of physical activity. It is particularly important to understand the differences in risk factors for cardiovascular disease (CVD) between migrant (first-generation) and second-generation ethnic groups because the second-generation of some ethnic groups is still relatively young and may still have a good chance of altering their CVD risk in later life [4]. A child- hood in the UK has exposed second-generation ethnic mi- norities to many experiences that differ from those of their parents, making it plausible that second-generation ethnic minorities also differ from their parents in their health behaviours. Physical activity is known to be low among South Asians compared to the rest of the popula- tion [5]. However, exposure to activity in school, through the media and the wider social environment may mean that second-generation ethnic groups will have physical Papers reporting only physiological data on fitness will be excluded. Papers reporting observational studies will be included. Papers reporting experimental studies will be excluded. For the qualitative literature: For the qualitative literature: Papers must report on the attitudes, influences, barriers or motivators to physical activity. barriers or motivators to physical activity. The search yielded 821 hits altogether. We reviewed the search results by first examining the titles and abstracts of Bhatnagar et al. International Journal of Behavioral Nutrition and Physical Activity (2015) 12:96 Page 3 of 19 (focus groups or interviews), ethnic group, location, num- ber of participants and analytical approach. papers that met the inclusion criteria. If the abstract met the inclusion criteria, we read the paper in full to decide whether or not to include it in the review. CF read through 20 % of the abstracts to judge against the inclu- sion/exclusion criteria and any disagreements were re- solved through discussion with PB. We searched the references of all papers read in full and the publication lists of included authors; we subjected these to the same title and abstract screening process. Figure 1 illustrates the review process. Reasons for exclusion at the title and ab- stract level included not reporting on the prevalence of physical activity, not reporting on South Asian ethnic groups and not reporting on a UK population. Study quality assessment We assessed the quality of the papers included in the re- view by using a quality appraisal tool from the National Institute for Health and Clinical Excellence (NICE) Pub- lic Health guidance [6]. Quality appraisal tools are intended as guides; we used the NICE checklist for stud- ies reporting correlations and associations in order to as- sess both the internal and external validity of the papers. Quantitative studies For the quantitative observational studies, we organised the reported prevalence of physical activity according to age-group. For the qualitative literature we synthesised the themes and also reported these according to age-group. Data extraction h 2008 Cross sectional Indian (218) Pakistani/ Bangladeshi (222) White UK (589), White Other (218), Black Caribbean (453), Black African (593), Mixed (279) [15] Hayes et al. 2002 Cross sectional Indian (249), Pakistani (287), Bangladeshi (117) European (749) [16] Hemmings et al. 2011 Cross sectional pilot South Asian (12) British White (11) [34] Hine et al. 1995 Cross sectional Indian (52), Pakistani (79), Bangladeshi (21) None [17] Karlsen et al. 2010 Cross sectional Muslim Indian (270), Pakistani (2,120), Bangladeshi (1,943), Sikh (657), Hindu (1,195) Christian by White British (10,577), Irish (1,729), Caribbean and No religion by White British (2,371), Caribbean (309) [18] Khunti et al. 2007 Cross sectional South Asian (2,732) White European (447) [19] Knight et al. 1993 Cross sectional Asian (128) Non-Asian (160) [20] Lean et al. 2001 Cross sectional South Asian (119) Italian (90), General Population (50) [21] McMinn et al. 2011 Cross sectional South Asian (487) White European (508), Black African-Caribbean (576) [22] De Munter et al. 2012 Cross sectional Indian (1,264) English European (14,723), English African (1,112) Table 1 Characteristics of quantitative studies Author and year Study design South Asian Ethnic groups (N) Comparator (N) Sex Age-group Location Physical activity measurement instrument [7] Duncan et al. 2006 Cross sectional Asian (67) White (176), Black (33) Male and Female 11 to 14 Birmingham Self-report, validated questionnaire - Four by one day [8] Duncan et al. 2008 Cross sectional South Asian (209) White (397) Male and Female 11 to 14 Birmingham Self-report, validated questionnaire - Four by one day [9] Duncan et al. 2012 Cross sectional South Asian (67) White (469) Male and Female 8 to 11 Coventry Pedometers worn over 4 days [10] Eyre et al. 2013 Cross sectional South Asian (65) White European (96) Male and Female 8 to 9 Coventry Physical activity and heart rate worn monitor for 7 days [11] Falconer et al. 2014 Cross sectional South Asian (607) White (1,904), Black/Black British (226) Male and Female 4 to 5 and 10 to 11 5 PCTs in England Self-report questionnaires [12] Ghouri et al. 2013 Cross sectional South Asian (87) European (99) Male 40 to 70 Scotland Accelerometers worn for 7 days [13] Griffiths et al. 2013 Prospective cohort Indian (139), Pakistani (177), Bangladeshi (70) White (5,710), Mixed (168), Black (142), Other (90) Male and Female Age 7 UK Accelerometer worn for 7 days, during waking hours. [14] Harding et al. Data extraction h For the quantitative studies, we extracted data on ethnic group, whether there was a comparison to the White British or general population, type of measurement, loca- tion of study and sample size. For the qualitative studies we extracted information on data collection method Table 1 describes the basic characteristics of the in- cluded papers, of which there were 29 [7–35]. One study was a prospective cohort [13] and the rest were cross- Total hits from database searches = 821 Embase = 453 Medline = 273 Psychinfo = 95 Excluded on basis of full paper = 18 Quantitative:14 Qualitative:4 Reasons for exclusion Conference paper =4 Results not reported by ethnic group =7 Physical activity prevalence not reported =2 Sample not representative =1 Findings still being analysed =1 Only attitudes to measurement tool reported =1 Paper not available =1 Patient group =1 Excluded on basis of title and abstract = 589 Studies for full screening = 58 Quantitative:40 Qualitative:18 Papers included in final review = 46 Quantitative:29 Qualitative:17 Duplicates removed = 176 Added in from reference list and author searches = 6 Quantitative:3 Qualitative:3 Added in from grey literature = 2 Fig. 1 Flow chart of review process Duplicates removed = 176 Excluded on basis of title and abstract = 589 Studies for full screening = 58 Quantitative:40 Qualitative:18 Added in from reference list and author searches = 6 Quantitative:3 Qualitative:3 Quantitative:29 Qualitative:17 Fig. 1 Flow chart of review process Bhatnagar et al. International Journal of Behavioral Nutrition and Physical Activity (2015) 12:96 Page 4 of 19 Page 4 of 19 Table 1 Characteristics of quantitative studies Author and year Study design South Asian Ethnic groups (N) Comparator (N) [7] Duncan et al. 2006 Cross sectional Asian (67) White (176), Black (33) [8] Duncan et al. 2008 Cross sectional South Asian (209) White (397) [9] Duncan et al. 2012 Cross sectional South Asian (67) White (469) [10] Eyre et al. 2013 Cross sectional South Asian (65) White European (96) [11] Falconer et al. 2014 Cross sectional South Asian (607) White (1,904), Black/Black British (226) [12] Ghouri et al. 2013 Cross sectional South Asian (87) European (99) [13] Griffiths et al. 2013 Prospective cohort Indian (139), Pakistani (177), Bangladeshi (70) White (5,710), Mixed (168), Black (142), Other (90) [14] Harding et al. Data extraction h 2012 Cross sectional British Pakistani (67) White British (70) Female 9 to 11 North East England Accelerometer worn for 2 school days [26]Pomerleau et al. 1999 Cross sectional South Asian (291) European (559), Afro- Caribbean (303) Female 40 to 69 London Self-report questionnaire [27] Riste et al. 2001 Cross sectional Pakistani (132) European (471), African- Caribbean (316) Male and Female 35 to 79 Manchester Self-report validated questionnaire [28] Smith et al. 2012 Cross sectional Indian, Pakistani, Bangladeshi White Male and Female 16 to 55 England Self-report questionnaire based on Allied Dunbar National Fitness Survey [29] Williams et al. 1994 Cross sectional South Asian (173) General population (344) Male and Female 30 to 40 Glasgow Self-report questionnaire. [30] Williams et al. 1998 Cross sectional British Asian (334) Other origin (490) Male and Female 14 to 15 in South Asians Age 35 in General population Glasgow Self-report questionnaire. [35] Williams et al. 2010 Cross sectional South Asian Sikhs (571), Muslims (179), Hindus (315) Whites (818) Male and Female 35 to 75 London Self-report, validated questionnaire -International Physical Activity Questionnaire [32] Williams et al. 2011 - JECH Cross sectional South Asian (5,421) Whites (8,974) Male and Female Over 16 England Self-report questionnaire based on Allied Dunbar National Fitness Survey [31] Williams et al. 2011 - Heart Cross sectional Indian (1,244), Pakistani/Bangladeshi (876) White (13,293) Male and Female Over 16 England Self-report questionnaire based on Allied Dunbar National Fitness Survey [33] Yates et al. 2010 Cross sectional South Asian (1,164) White European (4,310) Male and Female 25 to 75 in South Asians 40 to 75 in White European Leicester Self-report, validated questionnaire -International Physical Activity Questionnaire Table 1 Characteristics of quantitative studies (Continued) sectional. Eight studies had participants from central England, six were in London, five were based in the North of England, four in Scotland, one in Bristol and the rest were across England or the UK. The age of participants ranged from two to 79 years, and we present the results for children and adults separately. Twelve of the 29 papers reported by sub-group of South Asian ethnicity or reli- gion, with the rest grouping Indians, Pakistanis and Bangladeshis into the broader South Asian category. Data extraction h 2008 Cross sectional Indian (218) Pakistani/ Bangladeshi (222) White UK (589), White Other (218), Black Caribbean (453), Black African (593), Mixed (279) Male and Female 11 to 13 London Self-report physical activity questions on vigorous sports [15] Hayes et al. 2002 Cross sectional Indian (249), Pakistani (287), Bangladeshi (117) European (749) Male and Female 25 to 74 Newcastle Self-report physical activity questionnaire, then created an index [16] Hemmings et al. 2011 Cross sectional pilot South Asian (12) British White (11) Male 14 to 15 London Accelerometer worn for 7 days [34] Hine et al. 1995 Cross sectional Indian (52), Pakistani (79), Bangladeshi (21) None Female 18 to 74 Bristol Self-report questionnaire [17] Karlsen et al. 2010 Cross sectional Muslim Indian (270), Pakistani (2,120), Bangladeshi (1,943), Sikh (657), Hindu (1,195) Christian by White British (10,577), Irish (1,729), Caribbean and No religion by White British (2,371), Caribbean (309) Male and Female Above 2 England Self-report question on taking no regular physical activity [18] Khunti et al. 2007 Cross sectional South Asian (2,732) White European (447) Male and Female 11 to 16 Leicester Self-report questionnaire derived from Four by one day [19] Knight et al. 1993 Cross sectional Asian (128) Non-Asian (160) Male 20 to 65 Bradford Self-report lifestyle questionnaire including exercise [20] Lean et al. 2001 Cross sectional South Asian (119) Italian (90), General Population (50) Female 20 to 42 Glasgow Self-report question on sport and recreational exercise [21] McMinn et al. 2011 Cross sectional South Asian (487) White European (508), Black African-Caribbean (576) Male and Female 9 to 10 London, Birmingham, Leicester Accelerometer worn for 7 days, during waking hours. [22] De M l Cross i l Indian (1,264) English European (14,723), E li h Af i (1 112) Male d 35 to 64 England Self-report i i b d Page 5 of 19 Page 5 of 19 Bhatnagar et al. International Journal of Behavioral Nutrition and Physical Activity (2015) 12:96 Table 1 Characteristics of quantitative studies (Continued) [23] Owen et al. 2009 Cross sectional South Asian (494) White European (562), Black African-Caribbean (607) Male and Female 9 to 10 London, Birmingham, Leicester Accelerometer worn for 7 days, during waking hours. [24] Pollard et al. 2008 Cross sectional British Pakistani (60) European (25) Female 20 to 40 North East England Self-report, validated questionnaire -International Physical Activity Questionnaire [25] Pollard et al. Data extraction h All but one of the included studies compared the physical activity prevalence of South Asians to the White British, therefore we have included the comparison to the White British group in order to contextualise the reported activ- ity of second-generation South Asian groups. Eight of the studies used accelerometers or pedometers to objectively measure physical activity levels, and six of these studies were carried out with school-children. Six studies reported using validated self-report questionnaires. However the remaining majority of papers used self-report, non-validated questionnaires to measure physical activity. Page 6 of 19 Bhatnagar et al. International Journal of Behavioral Nutrition and Physical Activity (2015) 12:96 Quality assessment Quantitative papers Of the papers reporting on children, only one reported a power calculation for the sample size [30]. The other 11 papers provided no justification for their sample size; the majority only reported the number of people they had re- cruited, implying that their aim was to recruit as many children as possible into their studies. Three of the studies on children had fewer than 70 South Asian participants [7, 9, 10]. Without a sample size calculation, we cannot know whether there are enough participants to detect a particular size of difference between the two ethnic groups and therefore whether to reject the null hypothesis of no difference between the two ethnic groups. The fact that all of the studies do report a difference, despite using differ- ent measurements, indicates that rejection of the null hy- pothesis may be the correction conclusion, but without a sample size calculation we cannot be sure. Qualitative studies are still potential pitfalls. The placement of the accelerom- eter on the body (at the hip, ankle or wrist) may affect the output of the device, and the devices cannot be worn dur- ing wet activities such as swimming. Although accelerom- eters and pedometers are able to provide objective data on physical activity, a criticism of them is that they are unable to measure the context in which the physical activity is carried out. Of the studies using questionnaires, three used a validated questionnaire [7, 8, 18] and one used a non-validated questionnaire on vigorous sports [14]. We included 17 studies in this review [36–52] (Table 2). The locations of the studies varied: four were based in London, four in the North West of England, four in cen- tral England and the remaining studies were conducted in Scotland, Wales or Great Britain in general. The age range of participants ranged from eight to 70 years, and we present the findings for children and second-generation adults, using the socioecological model; results for adults of any age are in the appendix. Six of the studies reported interviewing South Asians, with the other 11 presenting findings for Indians, Pakistanis or Bangladeshis separately. Interestingly, none of the papers included here specifically reported differences between South Asian groups, usually just stating when a finding or theme was relevant to one of the ethnic groups in particular. Of the 12 studies on children, only four included socio- economic or deprivation factors in their analyses [9, 11, 13, 14]. This is surprising given that socioeconomic factors and neighbourhood deprivation are common explanations for why South Asians do less physical activity than the White British population [54]. The exclusion of these fac- tors from analyses that account for other potential con- founding factors, such as age and sex, points to the lack of theoretical models in the papers. No paper, on either chil- dren or adults, reported using a theoretical model to describe the potential causes of physical inactivity in popu- lations. By not referring to any theoretical model, the au- thors have excluded a potential confounder from their analyses – which they had often measured as part of the study. The exclusion of socioeconomic status or deprivation means that it is possible that the differences found between ethnic groups could be due to their socioeconomic circum- stances rather than to their ethnic background. Second-generation adults Of h f Of the four papers reporting on second-generation South Asian adults, three lacked power calculations [24, 28, 32], rendering it impossible in these cases to determine whether the sample sizes obtained were large enough to detect a dif- ference in prevalence between generations, or between the UK-born ethnic groups and the White British ethnic groups. One of the four papers reported a sample size cal- culation [20]. The other three papers reported either using existing survey data or simply recruiting as many partici- pants as possible. Low sample sizes may mean that the samples are biased towards a particular age group, sex or other characteristic and may not give accurate snapshots of the prevalence of risk factors in second-generation ethnic minorities in the UK. Two of the papers used the snowball sampling technique to increase their sample size [20, 24]. Snowball sampling is commonly used in populations where it is difficult to recruit participants, but produces a biased sample because recruitment is done through the contacts of existing participants. This is likely to affect the generali- sabilty of the findings to the wider ethnic minority popula- tions that were studied, although the authors did not discuss this issue. There were also issues with the quality of measure- ment, both for ethnicity and physical activity. For the studies on children, ethnicity was measured through self-identification to pre-defined categories [9, 13, 14, 18, 21, 23, 25] (often through the parents) or through school records, where ethnicity is reported by the parents [7, 8, 10, 11, 30]. While self-identifying ethnicity is regarded as the best measure, assigning people to pre-defined cat- egories or asking them to choose from a pre-defined list restricts the process of self-identification, and therefore the meaning of ethnicity. Five of the 12 studies on children measured physical ac- tivity using an objective measurement tool, either an ac- celerometer or a pedometer [9, 10, 13, 21, 23], and the rest used a self-report physical activity questionnaire or, in one case, a single physical activity question [30]. While objective measures of physical activity are more reliable than questionnaires, which are subject to recall bias or so- cial desirability bias, particularly for children [53], there Three papers used non-validated self-report measures to estimate prevalence [20, 28, 32]. Self-report data is Table 2 Characteristics of qualitative studies Bhatnagar et al. Second-generation adults Of h f International Journal of Behavioral Nutrition and Physical Activity (2015) 12:96 Table 2 Characteristics of qualitative studies South Asian Ethnic group Location of study Main focus of study Data collection method Number of participants Sex of participants Age groups Reporting o second-gen information [36] Brophy et al. 2011 Bangladeshi South Wales Asking teenager to suggest recommendation to increase their physical activity 4 Focus groups 24 Male and Female 16 to 18 No [37] Eastwood et al. 2013 South Asian London Assessing the risk of CVD in religious and community settings Semi-structured interviews 24 Male and Female 30 to 67 No [38] Farooqi et al. 2000 South Asian Leicester Understanding attitudes and knowledge of lifestyle risk factors for CHD 6 Focus groups 44 Male and Female Over 40 Yes [39] Grace et al. 2008 Bangladeshi London Preventing diabetes 17 Focus groups and Semi-structured interviews 129 in Focus groups 8 interviews Male and Female Mean age 35 Yes [40] Horne et al. 2009 South Asian North West England Fall prevention in 60 to 70 year olds Participant observation, 15 Focus groups and Semi-structured interviews 87 in Focus groups 40 interviews Male and Female 60 to 70 No [41] Horne et al. 2010 South Asian North West England Influence of primary health-care professionals on physical activity Participant observation, 15 Focus groups and Semi-structured interviews 87 in Focus groups 40 interviews Male and Female 60 to 70 No [42] Horne et al. 2012 Indian and Pakistani North West England To identify the attitudes and beliefs associated with the uptake and adherence ofphysical activity among community-dwelling South Asians Focus groups and Semi-structured interviews 29 in Focus groups 17 interviews Male and Female In 60s No [43] Horne et al. 2013 Indian and Pakistani North West England Understanding the barriers to maintaining physical activity 15 Focus groups and Semi-structured interviews 29 in Focus groups 17 interviews Male and Female In 60s No [44] Jepson et al. 2008 (report) Indian, Pakistani, Bangladeshi Aberdeen, Glasgow, Edinburgh To explore the barriers, facilitators and motivators for South Asians 9 Focus groups and Semi-structured interviews 59 in Focus groups 10 interviews Male and Female Ages 20 to 40 No [45] Jepson et al. Second-generation adults open to recall bias and there is a real possibility of reporting an inaccurate prevalence. No study reporting on second-generation adults used an objective measure of physical activity. Three studies reported information on the attitudes, mo- tivations and barriers to physical activity in second- generation South Asian adults [38, 39, 49]. One of these was a report, rather than a peer-reviewed article and had limitations in the reporting of the methods, context and analysis [49]. The authors did not describe their sam- pling approach, the context in which focus groups were held or give detail of how they analysed the focus group data. Without this information it is difficult to assess the quality of the study accurately. Three papers measured ethnicity through people self- identifying to pre-defined categories. In the fourth paper, the authors obtained ethnicity information by identifying South Asian surnames in birth records [20]. The authors do not report confirming this ethnicity information with their participants, and as a result each participant has had their ethnicity assigned for them, based on ancestry. This is essentially only a physical measure of ethnicity; if people have not self-identified as South Asian, we can- not be sure that they have a cultural or social identifica- tion with the South Asian population. None of these studies reported how the researcher may have influenced data collection. Of the three studies, only Grace et al. 2008 reported receiving ethical approval for their study [39]. Ethical approval is important in any re- search project in order to protect research participants and ensure researchers and universities are carrying out appropriate research. All three studies neglected to discuss the limitations of their studies, a key element of high qual- ity research. Reporting the limitations of your study allows the reader to ascertain whether the authors themselves understands the extent to which their findings are valid, and also allows the reader to gauge the transferability of the findings. The analysis of two papers was insufficient [20, 24]. Where regression models were conducted, important confounders, such as socioeconomic measures, were not included, even where data were available in the study. As discussed with the studies on children, this highlights the absence of theoretical frameworks addressing all po- tential causes of the behaviour being studied in the con- struction of statistical models to explain the behaviour. Second-generation adults Not including known confounders affects the conclu- sions drawn from the study, as any associations found between variables may in fact be confounded by a third variable. The dearth of socioeconomic status measures in the regression models raises concerns about the methods and theories used to identify potential con- founders in the studies as a whole. The two peer-review articles provided only fleeting re- marks on adult second-generation physical activity atti- tudes. The report by Rai & Finch 1997 provided some more information, although the information was not re- ported separately for the second-generation [49]. Poor discussion of the study limitations, analytical approach and methods means that although the findings reported are rich in their description, the only study with detailed information on second-generation South Asian adults in the UK is low in quality. Qualitative papers Children Fi Five papers reported on motivations or barriers to phys- ical activity in South Asian children in the UK [36, 47, 48, 50, 51]. All of these papers were of reasonable qual- ity, but none of the papers discussed how the re- searchers themselves might have influenced the data collection or analysis, through their own background or attitudes. Reflecting on how an interviewer might influ- ence the interview or focus group is important because interviewees may offer more or less information accord- ing to how they perceive the interviewer [55]. For the rest of the papers, the overall quality was af- fected by the fact that all but one [45] failed to discuss the impact of the researcher on the data collection and analysis. Two papers were of fairly low quality [46, 51], with poor reporting of data collection methods, the con- text in which the research was carried out, the analytical techniques, and no discussion of the study limitations or reporting of whether ethical approval was received. Only one paper reported using a theoretical model [52]. Only one study on children reported using an existing theoretical model, which the authors used to guide their analysis of the data [39]. Theoretical models in qualitative research can be used to develop hypotheses, to develop interview guides or, as in this paper, used to guide the analysis [56]. Most papers conducted a the- matic analysis of their data, but placing these themes into a theoretical framework is useful for interpretation and can aid understanding of the practical value of the findings. Second-generation adults Of h f 2012 Indian, Pakistani, Bangladeshi Aberdeen, Glasgow, Edinburgh Describing the types of and motivators for physical activities that South Asians do 9 Focus groups and Semi-structured interviews 59 in Focus groups 10 interviews Male and Female Unknown No [46] Johnson 2000 Indian, Pakistani, Bangladeshi Midlands To fill the gap in knowledge about ethnic minority lifestyles and health Not reported Not reported Male Unknown No [47] Khunti et al. 2008 South Asian Leicester Understanding the impact of an Action Research Partnership to prevent diabetes 18 Focus groups Not reported Male and Female 11 to 15 No [48] Pallan et al. 2012 South Asian Birmingham Understanding the contextual influences on obesity 9 Focus groups 68 Male and Female Unknown (adults talking about children) No Table 2 Characteristics of qualitative studies (Continued) Bhatnagar et al. International Journal of Behavioral Nutrition and Physical Activity (2015) 12:96 q [49] Rai & Finch 1997 Indian, Pakistani, Bangladeshi England To investigate attitudes towards and barriers to physical activity in South Asian and Black communities in England 14 Focus groups 109 Male and Female 18 to 50 Yes [50] Rawlins et al. 2013 Indian, Pakistani, Bangladeshi London Perceptions of healthy eating and physical activity in young children 13 Focus groups 70 Male and Female 8 to 13 No [51] Rogers et al. 1997 Bangladeshi London To describe the contributing factors to variations in health-related behaviours and attitudes in 12 year olds Semi-structured interviews 41 Male and Female Age 12 No [52] Victor 2014 Bangladeshi and Pakistani Great Britain Physical activity during the daily life of elders Semi-structured interviews 109 Male and Female Over 50 No hatnagar et al. International Journal o Bhatnagar et al. International Journal of Behavioral Nutrition and Physical Activity (2015) 12:96 Page 9 of 19 Page 9 of 19 Differences between males and females Owen et al. (2009) used an accelerometer to measure physical activity in South Asian children and White European children, and also examined differences be- tween boys and girls. South Asians were less physically active than White European children for all measures, but there were large differences between girls and boys for both ethnic groups. South Asian girls were the least active, spending the largest number of minutes being sedentary and the smallest number of minutes being moderately or vigorously active. Harding et al. 2008 combined the Pakistani and Bangladeshi group and found that Pakistani/Bangladeshi girls were the least ac- tive compared to the White British and Indian groups. Interestingly, almost a third of Pakistani/Bangladeshi boys were in the most active quartile, compared to around a quarter of Indian and White British boys [14]. Only one study examined Indian, Pakistani and Bangladeshi groups separately [13]. Measured using ac- celerometers, in this study the Bangladeshi group was the least active and the Indian group the most active (33 % versus 40 % respectively meeting recommended physical activity levels). p p Lean et al. 2001 [20] and Smith et al. 2012 [28] both used a single question on physical inactivity to compare migrant South Asians, second-generation South Asians and White British or European origin populations. Both reported that adult second-generation South Asians were more active than the migrant generation, but still less active than the White British or European origin population. Smith et al. 2012 reported on physical activ- ity in Indians, Pakistanis and Bangladeshi separately. The pattern of lower physical inactivity in the second- generation compared to the first-generation was the same for all three ethnic groups, although the actual levels of inactivity were differed between the three groups. Within the South Asian ethnic group, the Indian ethnic group had the lowest prevalence of physical in- activity (31.5 %) and the Bangladeshi group had the highest prevalence of physical inactivity (49.4 %); the White ethnic group had the lowest prevalence of phys- ical inactivity overall (26.7 %). The difference in mean age between the first and second-generation was less than 10 years in both papers. Only one study examined Indian, Pakistani and Bangladeshi groups separately [13]. Measured using ac- celerometers, in this study the Bangladeshi group was the least active and the Indian group the most active (33 % versus 40 % respectively meeting recommended physical activity levels). Weekend and Weekday activity Weekend and Weekday activity While not reported in Table 3, Eyre et al. 2013 also found that South Asian children were less active than White European children at the weekends and after school [10]. Duncan et al. 2012 report on the average steps per day taken by South Asian children, at the weekend or a weekday. They found that South Asian children were less active than White children both on weekdays and at weekends, which is in line with the re- sults reported by Eyre et al. 2013 [9]. Studies reporting on physical activity prevalence among South Asian children all reported a lower preva- lence compared to White British children, irrespective of the measurement tool (Table 3). Studies that measured boys and girls separately consistently reported that girls were less physically active than boys, for both South Asian and White British groups. Second-generation South Asian adults Four studies reported on the physical activity of second- generation adult South Asian groups [20, 24, 28, 32]. Table 4 shows that while they used different measures, all four reported that second-generation South Asians were more active than the first-generation. The three studies comparing South Asian and White British groups showed that the second-generation is still less ac- tive than the White British population. Prevalence of physical activity South Asian children One study on children was a prospective cohort, which collected physical activity data from the children in year seven of their study. The rest of the studies on children were cross-sectional. Seven studies were located in the Midlands, with two of those also recruiting children from London. One study was exclusively based in London, one was based in the North East of England Page 10 of 19 Bhatnagar et al. International Journal of Behavioral Nutrition and Physical Activity (2015) 12:96 and another in Glasgow. Two studies were conducted throughout the UK or England. overall and during break-time, with South Asians being less active during break-time [10]. Pollard et al. 2012 ex- amined differences in activity during break time in school, and found that British Pakistani girls were less active than White British children, which concurs with the findings from Eyre et al. 2013 [25]. Information on the number of UK-born South Asian children is only available for three studies. Griffiths et al. 2013 used the Millennium Cohort Study, which recruited children born in the UK in the year 2000, therefore all the children included in their paper are second-generation (139 Indian, 177 Pakistani and 70 Bangladeshi) [13]. Harding et al. 2008 report on results from the DASH Study, which is based in London; they report that 76.1 % of the Indian group and 82.9 % of the Pakistani/Bangla- deshi group were born in the UK (166 and 184 respect- ively) [14]. Williams et al. 1998 report that 287 (86 %) of their South Asian sample were born in the UK [30]. [11] Falconer et al. 2014 4 to 5 and 10 to 11 5 PCTs in England Activity during break-time Eyre et al. 2013 used a pedometer and heart-rate moni- tor to measure activity levels in children aged between eight and nine years. The authors found that South Asian children were less physically active than White European children for all measures included in their study. They found significant differences between the two ethnic groups for the average counts per minute Pollard et al. 2008 used the International Physical Ac- tivity Questionnaire to measure physical activity in Pakistani women, which asks a range of questions [24]. As with the results from Lean et al. 2001 and Smith et al. 2012, the second-generation Pakistani women were more active than the migrant generation, but less active Bhatnagar et al. International Journal of Behavioral Nutrition and Physical Activity (2015) 12:96 Page 11 of 19 Page 11 of 19 Table 3 Physical activity prevalence in South Asian children Author and year Age- group Location Physical activity measurement instrument Main findings – all children Main findings – Male Main findings – Fem [13] Griffiths et al. 2013 Age 7 UK Accelerometer worn for 7 days, during waking hours. Meeting recommended activity levels: White: 51.4 % Indian: 40.0 % Pakistani: 45.2 % Bangladeshi: 32.8 % Overall counts/minute: White: 597 Indian: 511 Pakistani: 563 Bangladeshi: 538 Sedentary hours/day: White: 6.5 Indian: 6.9 Pakistani: 6.4 Bangladeshi: 6.5 Moderate and vigorous minutes/day: White: 60.2 Indian: 52.6 Pakistani: 58.2 Bangladeshi: 52.9 Steps/day: White: 10,343 Indian: 8,699 Pakistani: 9,419 Bangladeshi: 8,894 [11] Falconer et al. 2014 4 to 5 and 10 to 11 5 PCTs in England Self-report questionnaires Child does not achieve ≥1 hr of physical activity/ day White: 56.2 % Asian: 59.4 % [10] Eyre et al. 2013 8 to 9 Coventry Physical activity and heart rate worn monitor for 7 days Meeting WHO recommended activity levels: White European: 73 % South Asian: 35 % Wake hour average counts/ minute: Whi E 116 Table 3 Physical activity prevalence in South Asian children Bhatnagar et al. International Journal of Behavioral Nutrition and Physical Activity (2015) 12:96 Page 12 of 19 Page 12 of 19 Table 3 Physical activity prevalence in South Asian children (Continued) Table 3 Physical activity prevalence in South Asian children (Continued) White European: 1.3 South Asian: 1.0 Counts/minute during break-time: White European: 341 South Asian: 317 [21] McMinn et al. 2011 9 to 10 London, Birmingham, Leicester Accelerometer worn for 7 days, during waking hours. [25] Pollard et al. 2012 9 to 11 North East England Accelerometer worn for 2 school days [9] Duncan et al. 2012 8 to 11 Coventry Pedometers worn over 4 days Accelerometer worn for 7 days [16] Hemmings et al. 2011 14 to 15 London Activity during break-time Average counts/minute: White European: 481 South Asian: 452 [23] Owen et al. 2009 9 to 10 London, Birmingham, Leicester Accelerometer worn for 7 days, during waking hours. Average counts/minute: White European: 498 South Asian: 457 Sedentary minutes/day: White European: 554 South Asian: 593 Moderate and vigorous minutes/day: White European: 70 South Asian: 65 Mean number of steps: White European: 10,220 South Asian: 9,314 [25] Pollard et al. 2012 9 to 11 North East England Accelerometer worn for 2 school days [25] Pollard et al. 2012 9 to 11 North East England Accelerometer worn for 2 school days [9] Duncan et al. 2012 8 to 11 Coventry Pedometers worn over 4 days Average weekday steps/day: White: 14,734 South Asian: 13,023 Average weekend steps/ day: White: 11,135 South Asian: 10,383 Average total steps/day (PB calculation): White: 12,935 South Asian: 11,703 [14] Harding et al. 2008 11 to 13 London Self-report physical activity questions on vigorous sports [14] Harding et al. 2008 11 to 13 London Self-report physical activity questions on vigorous sports Percentage in most active 1st quartile: Percentage in most active 1st quartile: White UK: 23.9 % White UK: 12.6 % Indian: 23.8 % Indian: 16.5 % Pakistani/Bangladeshi: 31.3 % Pakistani/Bangladeshi: 14.9 % [14] Harding et al. 2008 11 to 13 London Percentage in most active 1st quartile: White UK: 12.6 % Indian: 16.5 % Pakistani/Bangladeshi: 14.9 % Self-report physical activity questions on vigorous sports Percentage in most active 1st quartile: White UK: 23.9 % Indian: 23.8 % Pakistani/Bangladeshi: 31.3 % Bhatnagar et al. International Journal of Behavioral Nutrition and Physical Activity (2015) 12:96 Bhatnagar et al. International Journal of Behavioral Nutrition and Physical Activity (2015) 12:96 Bhatnagar et al. International Journal of Behavioral Nutrition and Physical Activity (2015) 12:96 Page 13 of 19 Page 13 of 19 able 3 Physical activity prevalence in South Asian children (Continued) Percentage in least active 4th quartile:White UK: 21.4 % Percentage in least active 4th quartile:White UK:38.0 % Indian: 18.0 % Indian: 46.3 % Pakistani/Bangladeshi: 17.2 % Pakistani/Bangladeshi: 38.3 % 7] Duncan 11 to Birmingham Self report validated Very Inactive: Table 3 Physical activity prevalence in South Asian children (Continued) Table 3 Physical activity prevalence in South Asian children (Continued) Percentage in least active 4th quartile:White UK: 21.4 % Percentage in least active 4th quartile:White UK:38.0 % Indian: 18.0 % Indian: 46.3 % Pakistani/Bangladeshi: 17.2 % Pakistani/Bangladeshi: 38.3 % [7] Duncan et al. Activity during break-time International Journal of Behavioral Nutrition and Physical Activity (2015) 12:96 Page 14 of 19 Table 3 Physical activity prevalence in South Asian children (Continued) Table 3 Physical activity prevalence in South Asian children (Continued) British South Asian: 260.2 Moderate activity in counts/minute: British White: 70.5 British South Asian: 78.1 Vigorous activity in counts/ minute: British White: 5.2 British South Asian: 5.1 British South Asian: 260.2 Moderate activity in counts/minute: British White: 70.5 British South Asian: 78.1 Vigorous activity in counts/ minute: British White: 5.2 British South Asian: 5.1 mean METs in the UK-born South Asians as compared to the migrant South Asians [32]. mean METs in the UK-born South Asians as compared to the migrant South Asians [32]. mean METs in the UK-born South Asians as compared to the migrant South Asians [32]. than the European origin population. Pollard et al. 2008 found a significant difference between the groups for median MET-minutes (p = 0.03) but not for median pedometer-counts (p = 0.12). The difference in mean age between the migrant and British-born generation was about 2 years. Williams et al. 2011 also calculated METs from a questionnaire and found significantly higher Attitudes, motivators and barriers to physical activity The review of quantitative observational papers re- vealed that there is not only evidence of variation in physical activity prevalence within UK South Asians, Table 4 Physical activity prevalence in second-generation South Asian adults Author and year Sex Age-group Location Physical activity measurement instrument Main findings [20] Lean et al. 2001 Female 20 to 42 Glasgow Self-report question on sport and recreational exercise Engaging in no sport or recreational exercise: Migrant South Asians: 82 % British-born South Asians: 77 % Italian/General Population: 50 % [24] Pollard et al. 2008 Female 20 to 40 North East England Self-report, validated questionnaire -International Physical Activity Questionnaire Median MET-minutes: Migrant British Pakistani: 1,040 British-Born British Pakistani: 1,626 European: 2,394 Median pedometer counts: Migrant British Pakistani: 3,371 British-Born British Pakistani: 3,506 European: 3,781 [32] Williams et al. 2011 - JECH Male and Female Over 16 England Self-report questionnaire based on Allied Dunbar National Fitness Survey Mean total METs: Male UK-born South Asian: 1,385.23 Male born outside UK South Asian: 935.53 Female UK-born South Asian: 972.50 Female born outside UK South Asian: 843.66 [28] Smith et al. [32] Williams et al. 2011 - JECH Male and Female Over 16 England Self-report questionnaire based on Allied Dunbar National Fitness Survey Activity during break-time 2006 11 to 14 Birmingham Self-report, validated questionnaire - Four by one day Very Inactive: White: 20.9 % Asian: 14.9 % Inactive: White: 43.5 % Asian: 44.8 % Moderately Active: White: 21.5 % Asian: 26.9 % Active: White: 14.1 % Asian: 13.4 % [8] Duncan et al. 2008 11 to 14 Birmingham Self-report, validated questionnaire - Four by one day Average daily minutes spent in moderate and vigorous physical activity: White: 90.0 South Asian: 68.2 [18] Khunti et al. 2007 11 to 16 Leicester Self-report questionnaire derived from Four by one day Light aerobic exercise on six or more days during previous two weeks: Light aerobic exercise on six or more days during previous two weeks: Light aerobic exercise on six or more days during previous two weeks: White European: 39 % White European: 42 % White European: 37 % South Asians:40 % South Asians: 40 % South Asians: 39 % Hard aerobic exercise on six or more days during previous two weeks: Hard aerobic exercise on six or more days during previous two weeks: Hard aerobic exercise on six or more days during previous two weeks: White European: 41 % White European: 52 % White European: 32 % South Asians: 37 % South Asians: 48 % South Asians: 25 % [30] Williams et al. 1998 14 to 15 Glasgow Self-report questionnaire Physical exercise for 20 minutes once a week or less: Physical exercise for 20 minutes once a week or less: Other origin: 8 % Other origin: 15 % British Asian: 18 % British Asian: 16 % Physical exercise for 20 minutes 2–3 times/ week: Physical exercise for 20 minutes 2–3 times/ week: Other origin: 22 % Other origin: 39 % British Asian: 29 % British Asian: 53 % Physical exercise for Physical exercise for [30] Williams et al. 1998 14 to 15 Glasgow Self-report questionnaire [30] Williams et al. 1998 14 to 15 Glasgow Self-report questionnaire Bhatnagar et al. International Journal of Behavioral Nutrition and Physical Activity (2015) 12:96 Bhatnagar et al. Activity during break-time 2012 Male and Female 16 to 55 England Self-report questionnaire based on Allied Dunbar National Fitness Survey Three or fewer occasions of moderate/vigorous activity in the past four weeks: White: 26.7 % First-generation Indian: 43 % Second-generation Indian: 31.5 % First-generation Pakistani: 50.2 % Second-generation Pakistani: 38.5 % First-generation Bangladeshi: 60.8 % Second-generation Bangladeshi: 49.4 % [32] Williams et al. 2011 - JECH Male and Female Over 16 England [28] Smith et al. 2012 Male and Female 16 to 55 England Bhatnagar et al. International Journal of Behavioral Nutrition and Physical Activity (2015) 12:96 Bhatnagar et al. International Journal of Behavioral Nutrition and Physical Activity (2015) 12:96 Bhatnagar et al. International Journal of Behavioral Nutrition and Physical Activity (2015) 12:96 Page 15 of 19 Page 15 of 19 One paper also reported that family activities would be more appealing to Asian people and so would help South Asian children be more physically active. but also between the first and second-generations. The quantitative papers had limited information on poten- tial explanations for those differences. With our review of the qualitative literature we therefore aimed to ascer- tain what studies had been done exploring the atti- tudes, beliefs, motivators and barriers towards physical activity in UK South Asians, with a particular focus on the second-generation. but also between the first and second-generations. The quantitative papers had limited information on poten- tial explanations for those differences. With our review of the qualitative literature we therefore aimed to ascer- tain what studies had been done exploring the atti- tudes, beliefs, motivators and barriers towards physical activity in UK South Asians, with a particular focus on the second-generation. Attitudes, motivators and barriers around physical activity in second-generation adults Twelve papers reported on motivators and barriers to physical activity in South Asian adults in the UK, al- though only three of these reported any information on the second-generation. Two papers and one report de- scribed a change in behaviour or attitudes in the second- generation, but gave further no detail. Grace et al. 2008 report that in focus groups, younger and second- generation Bangladeshi women supported resisting the traditional norms and expectations of women in Bangladeshi culture [39]. Farooqi et al. 2000 report that some participants in focus groups commented that, in contrast to the first-generation women being quoted, at- titudes among younger Bangladeshi women are chan- ging; for example a participant’s daughter-in-law takes her own children swimming [38]. While only brief, both these papers indicate that there may be a positive change in attitude towards physical activity in the second- generation of South Asian women. Attitudes, motivators and barriers for physical activity in children Five papers reported on motivations and barriers to physical activity in South Asian children, but only two of these discussed generational differences in health behav- iours within South Asian groups. Table 5 summarises these themes using the socioecological framework as a guide. Placing the themes into a socioeconomic frame- work helps to understand if the motivations and barriers are at an individual-level, social-level or neighbourhood- level; once we know this, we can begin to understand in which areas interventions might be necessary. The ma- jority of the themes focused on barriers, which were sit- uated at all levels of the socioeconomic model, including Asian cultural factors, school facilities, the neighbour- hood environment and parental concerns over the cost of physical activity. The report by Rai and Finch 1997 describes differences in attitudes between younger and older people; all of their study participants aged under 30 were born in the UK [49]. Rai and Finch 1997 studied both Black and South Asian people and combine the findings from these two ethnic groups in their discussion. They note that younger people do not share some of the beliefs of older Only three themes relating to motivations are present in the literature for children, with two of these focusing on boys. One of the themes was a motivator for boys but a barrier for girls, in that boys were described in one paper as being more interested sports than girls are. Table 5 Motivators and barriers to South Asian children in the UK Religious factors Asian cultural factors Western gender factors Other individual factors School Facilities Neighbourhood environment Economic Motivators Family activities Boys are motivated by their peers and siblings (Bangladeshi boys) Boys are more interested in sports than girls are Barriers Attending the Mosque after school limits time Pakistani and Bangladeshi parents themselves are inactive It’s embarrassing to exercise (Bangladeshi girls) Parents have limited awareness of physical activity recommendations Lack of changing rooms and storage facilities Fear of unsafe roads in high socioeconomic groups Concern about the cost of physical activities Lack of interest in PE classes in girls, for all ethnic groups Concern over security of their children playing outside Being physically active might have a negative effect on their schoolwork (Bangladeshi girls) Parents who work do not have time to take their children to leisure activities It is quicker and easier to use the car for parents. Attitudes, motivators and barriers for physical activity in children Lack of facilities in the local area Girls not encouraged to play out because of people looking at them (Bangladeshi) Table 5 Motivators and barriers to South Asian children in the UK Girls not encouraged to play out because of people looking at them (Bangladeshi) Girls not encouraged to play out because of people looking at them (Bangladeshi) Bhatnagar et al. International Journal of Behavioral Nutrition and Physical Activity (2015) 12:96 Page 16 of 19 people and that their views are more shaped by the media as compared with older people. The authors state that the experience of early life in the UK underpins the differences between the younger and older people, who had grown up in other countries. Comparison to existing literature Previous reviews on physical activity in South Asians have shown that physical activity prevalence is low in these ethnic groups, but have not reported on variation in prevalence levels within these ethnic groups. While there have been no other reviews on this topic within the UK, there is evidence from the Netherlands to support generational differences in CVD risk factors in ethnic mi- nority groups [57, 58]. Hosper et al. find differences in levels of obesity, smoking, physical activity and alcohol consumption in Turkish and Moroccan migrants to the Netherlands. They also find that socioeconomic status in women was higher in the second-generation compared to the first-generation. While caution must be made in com- paring the socioeconomic status changes of two different ethnic groups in two different countries, it is plausible that the advantages of being born in a country compared to migrating to it would benefit the second-generation of an ethnic minority, as appears to be the case for Turkish and Moroccan immigrant women to the Netherlands. This finding corroborates the findings of Smith et al. 2012, who use Health Survey for England data in their paper and also report higher socioeconomic positions in the UK-born ethnic minority groups compared to the first-generation [28]. Unfortunately there was no information available by gender from the papers included in this review to com- pare to Hosper et al’s findings. While Rai and Finch 1997 have not explicitly reported the differences between second-generation and first- generation South Asians within their report, we were able to analyse the quotes by South Asians aged under 30 (reported as all being born in the UK). Attitudes, motivators and barriers for physical activity in children Table 6 sum- marises the themes discussed by participants in both the report by Rai and Finch, and in the two peer-reviewed papers. While there were some Asian cultural and reli- gious barriers, there is an indication that there may be a change in attitudes among the younger and second- generation towards the commonly reported barriers to physical activity found in South Asian women [2] Summary of findings This review demonstrated that there is some evidence of differences in the prevalence of physical activity be- tween first and second-generation South Asians in the UK, and between second-generation South Asians and the White British population. There is also limited evi- dence that second-generation South Asian adults have different attitudes to physical activity as compared to the first-generation. The studies that have explored physical activity in children have predominantly fo- cused on barriers, but do also show that factors in the neighbourhood and school environment affect the physical activity of South Asian children in the UK. This is an important finding, as it shows that factors other than ethnic background are affecting the physical activity of South Asian children. Small samples and failure to adjust for important con- founding variables limited the generalisability of results that would have otherwise been highly valuable in ethni- city and physical activity research. Papers studying eth- nic minorities can suffer from low sample sizes due to the smaller proportion of people from ethnic minorities in the general population. It is therefore, perhaps, unsur- prising if studies reporting on subsections of the UK ethnic minority population struggle to recruit enough Table 6 Motivators and barriers to adult second-generation South Asians in the UK Religious factors Asian cultural factors Other individual factors Local facilities Economic factors Motivators Younger and second-generation women resist traditional norms and expectations of women in Bangladeshi culture South Asian men described having positive role model as children People like challenges Women want to look good Men want to socialise through activity Barriers Religious activities such as Namaz restricts time Asian women are more reluctant to use child-minders and so caring for children means they have less time. Tiredness after work Lack of facilities in local area Cost of using facilities Young people don’t think about being physically active for health Islam restricts clothing women can wear Experience of racism at gyms Muslim women do not want to use mixed-sex facilities People like challenges Women want to look good Men want to socialise through activity Barriers Religious activities such as Namaz restricts time Asian women are more reluctant to use child-minders and so caring for children means they have less time. Strengths and limitations of the review Strengths and limitations of the review As far as we are aware, this is the first paper to review and assess studies reporting on physical activity in second-generation UK South Asians. We conducted a systematic searched of the literature although we may have missed research published in the ‘grey’ literature. Ideally, systematic reviews are conducted independently by two researchers whose their findings are compared, but one author carried out the systematic search for this re- view. We are confident that all relevant papers were in- cluded, but because only one person screened the papers for inclusion we cannot rule out the possibility that extra papers may have been eligible for inclusion in the study. At present a limited amount is known about the epi- demiology of physical activity in second-generation South Asians, although it is apparent that they are more active than the migrant generation; even less is known about the reasons for this increase and in what ways physical activity behaviour has changed. More theoretically-informed quantitative research with adequate sample sizes needs to be carried out in order to establish firmly the differences in physical activity prevalence between the generations of ethnic minorities. Future research also needs to distin- guish different South Asian minorities to reflect the het- erogeneity of the groups comprising ‘South Asian’; appropriate strategies for physical activity improvement can only be developed if research results are presented separately for each ethnic group. Our analysis of the papers was hindered by the different groupings of ethnic minorities used in the paper. A num- ber of the papers used the broad categorisation of ‘South Asian’, which is not directly comparable to the more de- tailed categories of ‘Indian’, ‘Pakistani’ and ‘Bangladeshi’ as these three groups differ in their socioeconomic and CVD risk factor profiles [54]. While we attempted to compare all the papers, this should be taken into account. One strength of this paper is the inclusion of both quantitative and qualitative literature. Quantitative methods are limited in their ability to explain the causes of differences between populations. Qualitative methods are ideally suited to this, and the papers in this review indicate that second-generation South Asians have a dif- ferent attitude towards physical activity as compared to the first-generation, something which could not be gleaned from the quantitative literature. Grieser et al. Summary of findings Tiredness after work Lack of facilities in local area Cost of using facilities Young people don’t think about being physically active for health Islam restricts clothing women can wear Experience of racism at gyms Muslim women do not want to use mixed-sex facilities Bhatnagar et al. International Journal of Behavioral Nutrition and Physical Activity (2015) 12:96 Page 17 of 19 Page 17 of 19 participants for a quantitative examination of the pooled results. groups. Although Grieser et al. 2006 studied different eth- nic groups in a different country, the principle that ethnic minority adolescents may have similar physical activity at- titudes to the majority ethnic group, or ethnic groups is important to note. The report by Rai & Finch highlights the importance of a childhood in the UK, citing the im- portance of childhood experiences in developing attitudes towards physical activity [49]. Some of the motivators and barriers to physical activity described by Rai & Finch are similar to those described in a review by Allender et al. 2006. Their review on understanding participation in sport and physical activity in the UK reports issues such as cost and being motivated by wanting to maintain ap- pearance [56]. There is not enough information from this review to know whether second-generation South Asians have similar physical activity attitudes to the White British population in the UK, but if childhood and school experi- ences are relevant for physical activity attitudes, this is theoretically possible. The age structure of the second-generation may have made it difficult to obtain a large sample size for second-generation adults. However, some of these stud- ies were conducted over ten years ago and there may now be enough adults in the UK-born ethnic minority groups to improve sample sizes. It should also be noted that the age structure of ethnic minority populations varies for each group, with the average age at migration to the UK and time since the majority of migration took place for each ethnic group affecting the current age structure of the second-generation. Results from this review indicate that there is some evidence of UK-born ethnic minorities obtaining a higher socioeconomic position than their parents. This is coupled with an increase in physical activity preva- lence between the generations. Strengths and limitations of the review 2006 explored the physical activity atti- tudes, preferences and practices of African American, His- panic and Caucasian girls aged 11 to 13 in the United States [59]. While the authors do not state whether the participants were born in the United States, we do know that the girls were attending school in the United States. The authors found few differences in attitudes towards physical activity between the ethnic groups, and thought that only a small number of these differences were related to ethnic background; for example, many Hispanic girls reported doing childcare in the past seven days. There were differences in the favoured activities of the ethnic Summary of findings Smith et al’s 2012 paper indicates that higher socioeconomic status may be the cause of changes in obesity prevalence for some ethnic minorities [28], but much more information is needed before accurate recommendations for policy can be made. What we do know is that the epidemiology of CVD in twenty to thirty years’ time is likely to be differ- ent for second-generation ethnic minorities if their phys- ical activity behaviours are not the same as those of the migrant generation. Conclusions This research highlights that there is variability in physical activity behaviour within South Asians in the UK. This Page 18 of 19 Page 18 of 19 Page 18 of 19 Bhatnagar et al. International Journal of Behavioral Nutrition and Physical Activity (2015) 12:96 review has shown that for second-generation South Asians, particularly adults, research that accurately mea- sures the level of physical activity is still to be done. Some work has begun on identifying factors that influence phys- ical activity, but in the majority of the quantitative litera- ture, socioeconomic factors are omitted from analysis, and we found no peer-reviewed qualitative literature studying physical activity in second-generation South Asian adults as a distinct group. There were some studies on South Asian children, but these were limited in that they mainly focused on barriers to physical activity. 9. Duncan MJ, Birch S, Al-Nakeeb Y, Nevill AM. Ambulatory physical activity levels of white and South Asian children in Central England. Acta Paediatr. 2012;101:e156–62. 10. Eyre EL, Duncan MJ, Smith EC, Matyka KA. Objectively measured patterns of physical activity in primary school children in Coventry: the influence of ethnicity. Diabet Med. 2013;30:939–45. 11. Falconer CL, Park MH, Croker H, Kessel AS, Saxena S, Viner RM, Kinra S (2014) Can the relationship between ethnicity and obesity-related behaviours among school-aged children be explained by deprivation? A cross-sectional study. BMJ Open. doi: http://dx.doi.org/10.1136/bmjopen-2013-003949 12. Ghouri N, Purves D, McConnachie A, Wilson J, Gill JMR, Sattar N. Lower cardiorespiratory fitness contributes to increased insulin resistance and fasting glycaemia in middle-aged South Asian compared with European men living in the UK. Diabetologia. 2013;56:2238–49. 13. Griffiths LJ, Cortina-Borja M, Sera F, et al. (2013) How active are our children? Findings from the Millennium cohort study. BMJ Open. doi: http:// dx.doi.org/10.1136/bmjopen-2013-002893 From the papers found in this review, it seems there is a significant gap in the literature on the perceptions, at- titudes and experiences of physical activity among second-generation South Asians in the UK, which po- tentially has importance for health and social inequalities policies in the UK. 14. Harding S, Teyhan A, Maynard MJ, Cruickshank JK. Ethnic differences in overweight and obesity in early adolescence in the MRC DASH study: The role of adolescent and parental lifestyle. Int J Epidemiol. 2008;37:162–72. 15. Hayes L, White M, Unwin N, Bhopal R, Fischbacher C, Harland J, et al. Competing interests The authors declare th Competing interests The authors declare that they have no competing interests. 18. Khunti K, Stone MA, Bankart J, Sinfield PK, Talbot D, Farooqi A, et al. Physical activity and sedentary behaviours of South Asian and white European children in inner city secondary schools in the UK. 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https://openalex.org/W1965942067	https://www.revistas.usp.br/rcf/article/download/85357/88183	Portuguese	null	Lobbying na Regulação Contábil: Evidências do Setor Petrolífero	Revista Contabilidade & Finanças	2,014	cc-by	18,897	ISSN 1808-057X ISSN 1808-057X Ariovaldo dos Santos Ariovaldo dos Santos Professor Titular, Departamento de Contabilidade e Atuária, Faculdade de Economia, Administração e Contabilidade, Universidade de São Paulo E-mail: arisanto@usp.br Professor Titular, Departamento de Contabilidade e Atuária, Faculdade de Economia, Administração e Contabilidade, Universidade de São Pa E-mail: arisanto@usp.br Recebido em 23.9.2013 – Aceito em 30.9.2013 – 3a. versão aprovada em 4.6.2014. RESUMO Este trabalho tem como objetivo identificar os fatores determinantes à submissão de cartas comentários, como estratégia de lobbying no contexto da regulação contábil, à audiência pública do Discussion Paper Extractive Activities do International Accounting Standards Board (IASB). Os resultados mostram o tamanho como fator determinante, em todas as modelagens utilizadas, indicando que grandes empresas petrolíferas possuem maior probabilidade para realizar lobbying. Essa propensão é verificada para posicionamentos essencialmente desfa- voráveis às propostas apresentadas pelo IASB, o que implica em considerar que a revisão/substituição do International Financial Reporting Standard - IFRS 6 será um processo complexo e sujeito a pressões por parte das empresas petrolíferas para manter o status quo. Palavras-chave: Lobbying. Regulação contábil. Setor petrolífero. Escolhas contábeis. Lobbying na Regulação Contábil: Evidências do Setor Petrolífero* Lobbying on Accounting Regulation: Evidence from the Oil Industry Odilanei Morais dos Santos Professor Doutor, Departamento de Contabilidade, Instituto Brasileiro de Mercado de Capitais, Rio de Janeiro E-mail: odilaneisantos@terra.com.br R. Cont. Fin. – USP, São Paulo, v. 25, n. 65, p. 124-144, maio/jun./jul./ago. 2014 1 Introdução qual seja, tentar influenciar o IASB por meio da submissão de cartas comentários às audiências públicas. Olhando a regulação contábil das atividades de explora- ção e produção petrolífera, o debate em torno da definição do melhor modelo contábil capaz de capturar as transações econômicas do setor remonta às décadas de 1960 e 1970. Nos Estados Unidos, país pioneiro quando se trata do arcabouço teórico e normativo da contabilidade do setor petrolífero, tan- to a Security Exchange Commission (SEC) quanto o Financial Accounting Standards Board (FASB) divergiram entre si em relação às normas atinentes e permitiram a existência de dois métodos contábeis conflitantes denominados “esforços bem sucedidos” (successful efforts) e “capitalização total” (full cost) (Cortese, Irvine, & Kaidonis, 2009). Para atingir o objetivo proposto, verifica-se, por meio de técnicas multivariadas de análise de dados (regressões lo- gísticas, binomial e multinomial, e regressão de Poisson), se existe correlação entre as variáveis representativas das carac- terísticas das firmas e o fato de terem apresentado comentá- rios ao DPEA do IASB. Dessa forma, busca-se definir os fa- tores que determinam a adoção dessa estratégia de lobbying no processo regulatório contábil do setor petrolífero. Utiliza-se modelagem econométrica em amostra formada por empresas petrolíferas lobistas (aquelas que submeteram cartas comentários ao IASB) e por empresas petrolíferas não lobistas (aquelas que não submeteram cartas comentários) para identificar quais seriam os fatores econômicos preponderantes envolvidos na adoção da estratégia de lobbying por parte desse grupo de interesse (empresas petrolíferas) junto ao IASB. O cenário em que as empresas petrolíferas podem esco- lher livremente entre esses dois métodos contábeis foi fruto de um intenso jogo de interesses que colocou o FASB e a SEC em situações antagônicas, com o FASB, de um lado, defen- dendo a norma Statements of Financial Accounting Standard n. 19 – Financial Accounting and Reporting by Oil and Gas Producing Companies (SFAS 19) que regula o método dos esforços bem sucedidos; e de outro lado, a SEC, defendendo o método da capitalização total por meio de sua Regulation S-X 4-10, situação essa que ainda permanece vigente. 1 Introdução Essa estratégia de pesquisa tem sido comumente utili- zada com o objetivo de se identificar os motivos e as es- tratégias de lobbying empregadas pelos diversos grupos de interesse, a exemplo das pesquisas desenvolvidas por Dha- liwal (1982), Sutton (1984), Francis (1987), Deakin (1989), Kenny e Larson (1993), Tutticci, Dunstan, e Holmes (1994), Weetman, Davie, e Collins (1996), Georgiou e Roberts (2004), Asekomeh, Russel, e Tarbert (2006), Hansen (2011) e Ginner e Arce (2012), dentre outras. No âmbito do normatizador internacional também não foi diferente, dado que, desde 1998, tenta-se emitir uma norma contábil, completa, para o setor petrolífero, mas sem sucesso. Para permitir que as empresas europeias do setor extrativista pudessem adotar as normas internacionais em 2005, o International Accounting Standards Board (IASB) emitiu em 2004 a International Financial Reporting Standard n. 6 – Exploration for and Evaluation of Mineral Resources (IFRS 6) dando orientações gerais, mas sem entrar no mérito sobre qual método deveria ser utilizado pelas empresas. Nesse particular, faz-se necessário esclarecer que a uti- lização do conceito de lobbying empregado neste estudo apresenta uma visão bem particular e refere-se às manifes- tações explícitas contidas nas cartas comentários subme- tidas em audiências públicas de discussão de uma norma contábil, como é o caso do DPEA. Mais de seis anos se passaram desde a emissão do IFRS 6 e as discussões relacionadas à emissão da norma contábil internacional definitiva aplicável ao setor extrativista con- tinuam presentes com a publicação do Discussion Paper Extractive Activities (DPEA daqui em diante), decorren- te do projeto do IASB de revisão/substituição do IFRS 6, colocando em discussão dez questões chaves relacionadas ao reconhecimento, mensuração e divulgação dos eventos contábeis relativos às atividades extrativistas. Analisando o modus operandi do IASB para emissão de uma IFRS, por exemplo, é possível identificar que esse processo de influência não se dá apenas pela submissão de cartas comentários. ABSTRACT ABSTRACT This work aims to identify the determining factors in the submission of comment letters to the International Accounting Standards Board (IASB) on the discussion paper Extractive Activities as a lobbying strategy in the context of accounting regulation. The results show that size is a determining factor in all models used, indicating that large oil companies are more likely to lobby. This tendency is especially evident for companies that are predominantly opposed to the IASB proposals, which suggests that the IASB’s review/replacement of International Financial Reporting Standards 6 (IFRS 6) will be a complex process subject to pressure from oil companies to maintain the status quo. Keywords: Lobbying. Accounting regulation. Oil industry. Accounting choices. R. Cont. Fin. – USP, São Paulo, v. 25, n. 65, p. 124-144, maio/jun./jul./ago. 2014 124 * Artigo apresentado no VII Congresso Anpcont, Fortaleza, Brasil, 2013. * Artigo apresentado no VII Congresso Anpcont, Fortaleza, Brasil, 2013. R. Cont. Fin. – USP, São Paulo, v. 25, n. 65, p. 124-144, maio/jun./jul./ago. 2014 124 Lobbying na Regulação Contábil: Evidências do Setor Petrolífero 1 Introdução 1 Introdução Tabela 1 Métodos de participação para influenciar o IASB Métodos de Lobbying Formais Métodos de Lobbying Informais Métodos de Lobbying Direto Submissão de cartas comentários em resposta às audiências públicas para emissão de ◆◆ normas. Participação dos grupos de projetos como consultores. ◆◆ Participação das discussões em mesas redondas públicas. ◆◆ Participação em reuniões pri- ◆◆ vadas ou teleconferência com membros do Board. Métodos de Lobbying Indireto Submissão de comentários aos membros do IFRS ◆◆ Advisory Council, antigo Standards Advisory Council (SAC). Submissão de comentários ao ◆◆ European Financial Reporting Advisory Group (EFRAG). Intermediação via auditor ◆◆ externo. Intermediação via entidades de ◆◆ classe. Fonte: Orens, Jorissen, Lybaert, e Tas (2011). Fonte: Orens, Jorissen, Lybaert, e Tas (2011). probabilidade de utilizar outros métodos complementares do que companhias que não o faziam (Georgiou, 2004). Assim, sabendo-se que na prática se trata de conceito muito mais abrangente, opta-se, por restrições metodoló- gicas, como estratégia de lobbying aquela referente à uti- lização de cartas comentários em resposta às audiências públicas dos órgãos normatizadores. A escolha do setor petrolífero e, consequentemente, das normas contábeis a ele aplicáveis, decorre do fato de as evi- dências históricas trazidas por Collins, Dent, e O’Connor (1978); Solomons (1978); Deakin (1979); Collins, Rozeff, e Salatka (1982); Deakin (1989); Zeff (2005); King (2006), dentre outros, revelarem que o processo de regulação con- tábil norte-americano aplicável ao setor petrolífero decor- reu de um forte sistema de lobbying, pressões e influência sobre a SEC e FASB. Esse fato também foi observado no âmbito do IASB, por ocasião da emissão do IFRS 6, como mostram as evidências trazidas por Cortese, Irvine, e Kai- donis (2010) de que o órgão internacional foi capturado pelos regulados, resultando em uma norma que manteve o status quo (escolha livre do método contábil), atendendo claramente aos interesses das empresas petrolíferas. Em defesa da utilização das cartas comentários, Asekomeh et al. (2006) argumentam que, diferentemente das respostas dadas a perguntas feitas em pesquisas que se utilizam de ques- tionários, que podem ser enviesadas/tendenciosas, os comen- tários às questões colocadas em audiência pública e respon- didas via carta comentário mostram de forma mais fidedigna as ações de lobbying, contribuindo significativamente para o entendimento das posições de cada agente do processo. 1 Introdução Ainda na direção da utilização das cartas comentários, Georgiou (2004) investigou a efetividade dos métodos de lobbying não apenas por meio de cartas comentários, e os resultados mostraram que, mesmo utilizando outros méto- dos, como intermediação da firma de auditoria e encontros com membros da ASB (U.K’s Accounting Standards Board), essas estratégias estavam associadas significativamente ao uso de cartas comentários. O autor concluiu que compa- nhias que submetiam cartas comentários tinham maior Nesse contexto, com a emissão do DPEA, tem-se no- vamente um cenário para se observar o lobbying exercido pelas empresas petrolíferas no que se refere à normatização contábil do setor no âmbito do IASB, motivo pelo qual este estudo ganha importância e se fundamenta. 1 Introdução Diversas são as estratégias de lobbying possíveis, como as listadas a seguir: (a) participações em reuniões com membros do board ou do staff, sejam formais ou informais, presenciais ou à distância (videoconferência); (b) participação em seções públicas (mesas redondas) ou visitas de campo (outreach) realizadas durante o perío- do de audiência estabelecido; No que tange ao processo de regulação contábil, recorre- se à teoria econômica da regulação e às proposições de Watts e Zimmerman (1978) de que os agentes possuem incentivos econômicos para influenciar os órgãos reguladores, via lo- bbying, com o objetivo de obter normas que atendam a seus interesses. Com isso, busca-se responder a seguinte questão de pesquisa: quais os fatores determinantes à realização de lobbying na regulação contábil do setor petrolífero, tendo por base a estratégia de submissão de cartas comentários? (c) indicação de membros para a fundação mantenedo- ra e seus diversos organismos, como o board, comitês técnicos ou de assessoria, a exemplo do IFRS Advisory (c) indicação de membros para a fundação mantenedo- ra e seus diversos organismos, como o board, comitês técnicos ou de assessoria, a exemplo do IFRS Advisory Council, do IFRS Interpretation Committee e do novís- simo Accounting Standards Advisory Forum (ASAF) Council, do IFRS Interpretation Committee e do novís- simo Accounting Standards Advisory Forum (ASAF) – fórum consultivo em que se busca a contribuição de seus membros para o desenvolvimento de padrões nor- mativos aceitos globalmente e de elevada qualidade ou, ainda, para os diversos grupos de trabalhos; O objetivo consiste em identificar os fatores determi- nantes da adoção de estratégias de lobbying sobre a regu- lação contábil do setor petrolífero de modo que se possam avaliar as características marcantes que levam o grupo de interesse formado pelos preparadores de demonstrações fi- nanceiras de empresas petrolíferas a realizar essa atividade, ainda, para os diversos grupos de trabalhos; (d) financiamento para as mantenedoras dos órgãos re- guladores; (e) utilização de firmas de auditorias e entidades de classe como mediadoras junto ao board; R. Cont. Fin. – USP, São Paulo, v. 25, n. 65, p. 124-144, maio/jun./jul./ago. 2014 125 Odilanei Morais dos Santos e Ariovaldo dos Santos ma que demonstra um conjunto de estratégias de lobbying que podem ser utilizadas durante os diferentes estágios do proces- so de emissão das normas pelo IASB, conforme Tabela 1. 2 Fundamentação Teórica Assim, admitindo que o processo seja político, de acordo com Solomons (1978), as normas contábeis não estariam pautadas necessariamen- te por questões teóricas ou técnicas, mas sim pelos diferen- tes interesses das partes envolvidas que exercerem pressão para obter ganhos em particular. Segundo Kothari, Ramanna, e Skinner (2010), os reguladores são dotados de uma ideologia própria e as normas seriam fruto de um conjunto de ideologias e dos efeitos proporcionados pela pressão (lobby) reali- zada por alguns grupos de interesse junto ao regulador. Nessa concepção, o lobby não é visto como uma atitude ilegal ou imoral, mas um mecanismo utilizado pelo re- gulador para se informar sobre as práticas e políticas adotadas pelas empresas. Sob a hipótese dos custos políticos, os autores estabe- lecem que grandes empresas tendem a usar métodos con- tábeis para reduzir os lucros mais frequentemente do que pequenas empresas, sendo o “tamanho” um estimulador de atenção política que a empresa recebe. O pressuposto é o de que lucros mais elevados podem atrair atenção adversa de órgãos reguladores, entidades de classe, imprensa, ambien- talistas, grupos de defesa dos consumidores etc. (Watts & Zimmerman, 1986). As modelagens de diversas pesquisas que tiveram como objeto de estudo o lobbying na regulação contábil podem ser vistas como um subconjunto de pesquisas sobre esco- lhas contábeis (Francis, 1987; Kenny & Larson, 1993), uma vez que as atividades de lobbying referem-se apropriada- mente a uma das dimensões das escolhas contábeis, con- forme Francis (2001). Watts e Zimmerman (1978, 1986), em consonância com a teoria econômica da regulação (Stigler, 1971; Peltzman, 1976; Posner, 1974; Becker, 1983), argumen- tam que a regulação é feita de acordo com os interesses dos grupos que forem mais efetivos politicamente em convencer o regulador/normatizador a agir em bene- fício deles. Sendo uma escolha contábil, conforme Fields, Lys, e Vicent (2001), qualquer decisão cujo propósito primário seja influenciar (na forma ou na substância) as saídas do sistema contábil de um modo particular, a teoria das escolhas contábeis se conecta com a teoria dos grupos de interesses pelo fato de as políticas contábeis serem estabelecidas tendo por base os diversos incentivos eco- nômicos existentes e que tais incentivos também se fa- zem presentes no próprio processo de regulação. Assim, os gestores realizam lobbying para tentar influenciar o órgão normatizador na origem da elaboração da norma, ou seja, para obter uma regulação que atenda a seus in- teresses no seu nascedouro. 2 Fundamentação Teórica Alinhando as escolhas contábeis com o processo de regulação contábil, os gestores teriam incentivos econômi- cos para realizar lobbying contra ou a favor de uma regu- lamentação contábil de modo a tentar influenciar o órgão emissor a optar por modelos contábeis que lhes permitam, por exemplo: reduzir ou diferir o pagamento de tributos; diminuir os custos políticos e a produção de informações (divulgação); ou aumentar o recebimento de bônus. Watts e Zimmerman (1978) testaram empiricamente as hipóteses formuladas utilizando análise discriminante em uma amostra com 52 cartas comentários submetidas por empresas ao Discussion Memorandum da norma contábil sobre os efeitos das mudanças no nível geral de preços do FASB e concluíram que os gestores possuem incentivos econômicos para participarem do processo de elaboração das normas realizando lobbying junto ao regulador. Watts e Zimmerman (1986) definiram três hipóteses para os motivos das escolhas contábeis e que também podem ser entendidas no processo de regulação con- tábil: (1) hipótese da remuneração, na qual os gestores teriam incentivos para realizar escolhas que maximizem suas remunerações; (2) hipótese do endividamento, em que os gestores tendem a fazer escolhas que evitem a violação das cláusulas de empréstimo; e (3) hipótese do custo político, pelo qual os gestores fazem suas escolhas contábeis para evitar a visibilidade política da empresa perante a sociedade. y g j g A partir dos estudos de Watts e Zimmerman (1978), diversas outras pesquisas foram conduzidas sob a temá- tica do lobbying na regulação contábil. Francis (1987) e, posteriormente, Ndubizu, Choi, e Jain (1993) inves- tigaram o processo de emissão da norma SFAS 87 (Con- tabilidade dos Fundos de Pensão dos Empregados). Na primeira pesquisa, Francis (1987) formulou um modelo logit e o empregou numa amostra composta por 218 em- presas que submeteram cartas comentários ao Prelimina- ry Views do SFAS 87 (empresas lobistas) mais 582 outras empresas (as não lobistas). 2 Fundamentação Teórica (1974), Peltzman (1976) e Becker (1983). (1974), Peltzman (1976) e Becker (1983). A teoria da regulação, conforme Viscusi, Harrington, e Vernon (2005), pode ser vista sob três prismas: a teoria do interesse público; a teoria da captura; e a teoria econômi- ca da regulação ou teoria dos grupos de interesse. Para os propósitos deste estudo, este se enquadra no prisma da te- oria econômica da regulação ou dos grupos de interesse. No contexto dessa teoria, a regulação é feita para aten- der aos interesses dos grupos que forem mais politicamen- te efetivos em convencer o regulador a agir em benefício deles. Watts e Zimmerman (1978, 1986), em consonância com Stigler (1971), Peltzman (1976) e Posner (1974), assu- mem que os indivíduos agem para maximizar sua própria utilidade e, sendo assim, o processo de regulação (normati- zação) contábil é resultado de um processo político em que os indivíduos e grupos competem entre si por transferência de riqueza em seu próprio interesse. O pressuposto básico da teoria econômica da regula- ção corresponde ao fato de que a regulação é exercida de forma a atender as necessidades e o bem-estar do grupo de interesse que exercer maior pressão relativa sobre o normatizador (Viscusi, Harrington, & Vernon, 2005). Em função desse pressuposto, a teoria também é conhecida como teoria dos grupos de interesse, tendo como expo- entes principais os pesquisadores Stigler (1971), Posner Stigler (1971) e Peltzman (1976) argumentam que a teo- ria econômica no processo político foca nos incentivos aos indivíduos para se unirem em grupos com o objetivo de re- R. Cont. Fin. – USP, São Paulo, v. 25, n. 65, p. 124-144, maio/jun./jul./ago. 2014 126 Lobbying na Regulação Contábil: Evidências do Setor Petrolífero sujeitos a usar métodos contábeis que aumentem os lucros. O pressuposto é o de que quanto maior o grau de endi- vidamento, mais sufocada a empresa estará pelas restri- ções e condições impostas pelos credores. E, ainda, quanto mais rigorosas forem as restrições impostas pelos credores, maior será a probabilidade de a empresa violar as restri- ções. Assim, os gestores, aumentando os lucros, acabam por relaxar as restrições impostas pelos credores (Watts & Zimmerman, 1986). alizar lobby (influenciar reguladores) visando à transferên- cia de riquezas em interesse próprio. 2 Fundamentação Teórica A teoria institucional coloca as organizações dentro de um cenário social e explicitamente reconhece as influên- cias e interações do ambiente social externo nas atividades internas das organizações, as quais buscam a legitimidade ou a se manterem aceitas dentro do ambiente social (Kenny & Larson, 1993). Com a pesquisa desenvolvida por Deakin (1989), buscou-se estudar o processo de normatização contábil do setor petrolífero junto ao FASB. O autor utilizou uma amostra com empresas que adotavam o método da capi- talização total apenas, uma vez que essas seriam as mais afetadas pela proposta do FASB. Para definir a variável dependente, o autor classificou as empresas em lobistas (apresentaram comentário ao Discussion Memorandum ou ao Exposure Draft do FASB) e em não lobistas (não apresentaram qualquer comentário). Utilizou o modelo de regressão logística para testar as hipóteses relaciona- das à existência de cláusulas contábeis restritivas (cove- nant) e planos de compensação gerencial, as quais foram comprovadas empiricamente. Nesse contexto, organizações como o IASB buscam le- gitimidade por parte de seus constituintes para sobreviver, sendo a solicitação de comentários sobre os seus produ- tos (normas contábeis) diretamente às partes interessadas um dos caminhos seguidos nessa busca por legitimidade (Kenny & Larson, 1993). Consequentemente, os diversos grupos de interesses, também na busca por legitimidade e reconhecimento por parte dos seus pares, podem participar livremente do devi- do processo de emissão de normas desses órgãos, mesmo nos casos em que eles não sejam diretamente afetados por uma proposta normativa (Chatham et al., 2010). Destaque ainda para a pesquisa de Georgiou e Roberts (2004) que investigaram o comportamento das empresas em relação ao lobbying promovido sobre a norma “tribu- tos diferidos” do UK’ ASB. Os resultados indicaram que tamanho e o comportamento da empresa em audiências públicas passadas (frequência em realizar lobbying) foram os fatores chaves determinantes para a decisão de fornecer comentários ao UK’ ASB sobre tributos diferidos. Considerando que o processo de emissão de normas é um processo político, as organizações como o IASB pre- cisam continuamente monitorar as necessidades e influ- ências de seus constituintes para ajustar suas operações visando acomodar as necessidades demandadas do am- biente externo, alinhando-as às suas próprias necessida- des (Kenny & Larson, 1993; Larson & Kenny, 2011). 2 Fundamentação Teórica O autor formulou hipóteses para “tamanho”, “dívida” (como proxy para os efeitos da norma no balanço patrimonial) e “despesas com fundo de pensão” (como proxy para os efeitos da norma na de- monstração do resultado) e os resultados indicaram que não apenas o “tamanho” é importante na decisão de in- fluenciar o órgão normatizador, mas também a possibi- De forma mais explícita, a hipótese da remuneração, ou do plano de incentivo, estabelece que os gestores que recebem remuneração variável (bônus, ações ou opções de ações) usarão frequentemente métodos contábeis que aumentem o resultado da empresa no período e, dessa forma, a remuneração variável recebida, ao antecipar lucros de períodos futuros para o atual (Watts & Zim- merman, 1986). Em relação à hipótese do endividamento, Watts e Zim- merman (1986) argumentam que os gestores de empresas que possuem graus de endividamento maiores estão mais R. Cont. Fin. – USP, São Paulo, v. 25, n. 65, p. 124-144, maio/jun./jul./ago. 2014 127 Odilanei Morais dos Santos e Ariovaldo dos Santos lidade de a nova norma causar efeitos negativos adversos nas demonstrações contábeis. da credibilidade perante os agentes externos à organi- zação (Fogarty, 1992). da credibilidade perante os agentes externos à organi- zação (Fogarty, 1992). Ndubizu et al. (1993) investigaram a etapa do Expo- sure Draft do SFAS 87 do FASB, utilizando uma amostra com 1.802 empresas, sendo 156 empresas lobistas e que se manifestaram contrárias ao Exposure Draft e outras 1.646 empresas (as não lobistas). Os resultados de Ndu- bizu et al. (1993) divergiram daqueles apresentados por Francis (1987), uma vez que apenas a “volatilidade dos lucros”, novidade em relação ao estudo anterior, mos- trou-se relevante para explicar a decisão de apresentar comentários ao Exposure Draft, confirmando a hipótese formulada de que empresas com alta volatilidade nos lu- cros pré-adoção de uma nova norma teriam maior pro- babilidade de realizar lobbying. Tavares, Paulo, Anjos, e Carter (2013) recorrem à afirmação de Riahi-Belkaouri (2004) que diz que a promulgação de um padrão é uma escolha social que força os reguladores a adotar um processo político com o objetivo de encontrar acomodações para os diversos interesses, inclusive o seu próprio, ou seja, alinhado à teoria institucional, o regulador é motivado a adotar estratégias visando à manutenção do seu poder, da cre- dibilidade perante a comunidade ou da reeleição dos seus membros. 2 Fundamentação Teórica A teoria institucional, de acordo com Bengtsson (2011), tem sido usada em pesquisas sobre a emissão de normas contábeis para complementar os pressupostos da economia política, de onde decorre a teoria econômica da regulação, com foco no entendimento de como as pressões exercidas influenciam a adoção das normas contábeis. Os resultados indicam também que empresas que apre- sentaram comentários contrários à proposta de norma são as que, provavelmente, apresentam contratos com cove- nants, enquanto aquelas com posicionamentos favoráveis apresentaram maior probabilidade de não possuírem con- tratos com covenants. Tendo em vista que este estudo tem por base um documento para discussão (DPEA) e, por isso, não sen- do possível saber ainda a posição final do IASB sobre quais opiniões foram aceitas ou rejeitadas, para a com- preensão das acomodações realizadas, os pressupos- tos da teoria institucional aparecem neste estudo para complementar o entendimento de que a participação no processo de audiência pública (lobbying) é legítima e que as empresas buscam fazer valer no processo de regulação as suas escolhas contábeis, retornando à teo- ria econômica da regulação com vista a buscar identifi- car fatores que levam as empresas a participarem desse processo. Por fim, as evidências mostram que empresas possui- doras de planos de compensação gerencial têm maior pro- babilidade de oferecer comentários favoráveis à proposta colocada em audiência pública, enquanto que as empresas contrárias à minuta de norma em discussão seriam aque- las com maior probabilidade de não possuírem planos de compensação gerencial (Georgiou & Roberts, 2004). Mais recentemente, o processo de lobbying na regu- lação contábil também tem sido estudado sob a pers- pectiva da teoria institucional (Bengtsson, 2011; Cha- tham, Larson, & Vietze, 2010; Fogarty, 1992; Giner & Arce, 2012; Kenny & Larson, 1993; Koh, 2011; Larson, 2002, 2008; Larson & Kenny, 2011), a qual sugere que as ações das organizações devem ser entendidas como a busca por legitimidade na sociedade ou manutenção Um resumo dos principais estudos sobre o tema é apre- sentado na Tabela 2. R. Cont. Fin. – USP, São Paulo, v. 25, n. 65, p. 124-144, maio/jun./jul./ago. 2014 128 Lobbying na Regulação Contábil: Evidências do Setor Petrolífero Tabela 2 Pesquisas sobre lobbying na regulação contábil Pesquisa Objetivo MacArthur (1988) Investigar se a submissão de comentários ao UK’ ASB (28 propostas) tem relação com qualquer questão econômica (im- pacto nos números contábeis) ou política (hipótese dos custos políticos). 2 Fundamentação Teórica Fogarty (1992) Analisar a existência e o processo operativo do FASB por meio da teoria institucional. Kenny e Larson (1993) Investigar as atividades de lobbying ao Exposure Draft sobre joint venture. Meier, Alam, e Pearson (1993) Investigar o lobbying realizado pelas firmas de auditoria em sete propostas de normas com efeito sobre os bancos e sobre associação de crédito e empréstimos. Tutticci, Dustan, e Holmes (1994) Entender o devido processo de emissão de normas do Australian Accounting Standard-setting. Para tanto, utilizam a análise de conteúdo do exposure drafts relacionada ao Ativo Intangível. Weetman, Davie, e Collins (1996) Procura responder as questões como: (a) As estratégias dos lobistas podem ser identificadas? (b) Quais as possíveis razões para não se apresentar comentários pelas empresas não lobistas? Tandy e Wilburn (1996) Investigar a participação da comunidade acadêmica no processo de emissão das normas, analisando as cartas submetidas aos Discussion Memorandums e Exposure Drafts para os SFAS 1 a 117. Larson (1997) Investigar as características dos respondentes e analisar se existiam diferenças significativas entre as empresas lobistas e não lobistas no que se refere ao tamanho da empresa, país de origem do respondente (13 países) e mercado de negociação das ações (norte-americano). McLeay, Ordelheide, e Young (2000) Examinar o impacto das atividades de lobbying na regulação contábil alemã, tendo em vista a posição dos preparadores, auditores e acadêmicos. Hill, Shelton, e Stevens (2002) Investigar as atividades de lobbying no processo de emissão da norma SFAS 123 – Contabilização dos Pagamentos Basea- dos em Ação. Larson (2002) Estabelece, com base na teoria institucional, uma estrutura para avaliar se o Standing Interpretations Committee (SIC) tem sido efetivo e legítimo. Georgiou (2004) Definir a estratégia de lobbying mais favorável, se mais cedo (formação da agenda de discussão do board) ou mais tarde (período de audiência pública da minuta de norma). Georgiou (2005) Capturar a tendência na estratégia de lobbying pelas empresas numa perspectiva de longo prazo, analisando uma série de eventos (emissão de várias normas) ao longo do tempo. Larson (2008) Investigar se as pressões políticas ao invés das discussões eminentemente técnicas afetaram decisivamente a emissão do padrão contábil SIC 12, que trata da consolidação das empresas de propósitos específicos. Fundamenta-se na teoria institucional. 2 Fundamentação Teórica 2014 129 Odilanei Morais dos Santos e Ariovaldo dos Santos o objetivo deste trabalho, o qual envolve estabelecer os fatores determinantes que levaram as empresas petrolí- feras a oferecer comentários ao DPEA do IASB, ou seja, fazer lobbying. Das pesquisas comentadas, destaque para os estu- dos de Watts e Zimmerman (1978), Francis (1987), Deakin (1989), Ndubizu et al. (1993) e Georgiou e Ro- berts (2004) por se alinharem metodologicamente com 3 Metodologia meh et al. (2006), Chung (1999), Sutton (1984) e Georgiou (2004), e esse é o caminho adotado nesta pesquisa. Para que se possam identificar os determinantes da ati- vidade de lobbying na regulação contábil do setor petrolífero são utilizadas as estratégias de pesquisa descritas a seguir. Dessa forma, e tendo por base o tipo de regressão uti- lizada, a variável dependente apresenta as características constantes da Tabela 3. Com base nas 141 cartas comentários disponibilizadas pelo IASB em seu site na internet, primeiramente é reali- zada uma análise de conteúdo das cartas submetidas pelas empresas petrolíferas com vistas à mensuração da variável dependente utilizada nos modelos econométricos. Tabela 3 Características da variável dependente Regressão Logística Binominal Regressão de Poisson Intensidade Favorabilidade Intensidade Desfavorabilidade Código Frequência Código Frequência Código Frequência 0 127 0 127 0 127 1 25 1 0 1 2 Regressão Logística Multinominal 2 3 2 2 3 3 3 3 Código Frequência 4 9 4 5 0 127 5 4 5 3 1 13 6 2 6 6 2 12 7 1 7 1 8 2 8 3 9 1 9 0 10 0 10 0 Tabela 3 Características da variável dependente Regressão Logística Binominal Regressão de Poisson Intensidade Favorabilidade Intensidade Desfavorabilidade Código Frequência Código Frequência Código Frequência 0 127 0 127 0 127 1 25 1 0 1 2 Regressão Logística Multinominal 2 3 2 2 3 3 3 3 Código Frequência 4 9 4 5 0 127 5 4 5 3 1 13 6 2 6 6 2 12 7 1 7 1 8 2 8 3 9 1 9 0 10 0 10 0 A segunda etapa do estudo está baseada em análises quan- titativas, com a utilização de modelos econométricos para se estabelecer os fatores econômicos determinantes da adoção da estratégia de lobbying via submissão de cartas comentários. Em função das características da variável dependente, utilizam-se as regressões logísticas, binomial e multinomial e a regressão de Poisson. 2 Fundamentação Teórica No primeiro caso, a variável depen- dente assume valores “1” para o caso de se ter oferecido co- mentários ao documento do IASB ou “0” se for o contrário. Na regressão logística multinomial, a variável depen- dente é decomposta em três categorias: “0” para representar as empresas que não ofereceram comentários ao documen- to do IASB; “1” para representar as empresas que ofere- ceram comentários predominantemente favoráveis; e “2” para representar as empresas que ofereceram comentários predominantemente desfavoráveis às proposições contidas no documento do IASB. Em relação às variáveis independentes, o “tamanho”, como visto, tem sido comumente utilizado na literatura sobre lobbying na regulação contábil para expressar a ex- posição da empresa (hipótese do custo político) (Watts & Zimmerman, 1978; Francis, 1987; Deakin, 1989; Ndubizu, Choi, & Jain, 1993; Georgiou & Roberts, 2004). Por fim, emprega-se a regressão de Poisson em que a variável dependente pretende expressar o grau de concor- dância ou discordância em relação às dez questões coloca- das em discussão no documento do IASB. Assim, ela pode assumir valores de zero, para os casos de empresas que não ofereceram comentários, até dez, que representa a quanti- dade máxima de vezes em que a empresa foi favorável ou desfavorável às questões apresentadas no DPEA. O “tamanho” também tem sido associado com a hipó- tese de que uma estratégia de lobbying somente é adotada se os benefícios obtidos forem maiores que os custos do lo- bbying (Sutton, 1984). Nesse sentido, Koh (2011) expressa que grandes empresas, por possuírem mais recursos, usual- mente tendem a influenciar o resultado final e, consequen- temente, colhem favoravelmente os frutos do lobbying. 2 Fundamentação Teórica Chatham, Larson, e Vietze (2010) Analisar os comentários enviados ao discussion paper sobre instrumentos financeiros visando identificar os principais pon- tos da proposta, alinhado ao fato de que a União Europeia rejeitou a adoção integral do IAS 39, exigindo esforço adicional do IASB para reverter a situação. Fundamenta-se na teoria institucional. Georgiou (2010) Investigar a natureza e participação dos fundos de investimentos do Reino Unido nas audiências públicas realizadas pelo IASB no período de 2001 a 2006. Stenka e Taylor (2010) Compreender a complexidade das atividades de lobbying no âmbito de quatro exposure drafts. Os lobistas são classifica- dos em dois grupos: corporativos e não corporativos, e as análises são realizadas com o emprego da ANOVA e regressão univariada. Hansen (2011) Fornecer evidências de como o IASB gera seus padrões normativos na presença de lobistas com preferências distintas. Para tanto, utiliza-se das cartas comentários (629 cartas) de cinco audiências promovidas pelo IASB. Larson e Kenny (2011) Investigar o comportamento do IASB quando da emissão das normas contábeis frente às doações financeiras voluntárias recebidas de seus constituintes. Bengtsson (2011) Utiliza a teoria institucional para investigar como o IASB, após a crise financeira mundial, respondeu às pressões políticas promovidas pela União Europeia como a observada em relação à norma de instrumentos financeiros. Koh (2011) Examinar as características das firmas quanto à decisão de se fazer ou não fazer lobbying. Para aquelas que submeteram opiniões sobre a norma de stock option do FASB, analisou se o lobbying foi a favor ou contra a norma em discussão. Giner e Arce (2012) Analisar o comportamento dos lobistas, tendo por base a teoria institucional, e avaliar a influência destes sobre as decisões do IASB no que se refere à norma IFRS 2. Tavares, Anjo, Paulo, e Carter (2013) Investigar quais foram as opiniões mais frequentes submetidas ao IASB/FASB em relação ao Revised Exposure Draft Reve- nue from Contracts with Customers. Matos, Gonçalves, Niya- a e Mar ues (2013) Investigar se as normas que possuem maior nível de inconsistência com GAAPs locais foram alterados/aprovados de acor- do co a artici ação geográfica do e bro do conselho do IASB R. Cont. Fin. – USP, São Paulo, v. 25, n. 65, p. 124-144, maio/jun./jul./ago. 3.1 Definição das Variáveis e Hipóteses de Pesquisa. Como exposto anteriormente, a variável dependente utilizada como proxy para lobbying refere-se às manifesta- ções explícitas contidas nas cartas comentários submetidas à audiência pública de discussão do DPEA. Trata-se de mo- delagem amplamente utilizada na literatura contábil sobre lobbying, a exemplo de Watts e Zimmerman (1978), Francis (1987), Deakin (1989), Ndubizu et al. (1993) e Georgiou e Roberts (2004). Para operacionalizar a variável tamanho, foram utili- zadas quatro proxies, a saber: receitas líquidas do período (RECLIQ); ativo total do período (ATIVTOT); lucro líqui- do médio dos últimos três períodos (LUCMED); e gastos de exploração e desenvolvimento incorridos no período (UPSTREAM). A hipótese de teste subjacente ao tamanho é definida como: H1: Grandes empresas petrolíferas possuem maior pro- babilidade de realizarem lobbying, favorável ou desfavorá- vel, do que as demais empresas petrolíferas. Tendo em vista que a adoção de outras estratégias para mensuração da variável dependente envolve, na maioria das vezes, informações privadas, o que torna a modelagem de difícil execução (Holthausen & Leftwich, 1983), tem-se uma limitação inerente ao estudo pelo fato de que as estratégias de lobbying estão limitadas às cartas comentários. Diversos são os estudos que defendem essa modelagem, como Aseko- A literatura vigente expõe ainda duas outras variáveis representativas dos incentivos econômicos para as esco- lhas contábeis (no caso, a possibilidade de influenciar uma norma junto a órgãos normatizadores). Pressupõe-se que R. Cont. Fin. – USP, São Paulo, v. 25, n. 65, p. 124-144, maio/jun./jul./ago. 2014 130 Lobbying na Regulação Contábil: Evidências do Setor Petrolífero pelos Estados Unidos, quais sejam, os métodos dos esfor- ços bem sucedidos e da capitalização total. gestores de empresas que possuem compensação remune- ratória baseada em desempenho (medidos com base em in- dicadores derivados das informações contábeis) tenderão a escolher os modelos contábeis para beneficiarem-se, ou seja, optarão por propostas de normas que reflitam mé- todos contábeis que aumentem ou diminuam o lucro, de acordo com o seu interesse. Tal atitude pode ser entendida sob a perspectiva da teoria institucional, notadamente quanto à concepção do isomorfismo mimético, no qual as organizações copiam as práticas umas das outras, particularmente dentro do mes- mo setor, mesmo não sendo obrigadas a seguir normas es- pecíficas (Koh, 2011). A existência de cláusulas contábeis restritivas (cove- nants) em contratos de financiamento e/ou captação de recursos é a outra característica. 3.1 Definição das Variáveis e Hipóteses de Pesquisa. A teoria prega que os ges- tores tenderão a escolher modelos contábeis que levem a não violação dessas cláusulas restritivas, ou seja, quanto mais próxima a empresa estiver de um limite fixado em um covenant, baseado em números contábeis, maior será a probabilidade de o administrador utilizar procedimentos que aumentem o resultado e/ou reduzam o nível de endivi- damento (Holthausen & Leftwich, 1983). Com isso, a adoção dos métodos dos esforços bem su- cedidos ou da capitalização total é uma realidade mundial, inclusive para facilitar as empresas não norte-americanas a publicarem suas demonstrações financeiras naquele país por ocasião da listagem da empresa para negociação de suas ações na bolsa de valores de Nova Iorque, por exemplo. A utilização da variável USLISTING teve por objetivo capturar o risco da possível mudança na regulação contá- bil sob duas perspectivas: uma considerando as empresas norte-americanas e outra considerando as empresas es- trangeiras, mas com ações negociadas no mercado norte- americano. Para operacionalizar essas duas características, são uti- lizadas as variáveis binárias COMPGEN e COVENANT, que assumem valor “0” para os casos em que a empresa não apresente a característica de interesse, e valor “1” para os casos em que a empresa possua a característica testável. Utilizaram-se diretamente as notas explicativas das de- monstrações financeiras das empresas disponíveis em seus websites para mensurar essas variáveis, pesquisando refe- rências à existência ou não de compensação gerencial e de cláusulas restritivas. No primeiro caso, mesmo que não se tenha um hori- zonte claro de que os Estados Unidos venham a adotar as normas internacionais (IFRS) para as empresas estaduni- denses, evidências mostram que a participação dos norte- americanos no processo de emissão de normas pelo IASB é relevante, chegando, em alguns casos, a serem os mais par- ticipativos nas audiências públicas (Larson, 1997; Larson & Kenny, 2011; Giner & Arce, 2012). Reconhece-se que é possível operacionalizar essas variá- veis de diversas maneiras e assume-se a atribuição binária de “0” ou “1” para essas variáveis a partir da existência ou não da característica testável como uma limitação da pesquisa. Isso se torna relevante pelo fato de que as variáveis binárias, da forma como foram operacionalizadas, podem não capturar a magnitude da característica que se quer investigar de maneira apropriada. Deakin (1989), Georgiou e Roberts (2004), Ge- orgiou (2005) e Koh (2011), por exemplo, apresentam outros caminhos para se operacionalizar essas variáveis. 3.1 Definição das Variáveis e Hipóteses de Pesquisa. No presente estudo, das 141 cartas recebidas pelo IASB na audiência pública do DPEA, 27 cartas (ou 19%) foram de interessados norte-americanos, atrás apenas dos euro- peus, que submeteram 76 cartas. Analisando o processo de emissão do IFRS 2, por exem- plo, Giner e Arce (2012) evidenciam que, das 539 cartas recebidas pelo IASB, 264 foram encaminhadas por norte- americanos e argumentam que essa forte participação no devido processo de emissão de normas pelo IASB é mo- tivada por receios quanto à possibilidade de mudança na posição do FASB/SEC em relação à adoção das IFRS para as empresas nativas (Giner & Arce, 2012). As hipóteses testáveis correspondem a: As hipóteses testáveis correspondem a: H2: Empresas petrolíferas que apresentem planos de compensação gerencial possuem maior probabilidade de realizar lobbying, favorável ou desfavorável, do que empre- sas petrolíferas que não possuem planos de compensação gerencial. Sob a perspectiva das empresas não estadunidenses e que adotam as IFRS, tem-se que mudanças nas normas in- ternacionais podem elevar os custos de transação, neces- sitando de ajustes e informações adicionais para atender as necessidades dos investidores norte-americanos, como argumentam Larson (1997) e Georgiou (2005). Com isso, essas empresas teriam incentivos para fazer lobbying junto ao IASB visando à manutenção da situação vigente. H3: Empresas petrolíferas que apresentem cláusulas restritivas possuem maior probabilidade de realizarem lo- bbying, favorável ou desfavorável, do que empresas petrolí- feras que não possuem cláusulas restritivas. Visando capturar o risco de possíveis mudanças na re- gulação contábil do setor petrolífero, também são conside- radas no modelo de teste as variáveis USLISTING e MÉ- TODO. A atual regulação contábil internacional aplicável às atividades extrativistas baseia-se na norma IFRS 6. Tal padrão estabelece que as empresas extrativistas podem de- finir sua política contábil livremente. Na prática, o IFRS 6 não definiu nenhum método específico de reconhecimen- to e mensuração e as empresas sujeitas às normas interna- cionais acabaram por escolher entre um dos dois métodos mais conhecidos mundialmente e que foram estabelecidos Dessa forma, considera-se que as empresas petrolíferas norte-americanas ou aquelas que possuem ações negociadas no mercado norte-americano possuem incentivos para rea- lizar lobbying junto ao IASB visando à manutenção do status quo, ou seja, em que se pode escolher entre os métodos dos esforços bem sucedidos ou da capitalização total para, dessa forma, evitar-se possíveis custos de transação com a conver- gência para novas regras. 3.1 Definição das Variáveis e Hipóteses de Pesquisa. A hipótese de teste corresponde a: H4: Empresas petrolíferas listadas no mercado norte- americano possuem maior probabilidade para realizar lo- H4: Empresas petrolíferas listadas no mercado norte- americano possuem maior probabilidade para realizar lo- R. Cont. Fin. – USP, São Paulo, v. 25, n. 65, p. 124-144, maio/jun./jul./ago. 2014 131 Odilanei Morais dos Santos e Ariovaldo dos Santos bbying desfavorável, do que empresas petrolíferas não lista- das no mercado norte-americano. a possibilidade de escolha por parte das empresas petro- líferas ao propor um método único de reconhecimento e mensuração e, dessa forma, as empresas, na concepção de Zeff (2002), teriam incentivos para realizar lobbying junto ao IASB visando manter o status quo. Tal incentivo também encontra respaldo em Watts e Zimmerman (1978, 1986), na medida em que preparadores levam em conta o poten- cial impacto de um novo tratamento contábil na expectati- va futura de fluxo de caixa para decidir sobre o lobbying. A proposta colocada em discussão pelo IASB no DPEA estabelece um novo método de reconhecimento e mensu- ração aplicável às atividades extrativistas e elimina a possi- bilidade de escolha por parte das empresas entre os méto- dos dos esforços bem sucedidos ou da capitalização. A proposta do IASB estabelece que os direitos legais para exploração dos recursos naturais é o ponto chave para o re- conhecimento do ativo e que os demais gastos necessários para confirmação dos recursos e garantir sua exploração co- mercial seriam extensões desse direito legal. Com essa pro- posta, tem-se um cenário totalmente diferente do ambiente atualmente institucionalizado e que compreende os métodos dos esforços bem sucedidos e da capitalização total. Como analisado em Santos, Lopes, e Silva (2010), empresas que seguem o método dos esforços bem sucedidos seriam as mais afetadas pela proposta apresentada pelo IASB no DPEA, com mudanças significativas em suas políticas contábeis. As- sim, espera-se que essas empresas tenham maior probabilida- de de realizar lobbying contrário à proposta do IASB. Zeff (2002) argumenta que, quando o normatizador prescreve uma nova norma que elimina a possibilidade de escolha ou impõe exigências para divulgações adicionais, isto se torna um gatilho para que os constituintes realizem pressões junto ao normatizador para fazer valer as suas ne- cessidades. Esse argumento também é encontrado em Sa- emann (1999), que complementa argumentando que, em geral, esse cenário leva os interessados a se oporem às pro- postas do normatizador. 3.1 Definição das Variáveis e Hipóteses de Pesquisa. Dessa forma, a variável MÉTODO presta-se a investigar se existe associação entre a escolha do método dos esforços bem sucedidos (identificado como “1”) e a probabilidade de se oferecer comentários contrários ao documento do IASB. A hipótese testável corresponde a: H5: Empresas petrolíferas que seguem o método dos esforços bem sucedidos possuem maior probabilidade de realizar lobbying desfavorável junto ao IASB do que as empresas petrolíferas que seguem o método da capitali- zação total. H5: Empresas petrolíferas que seguem o método dos esforços bem sucedidos possuem maior probabilidade de realizar lobbying desfavorável junto ao IASB do que as empresas petrolíferas que seguem o método da capitali- zação total. Tavares et al. (2013) explicam que regular é restringir as opções de escolhas contábeis na medida em que o regula- dor tem o poder de decidir sobre as políticas contábeis que os regulados devem seguir. LOBBYINGi = + u μLOBBYINGe - α1 + β1RECLIQi + β2ATIVTOTi + β3LUCMEDi + β4UPSTREAM + β5COMPGENi + β6COVENANTi + β7USLISTINGi + β8METODOi LOBBYING! LOBBYINGi = + μLOBBYINGe - α1 + β1RECLIQi + β2ATIVTOTi + β3LUCMEDi + β4UPSTREAM + β5COMPGENi + β6COVENANTi + β7USLISTINGi + β8METODOi LOBBYING! LOBBYINGi = ram ser aproveitadas em função da indisponibilidade das informações necessárias ao estudo. Em que LOBBYING assume valores que variam de “0” a “10”, sendo zero os casos de empresas que não oferece- ram comentários ao DPEA do IASB e dez o valor máximo possível de concordância (ou discordância) em relação ao documento do órgão internacional. Recorreu-se à base de dados da Evaluate Energy®, em- presa de consultoria líder no fornecimento de informações sobre o setor de óleo e gás. Como o DPEA ficou em audiên- cia pública no período de abril a julho de 2010, utilizaram- se as informações disponíveis do ano de 2009 e, para esse ano, a base de dados da Evaluate Energy® contava com in- formações de 262 empresas do setor petrolífero. Para exemplificar a utilização desses três modelos, su- ponha-se uma empresa que tenha oferecido comentários ao DPEA, sendo que das 10 perguntas apresentadas, 7 ob- tiveram respostas discordantes e 3 foram respondidas com comentários favoráveis. No primeiro modelo (regressão logística binomial), a empresa será codificada como “1” (ofereceu comentários); no modelo de regressão logística multinomial, receberá o código “2” (posição predominan- temente desfavorável) e, por fim, na regressão de Poisson será codificada como “3” no painel “lobbying favorável” e “7” no painel “lobbying desfavorável”. Como da base de dados da Evaluate Energy® não constam informações necessárias para operacionalizar as variáveis COMPGEN e COVENANT, optou-se por realizar uma se- leção aleatória dentre as 237 empresas da base de dados (ex- cluindo as 25 já pré-selecionadas e que representam as empre- sas lobistas) visando a escolha das empresas não lobistas. Para a definição do tamanho da amostra, visando à se- leção aleatória, utilizou-se a expressão, n = N x n0 / N + n0 em que n0 = 1 / E0 , sendo n0 = primeira aproximação do tamanho da amostra; E0 = erro amostral tolerável; N = tamanho da população alvo; e n = tamanho da amostra. As- sim, para uma população alvo (N) de 237 empresas e erro amostral tolerável (E0) de 6%, chegou-se a 127 empresas que, somadas às 25, resultaram em uma amostra final com 152 empresas petrolíferas. 2 2 2 3.2 Especificação dos Modelos Econométricos. Do exposto, chega-se aos seguintes modelos econo- métricos: Nesse contexto, a proposta contida no DPEA elimina Em que i denota empresa, e LOBBYING assume valor “1” nas empresas que ofereceram comentários ao DPEA e “0” para os casos de empresas que não ofereceram comen- tários. O termo de erro da regressão é indicado pelo parâ- metro ε; α é o intercepto e β1, β2, β3,..., β8 são os coeficientes estimados, dos quais se espera que sejam estatisticamente significantes e positivos. análise dos resultados reside nos coeficientes das variáveis independentes (β), que precisam ser estatisticamente rele- vantes e está baseada na ideia de que quando o coeficiente é maior que zero, maior é a probabilidade de ocorrência do evento de interesse (lobbying ao documento do IASB) e vice-versa. Em relação à regressão logística multinomial, tem-se a seguinte especificação: De acordo com Fávero, Belfiore, Silva, e Chan (2009), a ln = PROB (LOBBYING = 1\|X) PROB (LOBBYING = 0\|X) α1 + β1RECLIQi + β2ATIVTOTi + β3LUCMEDi + β4UPSTREAM + β5COMPGENi + β6COVENANTi + β7USLISTINGi + β8METODOi + εi ln = PROB (LOBBYING = 2\|X) PROB (LOBBYING = 0\|X) α1 + β1RECLIQi + β2ATIVTOTi + β3LUCMEDi + β4UPSTREAM + β5COMPGENi + β6COVENANTi + β7USLISTINGi + β8METODOi + εi ln = PROB (LOBBYING = 2\|X) PROB (LOBBYING = 0\|X) α1 + β1RECLIQi + β2ATIVTOTi + β3LUCMEDi + β4UPSTREAM + β5COMPGENi + β6COVENANTi + β7USLISTINGi + β8METODOi + εi Em que LOBBYING assume valores “0” nas empresas que não ofereceram comentários ao DPEA do IASB; “1” nas empresas que apresentaram comentários predominan- temente favoráveis ao documento do IASB; e “2” para os casos em que as empresas apresentaram comportamento predominantemente desfavorável ao documento do IASB. que ofereceram comentários predominantemente favorá- veis com aquelas que não ofereceram comentários e (2) as empresas que ofereceram comentários predominantemen- te desfavoráveis com aquelas que não ofereceram comentá- rios (Fávero, Belfiore, Silva, e Chan, 2009). No modelo de regressão de Poisson, utiliza-se a seguin- te especificação: As análises são realizadas comparando (1) as empresas R. Cont. Fin. – USP, São Paulo, v. 25, n. 65, p. 124-144, maio/jun./jul./ago. 2014 132 Lobbying na Regulação Contábil: Evidências do Setor Petrolífero 3.3 Características da Amostra. Como a amostra deve conter empresas lobistas e empre- sas não lobistas, inicialmente identificaram-se as empresas petrolíferas que participaram da audiência pública do DPEA. O IASB recebeu 141 cartas comentários, sendo que 39 (ou 28%) delas foram submetidas por empresas extrativistas e 33 (23%) por entidades não governamentais. Por fim, o grupo formado pelos emissores nacionais, entidades de classe con- tábil e reguladores do mercado de capitais contribuiu com 25 cartas (18%). Os grupos formados por investidores/usuários individuais, empresas de consultoria do setor/associações profissionais e firmas de auditoria submeteram 17, 12 e 8 cartas comentários, respectivamente. Outras 8 cartas foram submetidas por interessados diversos. A amostra utilizada se caracteriza por conter empresas de diferentes tamanhos, conforme pode ser observado na Ta- bela 4. Por exemplo: em média, a receita líquida (RECLIQ) obtida em 2009 foi (em valores logaritmados) de US$ 8,84 milhões. Contudo, tem-se uma variação considerável entre o valor mínimo de US$ 2,42 milhões e o valor máximo de US$ 12,54 milhões, o que proporciona um desvio padrão de US$ 1,95 milhão. Esse mesmo comportamento é observado nas demais variáveis, a exemplo do lucro líquido médio (LUC- MED), que variou de um valor mínimo de US$ 1,60 milhão para um máximo de US$ 20,7 milhões. Dentre as 39 cartas comentários submetidas ao IASB pelas empresas extrativistas, 28 delas são de empresas pe- trolíferas e 11 de mineradoras. Com isso, focou-se inicial- mente nessas 28 petrolíferas, sendo que 3 delas não pude- Tabela 4 Estatística descritiva das variáveis contínuas "Variável (em US$ milhões)" Média Mediana Mínimo Máximo Desvio Padrão "Teste KS p-valor" RECLIQ 8,84 9,00 2,42 12,54 1,95 0.953 UPSTREAM 0,95 6,34 -20,72 10,08 11,55 0,000 LUCMED 12,79 12,72 1,60 20,70 4,01 0,994 ATIVTOT 9,52 9,60 4,38 12,69 1,58 0,984 Nota: RECLIQ: receitas líquidas do período; UPSTREAM: gastos de exploração e desenvolvimento incorridos no período; LUCMED: lucro líquido médio dos últimos três períodos; ATIVTOT: ativo total do período. Teste KS: Teste de Kolmogorov-Smirnov para normalidade (p-valor > 0,05). Nota: RECLIQ: receitas líquidas do período; UPSTREAM: gastos de exploração e desenvolvimento incorridos no período; LUCME dos últimos três períodos; ATIVTOT: ativo total do período. Teste KS: Teste de Kolmogorov-Smirnov para normalidade (p-valor > Nota: RECLIQ: receitas líquidas do período; UPSTREAM: gastos de exploração e desenvolvimento incorridos no período; LUCMED: lucro líquido médio dos últimos três períodos; ATIVTOT: ativo total do período. Teste KS: Teste de Kolmogorov-Smirnov para normalidade (p-valor > 0,05). 3.3 Características da Amostra. Nota: LOBBYING: “0” se não apresentou comentários e “1” se apresentou comentários; RECLIQ: receitas líquidas do período; UPSTREAM: gastos de explo- ração e desenvolvimento incorridos no período; LUCMED: lucro líquido médio dos últimos três períodos; ATIVTOT: ativo total do período; COVENANT: “0” se não apresenta covenant e “1” se apresenta; COMPGEN: “0” se não apresenta compensação gerencial e “1” se apresenta; MÉTODO: “0” se adota o full cost e “1” se adota o successful efforts; USLISTING: “0” se negocia em outros mercados de capitais e “1” se negocia no mercado norte-americano. Significância: ** (1%) e * (5%). ticolinearidade. Posteriormente, outros modelos são elabo- rados apenas com uma dessas proxies, visando dar robustez às análises realizadas. Em função disso, é elaborado um modelo contendo to- das as proxies para tamanho e se realiza o teste Variance Inflation Factor (VIF) para análise do pressuposto da mul- 3.3 Características da Amostra. Nota: LOBBYING: “0” se não apresentou comentários e “1” se apresen- tou comentários; COVENANT: “0” se não apresenta covenant e “1” se apresenta; COMPGEN: “0” se não apresenta compensação gerencial e “1” se apresenta; MÉTODO: “0” se adota o full cost e “1” se adota o successful efforts; USLISTING: “0” se negocia em outros mercados de capitais e “1” se negocia no mercado norte-americano. Nota: LOBBYING: “0” se não apresentou comentários e “1” se apresen- tou comentários; COVENANT: “0” se não apresenta covenant e “1” se apresenta; COMPGEN: “0” se não apresenta compensação gerencial e “1” se apresenta; MÉTODO: “0” se adota o full cost e “1” se adota o successful efforts; USLISTING: “0” se negocia em outros mercados de capitais e “1” se negocia no mercado norte-americano. A correlação entre as variáveis explicativas permite que sejam feitas inferências a respeito do pressuposto da multicolinearidade. Nesse sentido, percebe-se que as vari- áveis relacionadas a “tamanho” apresentam altos graus de associações significativas, principalmente entre RECLIQ e LUCMED e ATIVTOT, variando de 77% a 89%, sendo um indicativo de possível quebra desse pressuposto. Quanto às variáveis COVENANT e COMPGEN, obser- va-se que as empresas da amostra possuem praticamente a mesma distribuição entre aquelas que possuem cláusulas Tabela 6 Matriz de correlação de Pearson LOBBYING RECLIQ UPSTREAM LUCMED ATIVTOT COVENANT COMPGEN METODO LOBBYING 1 RECLIQ 0,335 1 UPSTREAM 0,245 -0,004 1 LUCMED 0,422 0,767 0,363 1 ATIVTOT 0,400 0,894 0,269 0,882 1 COVENANT 0,070 -0,265 0,148 -0,203 * -0,194 * 1 COMPGEN 0,104 -0,197 * 0,084 -0,181 * -0,181 * 0,445 1 METODO 0,148 0,130 0,363 0,266 0,236 0,031 0,016 1 USLISTING 0,127 -0,236 0,226 -0,066 -0,155 0,412 0,489 0,075 Nota: LOBBYING: “0” se não apresentou comentários e “1” se apresentou comentários; RECLIQ: receitas líquidas do período; UPSTREAM: gastos de explo- ração e desenvolvimento incorridos no período; LUCMED: lucro líquido médio dos últimos três períodos; ATIVTOT: ativo total do período; COVENANT: “0” se não apresenta covenant e “1” se apresenta; COMPGEN: “0” se não apresenta compensação gerencial e “1” se apresenta; MÉTODO: “0” se adota o full cost e “1” se adota o successful efforts; USLISTING: “0” se negocia em outros mercados de capitais e “1” se negocia no mercado norte-americano. Significância: ** (1%) e * (5%). 3.3 Características da Amostra. AM permaneceu com distribuição não normal. Mesmo que o pressuposto da normalidade dos resíduos não seja uma exigência para a regressão logística, optou- se por calcular o logaritmo natural das variáveis contínuas, buscando uma distribuição normal para elas, visando miti- gar possível “efeito escala” nas regressões. Como consta na Tabela 4, após a transformação, apenas a variável UPSTRE- Em relação às variáveis dicotômicas (Tabela 5), das 152 empresas da amostra, 25 ofereceram comentários ao DPEA do IASB, correspondente a 16% da amostra. Essas empresas, para efeito deste estudo, são caracterizadas como empresas lobistas, enquanto as demais empresas da amostra são as não lobistas. R. Cont. Fin. – USP, São Paulo, v. 25, n. 65, p. 124-144, maio/jun./jul./ago. 2014 133 Odilanei Morais dos Santos e Ariovaldo dos Santos Tabela 5 Estatística descritiva das variáveis dicotômicas Variável Valor "0" Valor "1" Total LOBBYING 127 25 152 84% 16% 100% COVENANT 41 111 152 27% 73% 100% COMPGEN 40 112 152 26% 74% 100% METODO 74 78 152 49% 51% 100% US LISTING 64 88 152 42% 58% 100% Nota: LOBBYING: “0” se não apresentou comentários e “1” se apresen- tou comentários; COVENANT: “0” se não apresenta covenant e “1” se apresenta; COMPGEN: “0” se não apresenta compensação gerencial e “1” se apresenta; MÉTODO: “0” se adota o full cost e “1” se adota o successful efforts; USLISTING: “0” se negocia em outros mercados de capitais e “1” se negocia no mercado norte-americano. restritivas (73%) ou compensação gerencial (74%) e aque- las que não possuem esses atributos. Em relação aos métodos contábeis utilizados pelas em- presas da amostra, observa-se que existe equilíbrio, com 51% das empresas seguindo o método successful efforts e 49% o método full cost. Já em relação às empresas que possuem ações negociadas no mercado norte-americano, são maioria na amostra utilizada, com 88 empresas ou 58% da amostra. Quanto à correlação entre as variáveis, é possível ob- servar, na Tabela 6, que as variáveis relacionadas ao “ta- manho” apresentam razoável associação estatisticamente significante com a variável dependente LOBBYING, com correlação variando de 24% a 42%, o que não ocorre em relação às demais, diferentemente do esperado. 4 Análise dos Resultados A relação das questões consta do Anexo 1. Também concordaram, em sua maioria, com as propo- sições relacionadas à aplicabilidade da norma IAS 36 (7ª questão) e de quais deveriam ser os objetivos das demons- trações contábeis elaboradas pelas empresas extrativistas (8ª questão). Pode-se inferir, dessa forma, que o atual modelo de reconhecimento das transações econômicas das empresas petrolíferas, bem como o atual conjunto de informações divulgadas obrigatoriamente parece satisfazer as necessi- dades das empresas petrolíferas, o que implica em conside- rar, tendo por base a teoria institucional, que qualquer evo- lução no status quo vigente terá que ser bem “costurado” pelo IASB com vistas a acomodar as necessidades de seus diversos constituintes, inclusive dos mais afetados, como é o caso das empresas petrolíferas. Contudo, analisando a questão que implica na adoção de um novo e único método contábil, com a eliminação dos métodos dos esforços bem sucedidos e da capitalização total (4ª questão) ou, ainda, as questões que trazem como proposta o aumento do nível de divulgação em nota expli- cativa (9ª e 10ª questões), percebe-se um posicionamento amplamente desfavorável às propostas do IASB. Buscando atingir o objetivo de se encontrar quais as carac- terísticas preponderantes que levaram as empresas petrolífe- ras a submeterem cartas comentários à audiência pública do DPEA, passa-se aos resultados obtidos por meio da utilização das técnicas de análise multivariada de dados. Os resultados da regressão logística estão expressos em cinco regressões (Ta- bela 8), sendo a primeira considerando todas as variáveis do modelo. As demais regressões levam em conta cada uma das variáveis selecionadas para a hipótese do “tamanho”. Os principais argumentos utilizados foram o de que o conceito de ativo contido no DPEA fere a estrutura concei- tual, ao permitir que gastos não relacionados a benefícios econômicos futuros sejam ativados e de que as informações exigidas para divulgação obrigatória eram demasiadas e que os custos de produção de tais informações superariam o benefício atinente à divulgação destas. Olhando o conjunto das questões, percebe-se um quase empate, com 115 opiniões favoráveis às proposições conti- das no DPEA e 118 opiniões contrárias. 4 Análise dos Resultados como base de mensuração das atividades do setor extrativista em oposição à mensuração baseada no valor justo (6ª ques- tão). Importante destacar, nesse ponto, que o grupo de traba- lho argumentou no DPEA que o valor justo seria a melhor representação dos eventos econômicos relacionados e produ- ziria informação mais útil aos usuários. Contudo, a mensura- ção seria complexa e muito subjetiva, restando considerar que o custo histórico deveria, por falta de opção, ser a base utiliza- da pelos preparados das demonstrações financeiras. As empresas petrolíferas que participaram da audiência pública (empresas lobistas no caso deste estudo) se mani- festaram favoravelmente, pode-se dizer, em quatro das dez questões colocadas em discussão pelo IASB (1ª, 6ª, 7ª e 8ª questões), como pode ser visto na Tabela 7. As empresas são amplamente favoráveis à concepção de que o escopo da norma contábil do setor extrativista deveria abranger somente o segmento conhecido como upstream (1ª questão) e foram unânimes em considerar o custo histórico R. Cont. Fin. – USP, São Paulo, v. 25, n. 65, p. 124-144, maio/jun./jul./ago. 2014 134 Lobbying na Regulação Contábil: Evidências do Setor Petrolífero Tabela 7 Análise do posicionamento das empresas ao DPEA QUESTÃO Nº 1 QUESTÃO Nº 2 QUESTÃO Nº 3 Concorda Discorda NR Total Concorda Discorda NR Total Concorda Discorda NR Total 18 2 5 25 9 10 6 25 11 13 1 25 72% 8% 20% 100% 36% 40% 24% 100% 44% 52% 4% 100% QUESTÃO Nº 4 QUESTÃO Nº 5 QUESTÃO Nº 6 Concorda Discorda NR Total Concorda Discorda NR Total Concorda Discorda NR Total 6 19 0 25 12 12 1 25 25 0 0 25 24% 76% 0% 100% 48% 48% 4% 100% 100% 0% 0% 100% QUESTÃO Nº 7 QUESTÃO Nº 8 QUESTÃO Nº 9 Concorda Discorda NR Total Concorda Discorda NR Total Concorda Discorda NR Total 14 9 2 25 13 11 1 25 6 19 0 25 56% 36% 8% 100% 52% 44% 4% 100% 24% 76% 0% 100% QUESTÃO Nº 10 RESUMO MANUTENÇÃO DO STATUS QUO Concorda Discorda NR Total Concorda Discorda NR Total SIM NÃO NR Total 1 23 1 25 115 118 17 250 107 39 4 150 4% 92% 4% 100% 46% 47% 7% 100% 71% 26% 3% 100% Nota: NR – não respondeu a questão. A relação das questões consta do Anexo 1. Nota: NR – não respondeu a questão. 4 Análise dos Resultados Contudo, ao segre- gar apenas as proposições que implicam em mudanças no status quo vigente (estabelecimento de um novo método de reconhecimento; possibilidade de adoção do valor justo; ou aumento dos itens de divulgação obrigatória – 4ª, 5ª, 6ª, 7ª, 9ª e 10ª questões), é possível verificar que as empresas fize- ram lobbying no sentido de rejeitar qualquer possibilidade de mudança no status quo. Foram 107 opiniões (71%) que levam à manutenção da situação vigente e apenas 39 (26%) que argumentam favoravelmente às propostas de mudan- ças trazidas pelo IASB. Por meio da distribuição Qui-quadrado, teste análogo ao teste F para a regressão múltipla, é possível constatar que coletivamente as variáveis do modelo são significativas do ponto de vista estatístico, com razão de verossimilhança es- timada em 35,296 e p-valor de 0,000. Dessa forma, há pelo menos um coeficiente diferente de zero ao nível de 1%. A medida de Nagelkerke assemelha-se ao R2 da regressão múltipla e informa o grau de ajuste do modelo. No resulta- do obtido, o modelo possui um poder explicativo de 40,8%. Como o interesse deste estudo reside na significância estatís- tica dos coeficientes das variáveis utilizadas e não necessaria- mente em se realizar previsões, considera-se que esse grau de ajustamento está aderente aos objetivos propostos. Outra maneira de se analisar o ajuste do modelo é verifi- cando o quanto este classifica corretamente os eventos, con- siderando o ponto de corte utilizado, que neste estudo foi de 16,5%, ou seja, o percentual de empresas da amostra que apre- sentam o evento de interesse. Os resultados obtidos indicam que o modelo classificou corretamente 76,3% dos casos. Esses achados estão condizentes com os observados por Kenny e Larson (1993) de que as empresas lobistas agem no sentido de se evitar qualquer mudança no status quo vigente ou nos argumentos de Holthausen e Leftwich (1983) de que a possibilidade de mudança no status quo afeta a decisão do gestor em influenciar o processo de regulação contábil. R. Cont. Fin. – USP, São Paulo, v. 25, n. 65, p. 124-144, maio/jun./jul./ago. 2014 135 Odilanei Morais dos Santos e Ariovaldo dos Santos Tabela 8 Resultado da regressão logística binomial Tabela 8 Resultado da regressão logística binomial g g Coef. Variáveis "Sinal Esperado" Regressão 1 Regressão 2 Regressão 3 Regressão 4 Regressão 5 Coef. Sig. VIF Coef. Sig. Coef. Sig. Coef. Sig. Coef. Sig. 4 Análise dos Resultados Ainda sobre a qualidade de ajuste do modelo, tem-se a Cur- va ROC (Receiver Operating Characteristic) que mede o poder discriminatório do modelo. Com valor de 86,9%, a especificação final do modelo apresenta poder de discriminação que se pode dizer excelente, conforme referência apresentada em Fávero et al. (2009): para Curva ROC menor ou igual a 0,5 (50%), não há discriminação; entre 0,5 e 0,8 (80%), há discriminação aceitável; e Curva ROC maior que 0,8, há excelente discriminação. uma delas perca significância na explanação do comporta- mento do fenômeno (Corrar, Paulo, & Dias Filho, 2007). Como a presença da multicolineariedade tende a dis- torcer os coeficientes angulares estimados, prejudicando a compreensão do real efeito da variável independente para o entendimento do fenômeno investigado, os resultados da regressão do primeiro modelo estão enviesados, o que pode explicar a não relevância estatística de algumas delas e mesmo o sinal negativo de RECLIQ. Os resultados evidenciam que apenas a variável LUC- MED apresenta significância estatística dentro dos níveis normalmente utilizados. Como o coeficiente estimado mostrou-se maior que zero, a interpretação é a de que quanto maior for o lucro líquido médio da empresa, maior será a probabilidade de a empresa fazer lobbying via carta comentários. O coeficiente estimado indica que o aumento em US$ 1 milhão no lucro líquido médio, aumenta o logit em 0,348 ou, então, calculando-se o antilogaritmo, a pro- babilidade da realização de lobbying é aumentada por um fator de 1,416 com o aumento do lucro líquido. Para contornar essa questão, novos modelos são elabora- dos contendo apenas uma das variáveis para tamanho. Con- firmando o efeito causado pela multicolinearidade, quando as variáveis relacionadas a tamanho foram colocadas no modelo isoladamente, cada uma delas se mostrou estatisticamente sig- nificativa e o sinal do coeficiente atendeu ao esperado. Os parâmetros gerais das regressões mantiveram-se pra- ticamente estáveis qualitativamente, sendo que todas elas, olhando-se o conjunto das variáveis, indicam modelos signi- ficativos do ponto de vista estatístico, conforme os testes da razão de verossimilhança. O R2 de Nagelkerke dos modelos situou-se entre 17,5% e 39,6%, sendo que a Curva ROC des- ses modelos apresentou excelente discriminação (exceção ao modelo 2, com discriminação apenas aceitável – 66%). É possível identificar, ainda, que a variável COMPGEN apresentou significância estatística marginal de 14,4%, fora dos níveis usuais é verdade, mas que, a depender do risco que se quer correr, pode-se considerá-la relevante. 4 Análise dos Resultados α CONSTANTE -10,858 0,002 - -7,408 0,000 -2,264 0,007 -7,905 0,000 -11,359 0,000 β1 RECLIQ + -0,022 0,959 7,485 0,689 0,000**** - - - - - - β2 UPSTREAM + 0,047 0,452 1,881 - - 0,173 0,097* - - - - β3 LUCMED + 0,348 0,023 5,137 - - - - 0,468 0,000* - - β4 ATIVTOT + 0,258 0,590 10,857 - - - - - - 1,019 0,000* β5 COVENANT + 0,732 0,393 1,406 0,724 0,267 -0,017 0,978 0,767 0,262 0,731 0,280 β6 COMPGEN + 1,151 0,144 † 1,483 0,671 0,340 0,494 0,475 1,208 0,109 † 0,919 0,209 β7 METODO + -0,126 0,824 1,183 0,451 0,383 0,283 0,564 -0,029 0,958 0,164 0,762 β8 USLISTING + 0,209 0,744 1,522 0,553 0,347 0,261 0,653 0,255 0,676 0,376 0,534 Teste Qui-quadradado 41,946 0,000* 28,833 0,000* 16,568 0,005* 40,553 0,000* 35,932 0,000* R2 de Nagelkerke 40,8% 29,2% 17,5% 39,6% 35,6% Casos "corretamente previstos" 76,3% 76,3% 59,9% 75,7% 77,6% Curva ROC 86,9% 80,9% 81,2% 86,1% 84,5% Número de Observações 152 152 152 152 152 Nota: RECLIQ: receitas líquidas do período; UPSTREAM: gastos de exploração e desenvolvimento incorridos no período; LUCMED: lucro líquido médio dos últimos três períodos; ATIVTOT: ativo total do período; COVENANT: “0” se não apresenta covenant e “1” se apresenta; COMPGEN: “0” se não apresenta compensação gerencial e “1” se apresenta; MÉTODO: “0” se adota o full cost e “1” se adota o successful efforts; USLISTING: “0” se negocia em outros mercados de capitais e “1” se negocia no mercado norte-americano. VIF: teste variance inflation factor. Significância: * (1%), (5%),* (10%) e † (15%). PROB(LOBBYINGi) = 1 + e - (α0 + β1-4TAMANHOi + β5COMPGENi + β6COVENANTi + β7USLISTINGi + β8METODOi + εi ) 1 PROB(LOBBYINGi) = 1 + e - (α0 + β1-4TAMANHOi + β5COMPGENi + β6COVENANTi + β7USLISTINGi + β8METODOi + εi ) 1 Nota: RECLIQ: receitas líquidas do período; UPSTREAM: gastos de exploração e desenvolvimento incorridos no período; LUCMED: lucro líquido médio dos últimos três períodos; ATIVTOT: ativo total do período; COVENANT: “0” se não apresenta covenant e “1” se apresenta; COMPGEN: “0” se não apresenta compensação gerencial e “1” se apresenta; MÉTODO: “0” se adota o full cost e “1” se adota o successful efforts; USLISTING: “0” se negocia em outros mercados de capitais e “1” se negocia no mercado norte-americano. VIF: teste variance inflation factor. Significância: * (1%), (5%),* (10%) e † (15%). 4 Análise dos Resultados Para tratar essa questão, lança-se mão do modelo de regressão logística multi- nomial, cujo resultado é apresentado na Tabela 9. De forma menos contundente, e com base na regressão logística binomial, pode-se aceitar a hipótese teórica apresen- tada pela literatura de que gestores de empresas que possuem compensação remuneratória baseada em desempenho medi- do a partir de indicadores derivados de informações contábeis tenderão a realizar lobbying sobre uma regulamentação, visan- do escolher os modelos contábeis que melhor lhes beneficie. As regressões logísticas multinomiais são dispostas em dois blocos, em que o primeiro conjunto contém os resultados da comparação entre as empresas petrolíferas com posições favo- ráveis às questões do DPEA e as empresas petrolíferas que não apresentaram comentários ao documento do IASB, enquanto que os resultados do segundo bloco se referem à comparação entre empresas petrolíferas com posições desfavoráveis e em- presas petrolíferas que não apresentaram comentários. q A modelagem econométrica aqui utilizada e por boa parte dos estudos sobre lobbying (Francis, 1987; Deakin, 1989; Ndu- Tabela 9 Resultado da regressão logística multinomial Coef. Variáveis "Sinal Esperado" Regressão 6 Regressão 7 Regressão 8 Regressão 9 Coef. Sig. Coef. Sig. Coef. Sig. Coef. Sig. 4 Análise dos Resultados Observou-se novamente a relevância marginal da vari- ável COMPGEN (10,9%) no modelo 4 e que contou com a variável LUCMED como proxy para tamanho. Isso sugere que empresas petrolíferas que apresentam planos de compen- sação gerencial possuem maior probabilidade de realizarem lobbying, via carta comentários, do que empresas petrolíferas que não possuem planos de compensação gerencial. E mais, as que possuem plano de compensação gerencial aumentam a probabilidade de se fazer lobbying por um fator de 3,347 (anti- logaritmo de 1,208) em relação às que não têm qualquer plano de compensação gerencial. Tendo em vista o alto grau de correlação observado entre as variáveis independentes, em especial as represen- tativas do tamanho, conforme especificado anteriormente (Tabela 6), recorreu-se ao teste VIF para a avaliação formal do pressuposto da multicolineariedade. Como se suspeitou, observa-se o não atendimento desse pressuposto, com alto valor de VIF para ATIVTOT e RECLIQ. A implicação des- se problema reside no fato de que variáveis independentes altamente correlacionadas fornecem informações similares para explicar e predizer a variável dependente, tornando di- fícil a separação dos efeitos de cada uma e fazendo com que R. Cont. Fin. – USP, São Paulo, v. 25, n. 65, p. 124-144, maio/jun./jul./ago. 2014 136 Lobbying na Regulação Contábil: Evidências do Setor Petrolífero bizu et al., 1993; Georgiou & Roberts, 2004) é eventualmente criticada em função de a variável dependente binária utilizada não conseguir capturar a intensidade da posição do respon- dente (Holthausen & Leftwich, 1983), mas apenas se este apre- sentou (código 1) ou não (código 0) comentários, ou se foi a favor (1) ou contra (0) a proposta. Os resultados apresentados sustentam a aceitação da hipótese dos custos políticos desenvolvida por Watts e Zimmerman (1978) de que, dentro do plano das escolhas contábeis, grandes empresas tendem a realizar lobbying sobre uma regulamentação, visando obter resultados que lhes sejam mais favoráveis. Os resultados também estão condizentes com os achados de Francis (1987), Deakin (1989) e Georgiou e Roberts (2004) no que se refere a “tamanho”. Dessa forma, reportando-se às hipóteses de testes estabelecidas, não se tem evidências para rejeitar a hipótese H1, considerando-se a modelagem da regressão logística binomial. Como o DPEA possui dez questões, os respondentes po- dem oferecer comentários a todas as questões ou somente a algumas delas, ou, ainda, ser favoráveis a algumas das questões e desfavoráveis a outras. Essas situações não são capturadas pelo modelo de regressão logística binomial. Nota: RECLIQ: receitas líquidas do período; UPSTREAM: gastos de exploração e desenvolvimento incorridos no período; LUCMED: lucro líquido médio dos últimos três períodos; ATIVTOT: ativo total do período; COVENANT: “0” se não apresenta covenant e “1” se apresenta; COMPGEN: “0” se não apresenta compensação gerencial e “1” se apresenta; MÉTODO: “0” se adota o full cost e “1” se adota o successful efforts; USLISTING: “0” se negocia em outros mercados de capitais e “1” se negocia no mercado norte-americano. Significância: * (1%), (5%) e * (10%). 4 Análise dos Resultados Por fim, observa-se que mais de 85% dos casos foram corretamente previstos, o que aponta para um bom ajustamento das regressões realizadas. contrário às propostas apresentadas. Os resultados obtidos por meio da regressão logísti- ca multinomial dão robustez para considerar o “tamanho” como fator econômico determinante para as empresas rea- lizarem lobbying via carta comentários. Assim, não se tem evidências para se rejeitar novamente a hipótese H1. Para a modelagem utilizando a regressão de Poisson (Tabela 10), foram executadas regressões considerando a intensidade com que as empresas petrolíferas foram favo- ráveis às propostas contidas no DPEA contra a posição de não se ter realizado lobbying (painel A) e também a inten- sidade com que as empresas foram desfavoráveis contra a posição de não se realizar lobbying (painel B). Analisando os coeficientes estimados, constata-se que as variáveis de tamanho, em cada modelo, sem contar a constante, mostraram-se estatisticamente significantes, nos dois blocos de resultados (exceção de UPSTREAM no primeiro bloco), reforçando os achados sobre a hipótese dos custos políticos. A leitura que se faz é a de que gran- des empresas petrolíferas possuem maior probabilidade para fazerem lobbying via carta comentários do que as de- mais empresas petrolíferas. Quanto aos parâmetros gerais das regressões reali- zadas, os resultados contidos na Tabela 10 revelam que todas podem ser consideradas válidas, uma vez que a sig- nificância estatística do teste Qui-quadrado ficou dentro do nível de 1%. Além disso, o R2 de MacFadden variou de 13% a 35%, grau de ajustamento relativamente baixo, mas aderente aos objetivos deste estudo. Analisando os resultados considerando a intensidade com que a empresa foi favorável às propostas do DPEA (painel A), primeiramente, as variáveis receita líquida (RECLIQ), lucro líquido médio (LUCMED) e ativo total (ATIVTOT) se destacam por apresentarem significância estatística e mais uma vez dão robustez para considerar o “tamanho” como indicativo da posição das empresas para realizarem lobbying via carta comentários. Analisando mais detidamente a probabilidade de se re- alizar lobbying via carta comentários, observa-se que essa tendência é maior no sentido de se fazer lobbying con- trário à proposta apresentada, pois veja-se: o aumento na receita líquida (RECLIQ), por um lado, proporciona um aumento por um fator de 1,421 (antilogaritmo de 0,352) na probabilidade de se realizar lobbying favorável em rela- ção a não se realizar lobbying. 4 Análise dos Resultados α CONSTANTE Posição Predominantemente Favorável -6,952 0,002* -3,360 0,000* -8,285 0,000* -10,010 0,000* β1 RECLIQ + 0,352 0,067* - - - - - - β2 UPSTREAM + - - 0,073 0,153 - - - - β3 LUCMED + - - - - 0,323 0,003* - - β4 ATIVTOT + - - - - - - 0,639 0,012 β5 COVENANT + 0,838 0,341 0,485 0,581 0,952 0,281 0,869 0,321 β6 COMPGEN + 0,776 0,391 0,730 0,422 1,180 0,206 0,960 0,294 β7 METODO + 0,433 0,481 0,152 0,808 -0,015 0,982 0,154 0,808 β8 USLISTING + 0,026 0,970 -0,365 0,599 -0,164 0,813 -0,054 0,938 α CONSTANTE Posição Predominantemente Desfavorável -18,242 0,000* -14,568 0,000* -17,044 0,000* -23,972 0,000* β1 RECLIQ + 1,394 0,000* - - - - - - β2 UPSTREAM + - - 1,451 0,001* - - - - β3 LUCMED + - - - - 0,801 0,000* - - β4 ATIVTOT + - - - - - - 1,844 0,000* β5 COVENANT + 0,770 0,404 -0,270 0,772 0,800 0,411 0,775 0,434 β6 COMPGEN + 0,286 0,788 0,663 0,564 1,312 0,240 0,763 0,496 β7 METODO + 0,276 7,488 -0,330 0,700 -0,154 0,859 0,057 0,951 β8 USLISTING + 1,212 0,257 1,027 0,438 0,858 0,412 1,126 0,313 Teste Qui-quadradado 41,759 0,000* 39,107 0,000* 49,798 0,000* 47,089 0,000* R2 de Nagelkerke 35,6% 33,6% 41,4% 39,5% Casos "corretamente previstos" 84,9% 86,8% 86,8% 86,2% Número de Observações 152 152 152 152 ln = α1 + β1-4TAMANHOi + β5COMPGENi + β6COVENANTi + β7USLISTINGi + β8METODOi + εi PROB (LOBBYING = 1\|X) PROB (LOBBYING = 0\|X) ln = α1 + β1-4TAMANHOi + β5COMPGENi + β6COVENANTi + β7USLISTINGi + β8METODOi + εi PROB (LOBBYING = 2\|X) PROB (LOBBYING = 0\|X) Tabela 9 Resultado da regressão logística multinomial 137 R. Cont. Fin. – USP, São Paulo, v. 25, n. 65, p. 124-144, maio/jun./jul./ago. 2014 137 Odilanei Morais dos Santos e Ariovaldo dos Santos contrário às propostas apresentadas. Tendo em vista a questão relacionada à multicolinea- ridade, as regressões logísticas multinomiais foram reali- zadas considerando as variáveis independentes para tama- nho isoladamente. Os parâmetros gerais dos modelos das quatro regressões são válidos, uma vez que o teste Qui- quadrado revela significância estatística ao nível de 1%. O grau de ajustamento dos modelos, conforme pseudo R2 de Negelkerke, varia entre 33% e 41%, valores adequados para este estudo. R. Cont. Fin. – USP, São Paulo, v. 25, n. 65, p. 124-144, maio/jun./jul./ago. 2014 4 Análise dos Resultados Por outro lado, tem-se um fator de aumento de 4,031 na probabilidade de se realizar lobbying desfavorável em relação a não se realizar lobbying no caso de aumento na receita líquida. É possível destacar ainda, estatisticamente, a signifi- cância da variável COMPGEN na presença das variáveis RECLIQ, LUCMED e ATIVTOT. Esse resultado, de certo modo, já havia sido apontado na regressão logística bino- mial, confirmando as análises realizadas anteriormente, com a observação de que, via regressão de Poisson, essa característica (possuir plano de compensação gerencial) é relevante para a realização de lobbying favorável ao DPEA. O mesmo comportamento observa-se nos outros casos: LUCMED, aumento por um fator de 1,381 contra 2,227; ATIVTOT, aumento por um fator de 1,895 contra 6,321. No caso dos gastos exploratórios (UPSTREAM), a proba- bilidade de se realizar lobbying só é observada se este for Tabela 10 Resultado da regressão de Poisson g Painel A: Lobbying a Favor do DPEA Coef. Variáveis Sinal Esperado Regressão 10 Regressão 11 Regressão 12 Regressão 13 Coef. Sig. Coef. Sig. Coef. Sig. Coef. Sig. α CONSTANTE -5,089 0,002* -1,892 0,314 -5,740 0,000* -7,510 0,000* β1 RECLIQ + 0,373 0,007* - - - - - - β2 UPSTREAM + - - 0,159 0,441 - - - - β3 LUCMED + - - - - 0,290 0,000* - - β4 ATIVTOT + - - - - - - 0,589 0,000* β5 COVENANT + 0,551 0,378 0,150 0,773 0,624 0,284 0,602 0,320 β6 COMPGEN + 0,950 0,139 † 0,952 0,159 1,271 0,035** 1,096 0,070* β7 METODO + 0,182 0,707 -0,046 0,915 -0,224 0,648 -0,004 0,993 β8 USLISTING + 0,001 0,998 -0,243 0,603 -0,247 0,585 -0,146 0,757 LOBBYINGi = + ui μLOBBYINGe - (α1 + β1-4TAMANHOi + β5COMPGENi + β6COVENANTi + β7USLISTINGi + β8METODOi ) LOBBYING! continua LOBBYINGi = + ui μLOBBYINGe - (α1 + β1-4TAMANHOi + β5COMPGENi + β6COVENANTi + β7USLISTINGi + β8METODOi ) LOBBYING! R. Cont. Fin. – USP, São Paulo, v. 25, n. 65, p. 124-144, maio/jun./jul./ago. 2014 138 Lobbying na Regulação Contábil: Evidências do Setor Petrolífero Teste Qui-quadrado 49,003 0,000* 41,021 0,000* 34,856 0,000* 42,237 0,000* R2 de McFadden 13,2% 13,8% 23,4% 18,4% Número de Observações 152 152 152 152 Painel B: Lobbying Contra o DPEA Coef. Variáveis Sinal Esperado Regressão 14 Regressão 15 Regressão 16 Regressão 17 Coef. Sig. Coef. Sig. Coef. Sig. Coef. Sig. 4 Análise dos Resultados Os resultados da regressão de Poisson, quando se con- sidera a variável dependente na perspectiva da intensidade com que se foi desfavorável às questões do DPEA, também apontam para a relevância estatística das variáveis RECLIQ, LUCMED e ATIVTOT. empresas com essas características tenderiam a possuir uma postura lobista, seja contra ou a favor de uma re- gulamentação contábil a respeito das atividades extra- tivistas, suportando, portanto, a teoria desenvolvida ao longo deste estudo. A partir das hipóteses de testes formuladas, reforça-se a conclusão de que não se tem evidências para rejeitar a hipótese H1. Além disso, a hipótese H2 não pode ser inte- gralmente descartada. Trata-se de evidências que referenciam mais uma vez a hipótese dos custos políticos de Watts e Zimmer- man (1978) de que grandes empresas tendem a reali- zar lobbying visando obter vantagens por meio de re- gulamentação que lhes seja mais favorável. No caso da regressão de Poisson, a análise dos coeficientes indica que a propensão de rejeição da proposta contida no DPEA é maior do que a aceitação e, com isso, tem-se que o lobbying seria no sentido de se refutar qualquer mudança do status quo vigente. Tentando estabelecer uma análise conclusiva quanto às variáveis representativas do tamanho, dado que elas servem para explicar um mesmo fenômeno e estão al- tamente correlacionadas, como visto anteriormente, buscou-se, via técnica de análise fatorial (Tabela 11), o estabelecimento de um fator representativo para o tama- nho que pudesse ser utilizado como variável dependente nas regressões logísticas, binomial e multinomial; e na regressão de Poisson. Interessante observar que os gastos exploratórios (UPS- TREAM) não se mostraram significantes em nenhum dos modelos estabelecidos, utilizando-se da regressão de Pois- son (regressões 11 e 15), fato diferente do que se desenhou anteriormente. Os pressupostos da análise fatorial foram atendidos: normalidade das variáveis, exceto para UPSTREAM; correlações significativas entre as variáveis RECLIQ, UPSTREAM, LUCMED e ATIVTOT; adequação da amostra à análise fatorial, com medida de Kaiser- Meyer-Olkin (KMO) de 0,826 e, por fim, que a matriz de correlação não é igual à matriz identidade, com teste de esfericidade de Bartlett altamente significativo (p < 0,001). 4 Análise dos Resultados α CONSTANTE -7,224 0,000* -2,190 0,470 -6,844 0,000* -10,139 0,000* β1 RECLIQ + 0,608 0,000* - - - - - - β2 UPSTREAM + - - 0,232 0,537 - - - - β3 LUCMED + - - - - 0,373 0,000* - - β4 ATIVTOT + - - - - - - 0,855 0,000* β5 COVENANT + 0,413 0,401 -0,214 0,701 0,377 0,404 0,390 0,423 β6 COMPGEN + 0,111 0,853 0,045 0,946 0,525 0,338 0,311 0,553 β7 METODO + 0,278 0,536 0,282 0,528 -0,045 0,913 0,169 0,710 β8 USLISTING + 0,665 0,228 0,600 0,265 0,394 0,421 0,476 0,363 Teste Qui-quadrado 25,343 0,000* 45,651 0,000* 23,623 0,000* 19,001 0,000* R2 de McFadden 28,5% 16,5% 35,1% 33,9% Número de Observações 152 152 152 152 Nota: RECLIQ: receitas líquidas do período; UPSTREAM: gastos de exploração e desenvolvimento incorridos no período; LUCMED: lucro líquido médio dos últimos três períodos; ATIVTOT: ativo total do período; COVENANT: “0” se não apresenta covenant e “1” se apresenta; COMPGEN: “0” se não apresenta compensação gerencial e “1” se apresenta; MÉTODO: “0” se adota o full cost e “1” se adota o successful efforts; USLISTING: “0” se negocia em outros mercados de capitais e “1” se negocia no mercado norte-americano. Significância: * (1%), (5%),* (10%) e † (15%). continuação Nota: RECLIQ: receitas líquidas do período; UPSTREAM: gastos de exploração e desenvolvimento incorridos no período; LUCMED: lucro líquido médio dos últimos três períodos; ATIVTOT: ativo total do período; COVENANT: “0” se não apresenta covenant e “1” se apresenta; COMPGEN: “0” se não apresenta compensação gerencial e “1” se apresenta; MÉTODO: “0” se adota o full cost e “1” se adota o successful efforts; USLISTING: “0” se negocia em outros mercados de capitais e “1” se negocia no mercado norte-americano. Significância: * (1%), (5%),* (10%) e † (15%). Nota: RECLIQ: receitas líquidas do período; UPSTREAM: gastos de exploração e desenvolvimento incorridos no período; LUCMED: lucro líquido médio dos últimos três períodos; ATIVTOT: ativo total do período; COVENANT: “0” se não apresenta covenant e “1” se apresenta; COMPGEN: “0” se não apresenta compensação gerencial e “1” se apresenta; MÉTODO: “0” se adota o full cost e “1” se adota o successful efforts; USLISTING: “0” se negocia em outros mercados de capitais e “1” se negocia no mercado norte-americano. Significância: * (1%), (5%),* (10%) e † (15%). 4 Análise dos Resultados Nota: TAMANHO: variável decorrente da análise fatorial, elaborada com base nos valores dos coeficientes dos escores fatoriais de cada uma das variáveis RECLIQ, UPSTREAM, LUCMED e ATIVTOT; COVENANT: “0” se não apresenta covenant e “1” se apresenta; COMPGEN: “0” se não apresenta compensa- ção gerencial e “1” se apresenta; MÉTODO: “0” se adota o full cost e “1” se adota o successful efforts; USLISTING: “0” se negocia em outros mercados de capitais e “1” se negocia no mercado norte-americano. Significância: * (1%), (5%),* (10%) e † (15%). Nota: TAMANHO: variável decorrente da análise fatorial, elaborada com base nos valores dos coeficientes dos escores fatoriais de cada uma das variáveis RECLIQ, UPSTREAM, LUCMED e ATIVTOT; COVENANT: “0” se não apresenta covenant e “1” se apresenta; COMPGEN: “0” se não apresenta compensa- ção gerencial e “1” se apresenta; MÉTODO: “0” se adota o full cost e “1” se adota o successful efforts; USLISTING: “0” se negocia em outros mercados de capitais e “1” se negocia no mercado norte-americano. Significância: * (1%), (5%),* (10%) e † (15%). Para obtenção dos fatores, utilizou-se a análise de compo- nentes principais (ACP), a qual busca resumir a maior parte da variância das variáveis a um número mínimo de fatores. Pelo critério de Kaiser (descarte dos autovalores menores do que 1, pela sua insignificância), foi possível definir um único fator, que explica 86,1% da variância dos dados. Os resulta- dos da análise fatorial constam do Apêndice 1. anteriores de que o “tamanho” é uma característica rele- vante que aumenta a probabilidade de a empresa realizar lobbying via carta comentários. Em todas as situações (re- gressões 18 a 22), a variável “tamanho” foi estatisticamente significativa. Analisando os coeficientes obtidos, a proba- bilidade é maior para se realizar lobbying¸ de forma geral, contrária as proposições do DPEA. Não se confirmou, entretanto, nessa configuração, a rele- vância dos planos de compensação gerencial (COMPGEN) como característica da realização de lobbying via carta co- mentários. Nas situações testadas, apenas quando se testou a intensidade quanto à aceitabilidade da proposta do DPEA, via regressão de Poisson (regressão 21), é que se encontrou significância estatística, mas de forma marginal. Assim, com base nos valores dos coeficientes dos es- cores fatoriais de cada uma das quatro variáveis (RECLIQ, UPSTREAM, LUCMED e ATIVTOT), se estabeleceu uma nova variável preditora decorrente da análise fatorial que foi denominada de “TAMANHO”. 4 Análise dos Resultados Assim, novas regressões foram geradas e seus resultados constam da Tabela 11. Os resultados evidenciam e referendam as conclusões 4 Análise dos Resultados Depreende-se desses resultados (regressão de Pois- son) que, independentemente da intensidade com que a empresa foi favorável ou desfavorável às proposições do IASB no DPEA, o tamanho e a existência de plano de compensação gerencial (de forma marginal) repre- sentam fatores determinantes à realização de lobbying por parte das empresas petrolíferas, indicando que as R. Cont. Fin. – USP, São Paulo, v. 25, n. 65, p. 124-144, maio/jun./jul./ago. 2014 139 Odilanei Morais dos Santos e Ariovaldo dos Santos Tabela 11 Regressões após análise fatorial Regressão Logística Binomial Regressão Logística Multinomial Regressão de Poisson Posição Predominante- mente Favorável Posição Predominante- mente Desfavorável Lobbying a Favor do DPEA Lobbying Contra o DPEA Variáveis Sinal Esperado Regressão 18 Regressão 19 Regressão 20 Regressão 21 Regressão 22 Coef. Sig. Coef. Sig. Coef. Sig. Coef. Sig. Coef. Sig. CONSTANTE -1,379 0,000* -3,601 0,000* -4,643 0,000* -1,474 0,016 -1,255 0,047 TAMANHO + 1,076 0,000* 0,697 0,036 1,446 0,000* 0,445 0,000* 0,595 0,000* COVENANT + 0,776 0,294 0,778 0,380 0,772 0,481 0,550 0,337 0,418 0,420 COMPGEN + 0,884 0,251 0,904 0,326 0,672 0,571 0,949 0,141 † 0,072 0,902 METODO + 0,280 0,594 0,367 0,554 0,059 0,946 0,119 0,801 0,196 0,678 USLISTING + -0,022 0,971 -0,301 0,663 0,592 0,594 -0,338 0,509 0,267 0,635 Teste Qui-quadradado 30,194 0,000* 38,492 0,000* 50,017 0,000* 40,312 0,000* R2 de Nagelkerke 30,5% 33,2% - - R2 de McFadden - - 11,8% 25,1% Número de Observações 152 152 152 152 Nota: TAMANHO: variável decorrente da análise fatorial, elaborada com base nos valores dos coeficientes dos escores fatoriais de cada uma das variáveis RECLIQ, UPSTREAM, LUCMED e ATIVTOT; COVENANT: “0” se não apresenta covenant e “1” se apresenta; COMPGEN: “0” se não apresenta compensa- ção gerencial e “1” se apresenta; MÉTODO: “0” se adota o full cost e “1” se adota o successful efforts; USLISTING: “0” se negocia em outros mercados de capitais e “1” se negocia no mercado norte-americano. Significância: * (1%), (5%),* (10%) e † (15%). 5 Conclusões 5 Conclusões do setor extrativista. Os resultados apresentados são robustos para considerar que existem fatores determinantes à adoção de estratégias de lobbying em relação a uma dada regulamentação contábil (ou proposta de), quando se considera a realidade das empresas petrolíferas, e comprovam a hipótese dos custos políticos (ou do tamanho) defendida por Watts e Zimmerman (1978) de que grandes empresas tendem a realizar lobbying sobre uma regulamentação contábil visando influenciar o normatizador para obter uma norma que atenda a seus interesses. do setor extrativista. As evidências também mostram, de forma marginal, que empresas petrolíferas que apresentam planos de com- pensação gerencial possuem maior probabilidade de reali- zar lobbying do que as demais empresas petrolíferas, como mostraram Watts e Zimmerman (1978), Deakin (1989) e Georgiou e Roberts (2004). Ao conjugar os resultados, pode-se inferir que as estra- tégias de lobbying promovidas pelas grandes empresas do setor se farão presentes junto ao IASB numa eventual nova regulamentação contábil do setor petrolífero para não se permitir alterações na situação vigente, ou seja, pode-se di- zer que os preparadores são favoráveis à utilização do custo histórico como base de valor e a existência de dois modelos contábeis concorrentes (esforços bem sucedidos e capitali- zação total) e desfavoráveis às mudanças que levem ao au- O fator “tamanho”, modelado sob diversas perspectivas, mostrou-se relevante em todas as modelagens econométri- cas utilizadas, suportando a hipótese de que grandes empre- sas petrolíferas possuem maior probabilidade para realizar lobbying. Essa propensão foi verificada para posicionamen- tos essencialmente desfavoráveis às propostas apresentadas no Discussion Paper que trata da regulamentação contábil R. Cont. Fin. – USP, São Paulo, v. 25, n. 65, p. 124-144, maio/jun./jul./ago. 2014 140 Lobbying na Regulação Contábil: Evidências do Setor Petrolífero mento do nível de divulgação das informações. mento do nível de divulgação das informações. Apesar de ser amplamente utilizado na literatura contá- bil, o conceito de lobbying empregado é um limitador dos resultados encontrados, dado que se considerou apenas o ato de enviar cartas comentários às consultas públicas do IASB. Assim, outras modelagens de pesquisas são possíveis de ser realizadas com vistas a ampliar essa visão, notada- mente com o emprego de questionários e entrevistas com o objetivo de contemplar outras formas de lobbying, a exem- plo do realizado por Georgiou (2004). Sob a perspectiva da teoria institucional, o devido proces- so normativo do IASB busca uma grande participação de seus constituintes em todas as fases de elaboração da norma para tornar o processo mais neutro e legítimo. Ao mesmo tempo, pela teoria econômica da regulação, sabe-se que a participa- ção no devido processo normativo tem por objetivo tentar in- fluenciar as decisões do board em interesse próprio. do setor extrativista. Assim, considerando o processo de elaboração de nor- mas por parte do IASB um processo político e que um mo- saico de interesses conflitantes interage na fixação dos pa- drões contábeis do IASB, espera-se que a operacionalização de revisão/substituição do IFRS 6 seja complexa e sujeita a grandes pressões por parte das empresas petrolíferas no sentido de manutenção do status quo. Tudo isso reforça os argumentos de Larson (2002) de que, em decorrência de fatores externos e políticos, o IASB pode seguir uma linha menos neutra visando à acomodação de interesses ou, até mesmo, de que o IASB pode, novamente, ser capturado pela indústria como sugerido em Cortese, Irvine, e Kaido- nis (2010), por ocasião da emissão do IFRS 6. Ainda no estudo sobre lobbying, é importante destacar que as proxies utilizadas para definição das características das firmas não se limitam àquelas utilizadas, podendo exis- tir outros determinantes econômicos. Mesmo consideran- do-se as variáveis utilizadas, estas podem assumir outras dimensões, especialmente as variáveis relacionadas às cláu- sulas restritivas (COVENANT) e a compensação gerencial (COMPGEN). A título de exemplo, ao invés de se assumir apenas valores binários (0 ou 1), poder-se-ia modelar a va- riável COMPGEN como uma variável contínua como, por exemplo, o percentual que a remuneração variável do ges- tor representa na sua remuneração total, com a hipótese de que quanto maior a dependência da remuneração variável, mais propenso o gestor estaria para realizar escolhas con- tábeis em seu favor. Os resultados apresentados ao longo deste trabalho de- vem ser analisados sob o prisma das empresas petrolíferas, não podendo ser generalizados para outros setores econô- micos ou processo de normatização do IASB. No entanto, os modelos econométricos utilizados são generalizáveis e podem ser replicados em outras pesquisas, principalmente as modelagens que utilizam as regressões logística multino- mial e de Poisson, novidades em estudo sobre lobbying na regulação contábil. Por fim, não se pode deixar de relatar as limitações ine- rentes à utilização da base de dados da Evaluate Energy ®, pois há de se considerar, como toda base de dados, que er- ros em relação aos inputs dos dados podem existir. Assim, o julgamento quanto à integridade da base de dados con- templa um viés dos pesquisadores. Referências Referências Asekomeh, A. O., Russel, A., & Tarbert, H. (2006). 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Fin. – USP, São Paulo, v. 25, n. 65, p. 124-144, maio/jun./jul./ago. 2014 141 Odilanei Morais dos Santos e Ariovaldo dos Santos lobbying in private accounting standard setting: does the IASB have to reckon with national differences? do setor extrativista. Questão nº 3: Você concorda que a definição de reservas e recursos minerais deve seguir o estabelecido pelo Committee for Mineral Reserves International Reporting Standards e as definições de reservas e recursos de óleo e gás sejam aquelas definidas pela Society of Petroleum Engineers (em conjunto com outros órgãos do setor) na IFRS Extractive Activities? Questão nº 4: Você concorda com a análise de que os direitos legais, tais como os direitos de exploração e extração devem ser a base de reconhecimento para os ativos minerais ou de óleo e gás, sendo que as informações subsequentes quanto às atividades de exploração e avaliação e o trabalho para desenvolver o acesso aos depósitos minerais ou de óleo e gás devem ser tratados como uma extensão desses direitos legais? Questão nº 5: Você concorda que o limite para sumarizar as informações das atividades de exploração, avaliação e de- senvolvimento deve ser inicialmente definido de acordo com os direitos exploratórios mantidos, não sendo maior do que uma simples área ou grupos de áreas contínuas e de onde se esperam gerar fluxos de caixa independentes? p g p p g p Questão nº 6: Você concorda que os ativos minerais ou de óleo e gás devam ser mensurados com base no custo histó- rico e que informações adicionais devem ser detalhadamente divulgadas com o objetivo de fornecer uma maior relevância às demonstrações financeiras? Questão nº 6: Você concorda que os ativos minerais ou de óleo e gás devam ser mensurados com base no custo histó- rico e que informações adicionais devem ser detalhadamente divulgadas com o objetivo de fornecer uma maior relevância às demonstrações financeiras? Questão nº 7: Você concorda com a visão de que o IAS 36 não deve ser aplicado para os ativos vinculados aos gastos exploratórios, a não ser que evidências disponíveis sugiram que a totalidade desses gastos não serão recuperáveis? Questão nº 7: Você concorda com a visão de que o IAS 36 não deve ser aplicado para os ativos vinculados aos gastos exploratórios, a não ser que evidências disponíveis sugiram que a totalidade desses gastos não serão recuperáveis? do setor extrativista. L., & Zimmerman, J. L. (1978). Towards a positive theory of the determination of accounting standards. The Accounting Review, 53 (1), 112-134. Meier, H. H., Alam, P., & Pearson, M. A. (1993). Auditor lobbying for accounting standards: the case of banks and savings and loan associations. Accounting and Business Research, 23 (92), 477-487. Watts, R. L., & Zimmerman, J. L. (1986). Positive accounting theory. New Jersey: Prentice-Hall. Ndubizu, G. A., Choi, Y. C., & Jain, R. (1993). 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Orens, R., Jorissen, A., Lybaert, N., & Tas, L. V. D. (2011). Corporate Zeff, S. A. (2002). “Political” lobbying on proposed standards: a challenge to the IASB. Accounting Horizons, 16 (1), 43-54.fh Zeff, S. A. (2005 February). The evolution of U.S. GAAP: the political forces behind professional standards. The CPA Journal, 21-29. fi pp g Orens, R., Jorissen, A., Lybaert, N., & Tas, L. V. D. (2011). Corporate 142 R. Cont. Fin. – USP, São Paulo, v. 25, n. 65, p. 124-144, maio/jun./jul./ago. 2014 Lobbying na Regulação Contábil: Evidências do Setor Petrolífero Anexo 1 Questões apresentadas pelo IASB no Discussion Paper Extractive Activities Questão nº 1: Você concorda que o escopo da IFRS Extractive Activities deve incluir somente as atividades de upstream relacionadas às operações de mineração e de óleo e gás? Questão nº 2: Você concorda que deve haver um modelo de contabilização e divulgação único aplicado tanto para as atividades de mineração quanto de óleo e gás? do setor extrativista. Questão nº 8: Você concorda que os objetivos da divulgação das atividades extrativistas são para capacitar os usuá- rios dos relatórios financeiros em avaliar: (a) o valor atribuído aos ativos minerais ou de óleo e gás de uma entidade; (b) a contribuição desses ativos para o desempenho financeiro do período corrente; e (c) a natureza e extensão dos riscos e incertezas associados a esses ativos? Questão nº 8: Você concorda que os objetivos da divulgação das atividades extrativistas são para capacitar os usuá- rios dos relatórios financeiros em avaliar: (a) o valor atribuído aos ativos minerais ou de óleo e gás de uma entidade; (b) a contribuição desses ativos para o desempenho financeiro do período corrente; e (c) a natureza e extensão dos riscos e incertezas associados a esses ativos? Questão nº 9: Você concorda que as informações a serem divulgadas em notas explicativas das demonstrações finan- ceiras devem incluir: (a) a quantidade de reservas provadas mais as reservas provadas e prováveis, com divulgação das quantidades de reservas separadamente por commodity e área geográfica material; (b) as principais premissas usadas para estimar as quantidades de reservas e uma análise de sensibilidade; (c) uma reconciliação entre as mudanças de estimativas nas quantidades de reservas de um ano para outro; (d) uma mensuração a valores correntes das quantidades de reservas divulgadas e uma reconciliação em relação às mudanças ocorridas; (e) identificação separada dos fluxos de caixas da ex- ploração, desenvolvimento e operações do período corrente em uma série de tempo, como por exemplo, de cinco anos; e (f) identificação separada das receitas de produção por commodity? Questão nº 10: Você concorda que os pagamentos feitos aos governos devem ser uma exigência quanto à divulgação em nota explicativa às demonstrações financeiras, justificando os custos da divulgação frente aos benefícios da informação? R. Cont. Fin. – USP, São Paulo, v. 25, n. 65, p. 124-144, maio/jun./jul./ago. 2014 143 Odilanei Morais dos Santos e Ariovaldo dos Santos Apêndice 1 Apêndice 1 Tabela Teste KMO, Bartlett e Comunalidades Medidas Valor Comunalidades Variáveis Inicial Extração Teste Kaiser-Meyer-Olkin (KMO) RECLIQ 1,000 0,878 Adequação da Amostra 0,826 UPSTREAM 1,000 0,805 Teste de Esfericidade de Bartlett Qui-Quadrado 607,017 LUCMED 1,000 0,864 df 6 Significância 0,000 ATIVTOT 1,000 0,896 Tabela Autovalores e percentual de variância explicada pelos fatores Componentes Autovalores Inicial Percentual de Variância Explicada Total % de Variação % Acumulado Total % de Variação % Acumulado 1 3,443 86,080% 86,080% 3,443 86,080% 86,080% 2 0,270 6,749% 92,829% 3 0,192 4,797% 97,627% 4 0,095 2,373% 100,000% Tabela Cargas fatoriais e matriz de coeficientes dos escores fatoriais Cargas Fatoriais Matriz de Coeficientes dos Escores Fatoriais Variáveis Componente 1 Variáveis Componente 1 RECLIQ 0,937 RECLIQ 0,272 UPSTREAM 0,897 UPSTREAM 0,261 LUCMED 0,929 LUCMED 0,270 ATIVTOT 0,946 ATIVTOT 0,275 Tabela Teste KMO, Bartlett e Comunalidades Tabela Autovalores e percentual de variância explicada pelos fatores Componentes Autovalores Inicial Percentual de Variância Explicada Total % de Variação % Acumulado Total % de Variação % Acumulado 1 3,443 86,080% 86,080% 3,443 86,080% 86,080% 2 0,270 6,749% 92,829% 3 0,192 4,797% 97,627% 4 0,095 2,373% 100,000% Tabela Autovalores e percentual de variância explicada pelos fatores Tabela Cargas fatoriais e matriz de coeficientes dos escores fatoriais Cargas Fatoriais Matriz de Coeficientes dos Escores Fatoriais Variáveis Componente 1 Variáveis Componente 1 RECLIQ 0,937 RECLIQ 0,272 UPSTREAM 0,897 UPSTREAM 0,261 LUCMED 0,929 LUCMED 0,270 ATIVTOT 0,946 ATIVTOT 0,275 144 R. Cont. Fin. – USP, São Paulo, v. 25, n. 65, p. 124-144, maio/jun./jul./ago. 2014
https://openalex.org/W3160119089	https://amb-express.springeropen.com/track/pdf/10.1186/s13568-021-01231-7	English	null	Construction and application of a CRISPR/Cas9-assisted genomic editing system for Corynebacterium glutamicum	AMB express	2,021	cc-by	10,533	© The Author(s) 2021. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativeco mmons.org/licenses/by/4.0/. Abstract Corynebacterium glutamicum is widely used as microbial cell factory for various bioproducts, but its genomic editing efficiency needs to be improved. In this study, a highly efficient CRISPR/Cas9-assisted genomic editing system for C. glutamicum was constructed. This system mainly involves a plasmid and can be used for both gene insertion and deletion in the chromosome of C. glutamicum. The recombinant plasmid for the target gene containing all the editing elements, and first constructed it in E. coli, then purified and transformed it into C. glutamicum. This temperature-sen- sitive plasmid was cured at high temperature after the genomic editing was completed in C. glutamicum. Using this genetic editing system, the genetic editing efficiency in C. glutamicum ATCC 13032 could reach 95%. The whole work of editing could be done in 8–9 days and showed most time-saving compared to the reported. Using this system, the native promoter of gdhA1 in ATCC 13032 has been replaced with the strong promoter PtacM, and more than 10 genes in ATCC 13032 have been deleted. The results demonstrate that this CRISPR/Cas9-assisted system is highly efficient and very suitable for genome editing in C. glutamicum. Keywords: Corynebacterium glutamicum, CRISPR/Cas9, Metabolic engineering, Genomic editing, l-Glutamic acid fermentation orynebacterium glutamicum, CRISPR/Cas9, Metabolic engineering, Genomic editing, l-Glutamic acid C. glutamicum, for example, biofuels (Sasaki et al. 2019; Zhang et al. 2019b), organic acid (Fukui et al. 2019), enzyme preparations (Yang et al. 2019) and other natu- ral compounds (Braga et al. 2018; Cheng et al. 2019; Zha et al. 2018) and so on. The metabolic engineering of C. glutamicum to produce various bioproducts indicated its attractive prospect. C. glutamicum, for example, biofuels (Sasaki et al. 2019; Zhang et al. 2019b), organic acid (Fukui et al. 2019), enzyme preparations (Yang et al. 2019) and other natu- ral compounds (Braga et al. 2018; Cheng et al. 2019; Zha et al. 2018) and so on. The metabolic engineering of C. glutamicum to produce various bioproducts indicated its attractive prospect. Introduction Corynebacterium glutamicum is an ideal industrial strain on account of high growth velocity and food-safety (Liu et al. 2016). Its traditional utilization in industries are for various amino acids production such as l-glutamic acid (Wada et al. 2016; Wen and Bao 2019), l-threonine (Lv et al. 2012; Wei et al. 2018), branched-chain amino acid (Schwentner et al. 2018; Wang 2019; Wang et al. 2019), l-lysine (Xiao et al. 2020), nonprotein amino acid (Mindt et al. 2019; Shi et al. 2018) and so on. C. glutamicum are one of most important model organisms in microbial cell factories due to decade studies, increasing other bio- products metabolic pathway have been constructed in Metabolic engineering studies are most important part of synthetic biology for C. glutamicum. With the development of molecular biology, the cell factories’ establishment was inclined to rational design of meta- bolic pathway including Design, Construction, Evalua- tion, and Optimization (Chen et al. 2017). Hence, some more efficient molecular tools must be constructed and used to reestablish metabolic pathway. To date, although there are some gene editing methods of C. glutamicum have been reported, there were some obvious deficien- cies like low efficiency and time-consuming. W. Jager Correspondence: xwang@jiangnan.edu.cn 1 State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, China Full list of author information is available at the end of the article Yao et al. AMB Expr (2021) 11:70 https://doi.org/10.1186/s13568-021-01231-7 Yao et al. AMB Expr (2021) 11:70 https://doi.org/10.1186/s13568-021-01231-7 Open Access Construction and application of a CRISPR/ Cas9‑assisted genomic editing system for Corynebacterium glutamicum Chengzhen Yao1,3, Xiaoqing Hu1 and Xiaoyuan Wang1,2,3 Correspondence: xwang@jiangnan.edu.cn 1 State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, China Full list of author information is available at the end of the article Strains, plasmids, and growth conditions All bacterial strains and plasmids used in this study are listed in Table 1. E. coli DH5α was cloning host for vec- tors’ construction. C. glutamicum ATCC 13032 are mainly used strain for editing strategy, meanwhile, C. glutamicum ATCC 14067 and C. glutamicum ssp. lacto- fermentum as supplementary strains used in this study. E. coli DH5α were cultured in Luria–Bertani (LB) media (tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, pH 7.0–7.2, LB agar supplemented with 15 g/L agar) at 37 °C. C. glutamicum is cultured in LBHIS media (d-sorbitol 91 g/L, tryptone 5 g/L, yeast extract 2.5 g/L, NaCl 5 g/L, brain heart infusion 1.85 g/L, pH 7.2–7.4, LBHIS agar supplemented with 15 g/L agar) at 30 °C. C. glutamicum harboring pCCG1/pCCG2 was cultured at 28 °C, and 37 °C for plasmid curing. Adding kanamycin 30 mg/L for E. coli DH5α and 20 mg/L for C. glutamicum as needed. The seed media (g/L): glucose·H2O 25, corn extract 20, KH2PO4 1, MgSO4·7H2O 0.4, urea 5, pH 7.2–7.4. l-glu- tamic acid fermentation media (g/L): glucose·H2O 110, corn extract 1, KH2PO4 2, MgSO4·7H2O 0.8, MnSO4·H2O 0.01, FeSO4·7H2O 0.02, pH 7.2–7.4. y g Streptococcus pyogenes type II CRISPR/Cas9-assisted genomic editing and CRISPRi technology are generaliz- able to microbiologic cell factories, such as C. glutami- cum (Cameron Coates et al. 2019; Cleto et al. 2016; Liu et al. 2017), Escherichia coli (Jiang et al. 2015; Pyne et al. 2015), Bacillus subtilis (Altenbuchner 2016; Westbrook et al. 2016), Lactobacillus (Oh and van Pijkeren 2014; Song et al. 2017), Aspergillus (Nodvig et al. 2018; Zhang et al. 2016), Saccharomyces cerevisiae (Mitsui et al. 2019; Zhang et al. 2019a). The CRISPR/Cas9 mutation and CRISPRi (Cleto et al. 2016; Gauttam et al. 2019) can fine- tune gene expression level in genome and have been used to study metabolic engineering in C. glutamicum, hence, some strategies for C. glutamicum genomic editing had been documented (Cho et al. 2017; Peng et al. 2017; Wang et al. 2018). Jiang (Jiang et al. 2017) was failed to construct CRISPR/Cas9-assisted genomic editing sys- tem, he/she speculated Cas9 protein might toxic to C. glutamicum, and reported Francisella novicida CRISPR/ Cpf1-assisted genome editing system, the efficiency of codon saturation mutagenesis reached near 100% while shown low efficiency in large gene deletion (> 1 kb). Peng (Peng et al. 2017) optimized codon of Cas9 and limit its expression in C. Strains, plasmids, and growth conditions glutamicum to make the cells survived in media, he used two plasmids to express Cas9 nuclease and targeted sgRNA separately. The sgRNA vector was constructed as two-step method: link targeted sgRNA (including N20 sequence) and homologous repaired arm Yao et al. AMB Expr (2021) 11:70 Yao et al. AMB Expr (2021) 11:70 Page 2 of 15 et al. (Jager et al. 1992) was the first to report SacB- based genomic editing in C. glutamicum and Tan (Tan et al. 2012) improving this method and successfully con- structed plasmid pDXW-3, which is a traceless editing method and needs two-step homologous recombination. Cre/loxP-based recombination system was first found in a plasmid of prophage of bacteriophage P1 (Aus- tin et al. 1981) and Nobuaki Suzuki et al. (Suzuki et al. 2004) first reported the Cre/loxP-based method for edit- ing of C. glutamicum genome, this method was suitable to large region (56 kb) deletion. Jinyu Hu et al. (Hu et al. 2013) constructed multiple-gene-deletion system based on Cre/loxP-mediated method in C. glutamicum, this method need three plasmids to edit genome: pBluescript II SK, pDTW202 and pDTW109, pBluescript II SK was used as vector of homologous arms and kanamycin cas- sette, pDTW202 was kanamycin cassette carrier (lox66 and lox71 sequence were added to each end of kana- mycin resistance gene), temperature-sensitive plasmid pDTW109 was used to delete kanamycin cassette from targeted gene locus in chromosome. This method will leave residual sequence in the genomic loci of interest and require two electrical conversions. These methods above are often show low efficiency (< 5%) and need at least 20 days in C. glutamicum. to the vector pFST respectively, electroporation for twice to introduce vector pFSC and pFST into C. glutamicum, so the method was tiring and time-consuming in practi- cal usage. In this research, we succeed in construction of a single- plasmid CRISPR/Cas9-assisted genomic editing for C. glutamicum, all elements including gene cas9, sgRNA and homologous arms were designed in one temper- ature-sensitive plasmid pCCG1 or pCCG2, the whole editing work could be done in 8–9 days and 5 days for continuous editing. The editing efficiency was about 50%–95% in C. glutamicum ATCC 13032, this method was suitable to both small and large (> 3 kb) targeted gene mutation. Meanwhile, this strategy was extended exam- ined in C. glutamicum ATCC 14067 and C. glutamicum ssp. lactofermentum (Shi et al. 2019) (a strain of C. glu- tamicum for l-isoleucine production, genomic sequence is similar to C. glutamicum ATCC 13869). Up to now, our strategy demonstrated high efficiency in editing proce- dure and time-saving compared to the reported. Materials and methods Strains, plasmids, and growth conditions Plasmid constructionh The main primers used in this study are given in Table 2. Kanamycin resistance gene (kanr) with its native pro- moter was amplified from plasmid pEC-XK99E using primers P1/P2; temperature-sensitive replicon pBL1TS in C. glutamicum was amplified from plasmid pDTW109 using primers P3/P4; PUC ori replicon in E. coli was amplified from plasmid pBluescript II SK using primers Yao et al. AMB Expr (2021) 11:70 Yao et al. AMB Expr (2021) 11:70 Page 3 of 15 Yao et al. AMB Expr (2021) 11:70 Table 1 Bacterial strains and plasmids used in this study Strains/plasmids Characteristics Strains E. coli E. coli DH5α F−, Φ80d/lacZΔM15, Δ (lacZYA-argF)U169, deoR, recA1, endA1, hsdR17 supE44, λ−, thi-1, gyrA96, relA E. coli str. K-12 substr. W3110 Wild strain C. glutamicum C. glutamicum ATCC 13032 Wild strain C. glutamicum ATCC 14067 Wild strain C. glutamicum ssp. lactofermentum A strain of C. glutamicum for l-isoleucine production CGN001 C. glutamicum ATCC 13032, Δldh CGN002 C. glutamicum ATCC 13032, ΔeutD CGN003 C. glutamicum ATCC 13032, ΔgabP CGN004 C. glutamicum ATCC 13032, ΔglnA1 CGN005 C. glutamicum ATCC 13032, ΔglnA2 CGN006 C. glutamicum ATCC 13032, ΔalaT CGN007 C. glutamicum ATCC 13032, ΔargR CGN008 C. glutamicum ATCC 13032, ΔgabTD CGN009 C. glutamicum ATCC 13032, ΔaceAB CGN010 C. glutamicum ATCC 13032, ΔpoxB CGG001 C. glutamicum ATCC 13032, Φ(PtacM-gdhA1) CGG002 CGG1, ΔgabP::gadB CGG003 C. glutamicum ATCC 13032, ΔgabP::gadB CGG004 CGG003, ΔeutD::gdhA CGG005 CGG004, ΔgabTD CGC001 C. glutamicum ATCC 13032, ΔeutD::speC CGY001 C. glutamicum ATCC 14067, ΔgabP CGY002 C. glutamicum ssp. lactofermentum, ΔgabP Plasmids pDTW109 Temperature-sensitive plasmid in C. glutamicum, Cmr pFSC A plasmid harboring codon-optimized cas9 gene, kanr pBluescript II SK A plasmid containing PUC ori replicon, ampr pEC-XK99E E. coli—C. glutamicum shuttle plasmid, kanr pJYW-5-gdhA A recombination plasmid harboring gdhA under promoter PtacM, ka pJYW-5-speC A recombination plasmid harboring speC under promoter PtacM, kan pJYW-5-gadB A recombination plasmid harboring gadB under promoter PtacM, ka pJYW-5-gdhA1 A recombination plasmid harboring gdhA1 under promoter PtacM, k pCCG1 E. coli—C. Plasmid constructionh glutamicum shuttle plasmid, CRISPR/cas9 vector, pBL1TS, ka pCCG2 A vector derive from pCCG1, pBL1TS, kanr pBS-sgRNA pBluescript II SK harboring sgRNA sequence pCCG1-ldh pCCG1 harboring ldh deletion elements, kanr pCCG1-eutD pCCG1 harboring eutD deletion elements, kanr pCCG1-gabP pCCG1 harboring gabP deletion elements, kanr pCCG1-glnA1 pCCG1 harboring glnA1 deletion elements, kanr pCCG1-glnA2 pCCG1 harboring glnA2 deletion elements, kanr pCCG1-alaT pCCG1 harboring alaT deletion elements, kanr pCCG1-argR pCCG1 harboring argR deletion elements, kanr pCCG1-gabTD pCCG1 harboring gabTD deletion elements, kanr pCCG1-aceAB pCCG1 harboring aceAB deletion elements, kanr pCCG1-poxB pCCG1 harboring poxB deletion elements, kanr pCCG1-PtacM-gdhA1 The plasmid to replace gdhA1 native promoter with PtacM and artific Table 1 Bacterial strains and plasmids used in this study Strains/plasmids Characteristics Source or reference Strains E. coli E. coli DH5α F−, Φ80d/lacZΔM15, Δ (lacZYA-argF)U169, deoR, recA1, endA1, hsdR17(rk −mk +), phoA, supE44, λ−, thi-1, gyrA96, relA TaKaRa E. coli str. K-12 substr. W3110 Wild strain ND C. glutamicum C. glutamicum ATCC 13032 Wild strain ND C. glutamicum ATCC 14067 Wild strain ND C. glutamicum ssp. lactofermentum A strain of C. glutamicum for l-isoleucine production Shi et al. (2019) CGN001 C. glutamicum ATCC 13032, Δldh This study CGN002 C. glutamicum ATCC 13032, ΔeutD This study CGN003 C. glutamicum ATCC 13032, ΔgabP This study CGN004 C. glutamicum ATCC 13032, ΔglnA1 This study CGN005 C. glutamicum ATCC 13032, ΔglnA2 This study CGN006 C. glutamicum ATCC 13032, ΔalaT This study CGN007 C. glutamicum ATCC 13032, ΔargR This study CGN008 C. glutamicum ATCC 13032, ΔgabTD This study CGN009 C. glutamicum ATCC 13032, ΔaceAB This study CGN010 C. glutamicum ATCC 13032, ΔpoxB This study CGG001 C. glutamicum ATCC 13032, Φ(PtacM-gdhA1) This study CGG002 CGG1, ΔgabP::gadB This study CGG003 C. glutamicum ATCC 13032, ΔgabP::gadB This study CGG004 CGG003, ΔeutD::gdhA This study CGG005 CGG004, ΔgabTD This study CGC001 C. glutamicum ATCC 13032, ΔeutD::speC This study CGY001 C. glutamicum ATCC 14067, ΔgabP This study CGY002 C. glutamicum ssp. lactofermentum, ΔgabP This study Pl id Table 1 Bacterial strains and plasmids used in this study Strains/plasmids Characteristics Source or reference Temperature-sensitive plasmid in C. glutamicum, Cmr Hu et al. (2013) A plasmid harboring codon-optimized cas9 gene, kanr Peng et al. (2017) A plasmid containing PUC ori replicon, ampr Hu et al. (2013) E. coli—C. glutamicum shuttle plasmid, kanr Hu et al. Genomic editing procedureh P5/P6, fused three fragments above to single Fx1 frag- ment with overlap PCR method, lacIq-cas9-T1T2 ter- minator fragment (Fx2) was amplified from pFSC using primers P7/P8, the fragment Fx2 contain three units: DNA-binding transcriptional repressor (LacIq), cas9 gene (under inducible promoter Ptrc) and T1T2 terminator. Ligate NotI-ApaI-digested Fx1, NotI-SmaI-digested Fx2 and artificial synthetic PtacM promoter (annealed prim- ers P9/P10) with T4 ligase (Thermo, USA), the products were transformed into E. coli DH5α to obtain plasmid pCCG-zero. Extracted the plasmid pCCG-zero from E. coli DH5α using TIANprep Mini Plasmid Kit (TIAN- GEN, China), then inserted the synthetic multiple clon- ing site (MCS, annealed primers P11/P12) between BamHI-SmaI-digested pCCG-zero, the new plasmid was named as pCCG1. Amplified fragment Fx3 from plas- mid pCCG1 using primers P13/P14, amplified fragment Fx4 from plasmid pEC-XK99E using primers P15/P16, amplified Fx5 fragment from plasmid pDTW109 using primers P17/18, ligate NotI-XmaJI-digested Fx3, XbaI- XhoI-digested Fx4 and XhoI-NotI-digested Fx5 using T4 ligase, the products were transformed into E. coli DH5α to construct a new plasmid pCCG2. Both plasmids pCCG1 and pCCG2 can utilize to editing genome of C. glutamicum. The genomic editing relies on two processes: Cas9 nucle- ase activity and homologous recombination repair activ- ity. For construction of recombinant plasmid to targeted gene, three elements need to be amplified: sgRNA, up and down homologous sequence flanking the deletion target sequence. In this study, several genes were used as candidates to test this method (Table 3). Take poxB deletion as an example, use primers poxBsg-F/sgRNA-R to amplify sgRNA fragment (pBS-sgRNA as template), use primers poxB-U-F/poxB-U-R and poxB-D-F/poxB- D-R to amplify up and down homologous repair arms (C. glutamicum genome as template), fuse sgRNA, up and down homologous repair arms to one strand using primers poxB-F/poxB-D-R. N20 sequence was added at the 5’-end of primer poxBsg-F and vector’s homolo- gous sequence at the 5’-end of primer poxB-F, ligate the fused fragment to the BamHI-AflII-digested pCCG1 by the ClonExpress® II One Step Cloning Kit (Vazyme, China), then use chemical transformation to construct the recombinant plasmid pCCG1-poxB in E. coli DH5α. The sgRNA-homologous arm fragment also can be ligated to pCCG1/pCCG2 with restriction enzyme diges- tion-T4 ligase method. The design of 20-bp region (N20) with protospacer-adjacent motif (PAM, NGG sequence) matching the targeted gene was programmed artificially or CHOPCHOP/CRISPy-web on net (Blin et al. 2016). Plasmid constructionh lactofermentum was completed by our research group; Cmr, chloramphenicol resistance gene; ampr, ampicillin resistance gene; kanr, kanamycin resistance gene; pBL1TS, temperature-sensitive replicon in C. glutamicum, derived from the plasmid pBL1 (GeneBank GI: 164604819), the replicase is active when temperature between 25 and 28 °C. Targeted gene deletion elements including N20 sequence, sgRNA and homologous repair arms ND: no data; C. glutamicum ATCC 13032, GeneBank GI: 58036263; C. glutamicum ATCC 14067, GeneBank GI: 1229082175; the genomic sequencing of C. glutamicum ssp. lactofermentum was completed by our research group; Cmr, chloramphenicol resistance gene; ampr, ampicillin resistance gene; kanr, kanamycin resistance gene; pBL1TS, temperature-sensitive replicon in C. glutamicum, derived from the plasmid pBL1 (GeneBank GI: 164604819), the replicase is active when temperature between 25 and 28 °C. Targeted gene deletion elements including N20 sequence, sgRNA and homologous repair arms Plasmid constructionh (2014) A recombination plasmid harboring gdhA under promoter PtacM, kanr This study A recombination plasmid harboring speC under promoter PtacM, kanr This study A recombination plasmid harboring gadB under promoter PtacM, kanr This study A recombination plasmid harboring gdhA1 under promoter PtacM, kanr This study E. coli—C. glutamicum shuttle plasmid, CRISPR/cas9 vector, pBL1TS, kanr This study A vector derive from pCCG1, pBL1TS, kanr This study pBluescript II SK harboring sgRNA sequence This study pCCG1 harboring ldh deletion elements, kanr This study pCCG1 harboring eutD deletion elements, kanr This study pCCG1 harboring gabP deletion elements, kanr This study pCCG1 harboring glnA1 deletion elements, kanr This study pCCG1 harboring glnA2 deletion elements, kanr This study pCCG1 harboring alaT deletion elements, kanr This study pCCG1 harboring argR deletion elements, kanr This study pCCG1 harboring gabTD deletion elements, kanr This study pCCG1 harboring aceAB deletion elements, kanr This study pCCG1 harboring poxB deletion elements, kanr This study The plasmid to replace gdhA1 native promoter with PtacM and artificial RBS, kanr This study Yao et al. AMB Expr (2021) 11:70 Yao et al. AMB Expr (2021) 11:70 Page 4 of 15 Table 1 (continued) Strains/plasmids Characteristics Source or reference pCCG1-ΔalaT::gdhA The plasmid to insert gdhA into alaT, kanr This study pCCG2-ΔalaT::gdhA The plasmid to insert gdhA into alaT, kanr This study pCCG1-ΔeutD::speC The plasmid to insert speC into eutD, kanr This study pCCG1-ΔgabP::gadB The plasmid to insert gadB into gabP, kanr This study pCCG1-ΔgabP::gadB2-gadB1m The plasmid to insert cluster gadB2B1m into gabP, kanr This study pCCG1-ΔeutD::gdhA The plasmid to insert gdhA into eutD, kanr This study pCCG1-gabP (14067) pCCG1 harboring gabP deletion elements in ATCC 14067, kanr This study pCCG1-gabP (ssp. lactofermentum) pCCG1 harboring gabP deletion elements in ssp. lactofermentum, kanr This study ND: no data; C. glutamicum ATCC 13032, GeneBank GI: 58036263; C. glutamicum ATCC 14067, GeneBank GI: 1229082175; the genomic sequencing of C. glutamicum ssp. lactofermentum was completed by our research group; Cmr, chloramphenicol resistance gene; ampr, ampicillin resistance gene; kanr, kanamycin resistance gene; pBL1TS, temperature-sensitive replicon in C. glutamicum, derived from the plasmid pBL1 (GeneBank GI: 164604819), the replicase is active when temperature between 25 and 28 °C. Targeted gene deletion elements including N20 sequence, sgRNA and homologous repair arms ND: no data; C. glutamicum ATCC 13032, GeneBank GI: 58036263; C. glutamicum ATCC 14067, GeneBank GI: 1229082175; the genomic sequencing of C. glutamicum ssp. Genomic editing procedureh In this research, fifteen candidate genes were used to test this strategy, gene knockout: ldh, eutD, gabP, glnA1, glnA2, alaT, argR, gabTD, aceAB, poxB; substitution of gene native promoter: gdhA1; gene insertion at targeted loci of genome: eutD:: speC, gabP:: gadB, eutD:: gdhA, alaT:: gdhA. When insert gene speC, gadB and gdhA into eutD, gabP and eutD, cloning the gene (including pro- moter PtacM) using primers speC-F/speC-R, gadB-F/ gadB-R, gdhA-F/gdhA-R and plasmids pJYW-5-speC, pJYW-5-gadB, pJYW-5-gdhA as template respectively, In this research, tracrRNA and crRNA was fused to a single synthetic guide RNA (sgRNA), which is 82-bp frag- ment. In order to facilitate the editing procedure, another plasmid pBS-sgRNA was constructed, the procession was as followings: linearized plasmid pBluescript II SK by PCR with primers P19/20, purified the products with SanPrep Column DNA Gel Extraction Kit (Sangon Bio- tech, China) and digested with XbaI-NcoI, then ligated it with NheI-NcoI-digested sgRNA (artificial synthesis) using T4 ligase, the products were transformed into E. coli DH5α to construct plasmid pBS-sgRNA, which was used as vector for cloning sgRNA fragment. Page 5 of 15 Yao et al. AMB Expr (2021) 11:70 Yao et al. AMB Expr (2021) 11:70 Yao et al. AMB Expr (2021) 11:70 Table 2 The main primers used in this study Primers Sequence P1 CATGCGGCCGCTTAGCTTGCAGTGGGCTTACATGG P2 CGGTCCGGAAACCCCAGAGTCCCGCTCAG P3 CGGTCCGGAGCAGTCATGTCGTGCTAATGTGTAAAAC P4 CCGCTCGAGCATTGTCAACAACAAGACCCATCAT P5 CCGCTCGAGGGGATAACGCAGGAAAGAACATG P6 GCGGGCCCCCTTAACGTGAGTTTTCGTTCCACT P7 CATGCGGCCGCCCAGCTCATAGACCGTATCCAAAG P8 TCCCCCGGGTTTCCTGCGTTATCCCCTGATT P9 CTTGAGCTGTTGACAATTAATCATCGTGTGGTACCATACTAGTGGATCCTTCCTACCC P10 GGGTAGGAAGGATCCACTAGTATGGTACCACACGATGATTAATTGTCAACAGCTCAAGGGCC P11 GATCCTTCCTAGCTAGCTTGGTTGGCGCGCCAATACTTAAGCCC P12 GGGCTTAAGTATTGGCGCGCCAACCAAGCTAGCTAGGAAG P13 CATGCGGCCGCCAGTGGAACGAAAACTCACGTTAAG P14 GCTCCTAGGAGCTCATAGACCGTATCCAAAGCAT P15 GCTCTAGATAGCTTGCAGTGGGCTTACATGG P16 ATTCTCGAGTCACGTAGCGATAGCGGAGTGT P17 ATTCTCGAGCTGGCACGCATAGCCAAGCTAG P18 CATGCGGCCGCCATTGTCAACAACAAGACCCATCAT P19 GCTCTAGAGCCTGGGGTGCCTAATGAGT P20 CATGCCATGGTACAACGTCGTGACTGGGAAAAC ldhsg-F GTGGATCCCAGCATGTAGCGGAATCGAGGTTTTAGAGCTAGAAATAGCAAGTTAAAAT ldh-U-F CCGCTCGAGTGCTTCCAGACGGTTTCATC ldh-U-R CACCTTGCGATCATCGACATAAGGGCTCCACTTCCTACGG ldh-D-F CCGTAGGAAGTGGAGCCCTTATGTCGATGATCGCAAGGTG ldh-D-R TTCCTAGCTAGCATCGGCCAAGGTCAAAGTG eutDsg-F CGGGATCCGTCGCCGTTGTGGACCATCAGTTTTAGAGCTAGAAATAGCAAGTTAAAAT eutD-U-F CCGCTCGAGACAAATTCATGGGAGGTGCC eutD-U-R AAGCAAGGCAAGCACGTTGGCGCAAGAAGATGCCAGACT eutD-D-F AGTCTGGCATCTTCTTGCGCCAACGTGCTTGCCTTGCTT eutD-D-R GCTCTAGAGCGTAGATTCCTGCTGGACC gabPsg-F GTGGATCCGTGGAGGCATTGATCACCACGTTTTAGAGCTAGAAATAGCAAGTTAAAAT sgRNA-R CACGACAGGTTTCCCGACTG gabP-F GTGGTACCATACTAGTGGATCCGTGGAGGCATTGATC gabP-U-F CAGTCGGGAAACCTGTCGTGTGCCACTTTCACCGACTTGG gabP-U-R CACAAACGCCAGGGAGTACAGCAGGGATACTTCGGCGATG gabP-D-F CATCGCCGAAGTATCCCTGCTGTACTCCCTGGCGTTTGTG gabP-D-R GCCAACCAAGCTAGCTAGGAACCGTGATGCTGCCTCTTCTAG glnA1sg-F CGTGGATCCCGTGGAGCATGGTGTTGAAGGTTTTAGAGCTAGAAATAGCAAGTTAAAAT glnA1-U-F ATTCTCGAGACAATAGCAATAACCCAGGAAACAC glnA1-U-R AGGAGTTCAGGGTTGCGTTGCGAGGTCTGGCAGGAGATTC glnA1-D-F GAATCTCCTGCCAGACCTCGCAACGCAACCCTGAACTCCT glnA1-D-R TGCTCTAGACATCTGAACTGATCGGCATCTAG glnA2sg-F CGTGGATCCGAAATATCCGCCGTTGTCAGGTTTTAGAGCTAGAAATAGCAAGTTAAAAT glnA2sg-R GGATTTGTTGTGGGGCTTGTCACGACAGGTTTCCCGACTG glnA2-F CGTGGATCCGAAATATCCGCCGTTGTCAG glnA2-U-F CAGTCGGGAAACCTGTCGTGACAAGCCCCACAACAAATCC glnA2-U-R CCAGATCCATCGACATTCCATTCGTGTTCCTACCTACCGTTTG glnA2-D-F CAAACGGTAGGTAGGAACACGAATGGAATGTCGATGGATCTGG glnA2-D-R TTCCTAGCTAGCCTCATCGGAGCAGGAGTAAGC CATGCGGCCGCTTAGCTTGCAGTGGGCTTACATGG CGGTCCGGAAACCCCAGAGTCCCGCTCAG CGGTCCGGAGCAGTCATGTCGTGCTAATGTGTAAAAC CCGCTCGAGCATTGTCAACAACAAGACCCATCAT CCGCTCGAGGGGATAACGCAGGAAAGAACATG GCGGGCCCCCTTAACGTGAGTTTTCGTTCCACT CATGCGGCCGCCCAGCTCATAGACCGTATCCAAAG TCCCCCGGGTTTCCTGCGTTATCCCCTGATT CTTGAGCTGTTGACAATTAATCATCGTGTGGTACCATACTAGTGGATCCTTCCTACCC GGGTAGGAAGGATCCACTAGTATGGTACCACACGATGATTAATTGTCAACAGCTCAAGGGCC GATCCTTCCTAGCTAGCTTGGTTGGCGCGCCAATACTTAAGCCC GGGCTTAAGTATTGGCGCGCCAACCAAGCTAGCTAGGAAG CATGCGGCCGCCAGTGGAACGAAAACTCACGTTAAG GCTCCTAGGAGCTCATAGACCGTATCCAAAGCAT GCTCTAGATAGCTTGCAGTGGGCTTACATGG ATTCTCGAGTCACGTAGCGATAGCGGAGTGT ATTCTCGAGCTGGCACGCATAGCCAAGCTAG CATGCGGCCGCCATTGTCAACAACAAGACCCATCAT GCTCTAGAGCCTGGGGTGCCTAATGAGT CATGCCATGGTACAACGTCGTGACTGGGAAAAC GTGGATCCCAGCATGTAGCGGAATCGAGGTTTTAGAGCTAGAAATAGCAAGTTAAAAT CCGCTCGAGTGCTTCCAGACGGTTTCATC CACCTTGCGATCATCGACATAAGGGCTCCACTTCCTACGG CCGTAGGAAGTGGAGCCCTTATGTCGATGATCGCAAGGTG TTCCTAGCTAGCATCGGCCAAGGTCAAAGTG CGGGATCCGTCGCCGTTGTGGACCATCAGTTTTAGAGCTAGAAATAGCAAGTTAAAAT CCGCTCGAGACAAATTCATGGGAGGTGCC AAGCAAGGCAAGCACGTTGGCGCAAGAAGATGCCAGACT AGTCTGGCATCTTCTTGCGCCAACGTGCTTGCCTTGCTT GCTCTAGAGCGTAGATTCCTGCTGGACC GTGGATCCGTGGAGGCATTGATCACCACGTTTTAGAGCTAGAAATAGCAAGTTAAAAT CACGACAGGTTTCCCGACTG GTGGTACCATACTAGTGGATCCGTGGAGGCATTGATC CAGTCGGGAAACCTGTCGTGTGCCACTTTCACCGACTTGG CACAAACGCCAGGGAGTACAGCAGGGATACTTCGGCGATG CATCGCCGAAGTATCCCTGCTGTACTCCCTGGCGTTTGTG GCCAACCAAGCTAGCTAGGAACCGTGATGCTGCCTCTTCTAG CGTGGATCCCGTGGAGCATGGTGTTGAAGGTTTTAGAGCTAGAAATAGCAAGTTAAAAT ATTCTCGAGACAATAGCAATAACCCAGGAAACAC AGGAGTTCAGGGTTGCGTTGCGAGGTCTGGCAGGAGATTC GAATCTCCTGCCAGACCTCGCAACGCAACCCTGAACTCCT TGCTCTAGACATCTGAACTGATCGGCATCTAG CGTGGATCCGAAATATCCGCCGTTGTCAGGTTTTAGAGCTAGAAATAGCAAGTTAAAAT GGATTTGTTGTGGGGCTTGTCACGACAGGTTTCCCGACTG CGTGGATCCGAAATATCCGCCGTTGTCAG CAGTCGGGAAACCTGTCGTGACAAGCCCCACAACAAATCC CCAGATCCATCGACATTCCATTCGTGTTCCTACCTACCGTTTG CAAACGGTAGGTAGGAACACGAATGGAATGTCGATGGATCTGG TTCCTAGCTAGCCTCATCGGAGCAGGAGTAAGC Page 6 of 15 Yao et al. AMB Expr (2021) 11:70 Yao et al. AMB Expr (2021) 11:70 Yao et al. Genomic editing procedureh AMB Expr (2021) 11:70 Table 2 (continued) Primers Sequence gdhA1sg-F CGTGGATCCGCAAACGCCTAGGATGTACAGTTTTAGAGCTAGAAATAGCAAGTTAAAAT gdhA1-F GTGGTACCATACTAGTGGATCCGCAAACGCCTAGGATG gdhA1-U-F CAGTCGGGAAACCTGTCGTGTGATGCGGTAGCGGTTCCTTTG gdhA1-U-R GTTCACATCAACCGGCTTGTCATAC gdhA1-D-F GTATGACAAGCCGGTTGATGTGAACGGGGAAGAATTAGGCAGGCATC gdhA1-D-R AAGCTAGACCCGGGCTTAAGCTCACGAAGTAAACGCAGCCGTAG alaTsg-F CGTGGATCCGGTTGCCAGGCTGATGTGCTGTTTTAGAGCTAGAAATAGCAAGTTAAAAT alaTsg-R ATTCTCGAGCACGACAGGTTTCCCGACTG alaT-U-F ATTCTCGAGCGGGGTAATGCCATAACGAG alaT-U-R GAATAGCGTGCTGAGCTGGGCGGAATAATGCCTTTGGAGGT alaT-D-F ACCTCCAAAGGCATTATTCCGCCCAGCTCAGCACGCTATTC alaT-D-R TTCCTAGCTAGCGACGCAGCAAGACCTGACATAC argRsg-F CGTGGATCCTCGCGGGCGATGCTATCTACGTTTTAGAGCTAGAAATAGCAAGTTAAAAT argRsg-R CAAAGCCAATCATGTAGGAGTTGCACGACAGGTTTCCCGACTG argR-F TGGTACCATACTAGTGGATCCTCGCGGGCGATGCTATCTAC argR-U-F CAGTCGGGAAACCTGTCGTGCAACTCCTACATGATTGGCTTTG argR-U-R CGAGAACGAAAACGGTGTCATAGTTGTACCTGGCTGGTGACTT argR-D-F AAGTCACCAGCCAGGTACAACTATGACACCGTTTTCGTTCTCG argR-D-R TGGCGCGCCAACCAAGCTAGCAACCTGGTCGTTGCCCTTAC gabsg-F CGGGATCCCCAGTAATCAACCCCAGCGAGTTTTAGAGCTAGAAATAGCAAGTTAAAAT gab-U-F CCGCTCGAGACGAGTTCGCTGATTTGGATG gab-U-R CAAGTCGGTGAAAGTGGCAACATGGTGAGGTTGGTCCGTC gab-D-F GACGGACCAACCTCACCATGTTGCCACTTTCACCGACTTG gab-D-R GCTCTAGAGGTGCCTAAAACAAAGAATCCAAG aceABsg-F CGTGGATCCGGAAATCCTCGTACGCCTCTGTTTTAGAGCTAGAAATAGCAAGTTAAAAT aceABsg-R GCCTCATCGGTGTCGTTGTAACACGACAGGTTTCCCGACTG aceAB-F TGGTACCATACTAGTGGATCCGGAAATCCTCGTACG aceAB-U-F CAGTCGGGAAACCTGTCGTGTTACAACGACACCGATGAGGC aceAB-U-R GTATCCGAGGATGGACTGGCAGGAACTCGGCGCAATGGGCT aceAB-D-F GGAACTCGGCGCAATGGGCT aceAB-D-R TGGCGCGCCAACCAAGCTAGCCCAGGGTTCGCTACGGAATC poxBsg-F GTGGATCCGGTCACCGGATACTTCACCGGTTTTAGAGCTAGAAATAGCAAGTTAAAAT poxB-F GTGGTACCATACTAGTGGATCCGGTCACCGGATACTTC poxB-U-F CAGTCGGGAAACCTGTCGTGGTTGCACTGCATGATCGGTT poxB-U-R CGCTGAAGGCTGTGGTGTTT poxB-D-F AAACACCACAGCCTTCAGCGTCGCAGTAACCAGAGCATTCC poxB-D-R AAGCTAGACCCGGGCTTAAGGTTTTCGAGGCGACCAGACAG speC-F ATTCTCGAGTCACATGTTCTTTCCTGCGTTATC speC-R AAGCAAGGCAAGCACGTTGTTACTTCAACACATAACCGTACAACCG gdhA-F AGTCTGGCATCTTCTTGCGCGACGTTTGAGCTGTTGACAATTAATC gdhA-R GAGCTCGAATTCTTAAATCACACC gadB-F CATCGCCGAAGTATCCCTGCATTTTGGGGAAGAATTAGGCAG gadB-R CGCCAGGGAGTACATCAGGTATGTTTAAAGCTGTTCTGTT Italics is the site of restriction endonuclease or homologous sequence of vector pCCG1/pCCG2 for recombinant plasmid construction using one-step cloning ligation Sequence on endonuclease or homologous sequence of vector pCCG1/pCCG2 for recombinant plasmid construction using one-step cloning ligatio Italics is the site of restriction endonuclease or homologous sequence of vector pCCG1/pCCG2 for recombinant plasmid construction using one-step cloning ligation Italics is the site of restriction endonuclease or homologous sequence of vector pCCG1/pCCG2 for recombinant plasmid construction using one-step cloning ligation The recombinant plasmid was purified from E. coli DH5α and electroporated into C. glutamicum. The electrocompetent of C. glutamicum was prepared as reported (Jiang et al. 2017) with modification: inoculate then ligate the gene between homologous arms to con- struct the plasmids pCCG1-ΔeutD::speC, pCCG1- ΔgabP::gadB, pCCG1-ΔeutD::gdhA, pCCG1-ΔalaT::gdhA and pCCG2-ΔalaT::gdhA in E. coli DH5α. Page 7 of 15 Page 7 of 15 Yao et al. AMB Expr (2021) 11:70 Yao et al. AMB Expr (2021) 11:70 Table 3 Gene editing in this study Table 3 Gene editing in this study a, LA: the length of homologous arms (bp), which is the up and down homologous sequence flanking the deletion target sequence; b, ND: no data; c, Using the strong promoter PtacM and artificial RBS (GAAAGGACTTGAACG) sequence to replace the native promoter of gene gdhA1 in genome Strains and Genes Locus Plasmids N20 and PAM sites LA (bp)a Gene deletion C. glutamicum ATCC 13032 ldh NCgl2810 PCCG1-ldh CAGCATGTAGCGGAATCGAGCGG 916/989 eutD NCgl2657 pCCG1-eutD GTCGCCGTTGTGGACCATCATGG 800/839 gabP NCgl0464 pCCG1-gabP GTGGAGGCATTGATCACCACTGG 553/580 glnA1 NCgl2133 pCCG1-glnA1 CGTGGAGCATGGTGTTGAAGCGG 700/643 glnA2 NCgl2148 pCCG1-glnA2 GAAATATCCGCCGTTGTCAGTGG 609/630 alaT NCgl2747 pCCG1-alaT GGTTGCCAGGCTGATGTGCTCGG 739/765 argR NCgl1345 pCCG1-argR TCGCGGGCGATGCTATCTACTGG 564/625 gabTD NCgl0462, NCgl0463 pCCG1-gabTD CCAGTAATCAACCCCAGCGATGG 754/754 aceAB NCgl2248, NCgl2247 pCCG1-aceAB GGAAATCCTCGTACGCCTCTTGG 810/829 poxB NCgl2521 pCCG1-poxB GGTCACCGGATACTTCACCGTGG 644/654 C. glutamicum ATCC 14067 gabP CEY17_02745 pCCG1-gabP-2 GTGGAGGCATTGATCACCACTGG 553/580 C. glutamicum ssp. Plasmid curing l d Plasmid pCCG1/pCCG2 harbor temperature-sensitive replicon in C. glutamicum on account of replicase muta- tion pBL1P47S, when the cultivation temperature higher than 34 °C, the replicase turns to inactivated. For curing the plasmid in positive editing cells, inoculate the cells into 4 mL liquid LBHIS media, shake at 37 °C, 200 rpm for 16 h to obtain proper cell density, plate streaking and cultured at 30 °C for 30–36 h to obtain single colony. Col- ony PCR or/and kanamycin-resistance LBHIS plates were used to verify plasmid curing, universal primers cas9Test- F (5′-GAAAACCCAATCAACGCATCT-3′)/cas9Test-R (5′-GCTTAGGCAGCACTTTCTCGT-3′) was designed in cas9 region, which are suitable for all plasmids curing examination, the product is 940-bp-sized fragment if the plasmid existed in cells and nothing for correct curing. For electroporation, ~ 1 μg plasmid was mixed with 80 μL electrocompetent, adding the mixture into pre- cooling 1 mm Gene Pulser cuvette, bath it in ice for 10 min and electroporation at 1.8 kV for twice, the cells were immediately recovered in 0.9 mL pre-cool- ing liquid LBHIS media for 1–1.5 h in shaker at 30 °C, 150 rpm, then spread the cell on LBHIS agar (supple- mented with kanamycin 20 mg/L, isopropyl β-d-1- thiogalactopyranoside, IPTG, 0.01 mM) and culture for 2–3 days at 28 °C until obtain proper size colonies. The colonies were examined by colony PCR method. Genomic editing procedureh lactofermentum gabP NDb pCCG1-gabP-3 GTGGAGGCATTGATCACCACTGG 553/580 Gene insertion Φ(PtacM-gdhA1)c NCgl1999 pCCG1-PtacM-gdhA1 GCAAACGCCTAGGATGTACATGG 721/662 eutD::speC NCgl2657 pCCG1-ΔeutD::speC GTCGCCGTTGTGGACCATCATGG 800/839 gabP::gadB NCgl0464 pCCG1-ΔgabP::gadB GTGGAGGCATTGATCACCACTGG 553/580 gabP::gadB2B1m NCgl0464 pCCG1-ΔgabP::gadB2-gadB1m GTGGAGGCATTGATCACCACTGG 553/580 eutD::gdhA NCgl2657 pCCG1-ΔeutD::gdhA GTCGCCGTTGTGGACCATCATGG 800/849 alaT::gdhA NCgl2747 pCCG1-ΔalaT::gdhA GGTTGCCAGGCTGATGTGCTCGG 739/765 alaT::gdhA NCgl2747 pCCG2-ΔalaT::gdhA GGTTGCCAGGCTGATGTGCTCGG 739/765 N20 and PAM sites LA (bp)a LA (bp)a A: the length of homologous arms (bp), which is the up and down homologous sequence flanking the deletion target sequence; b, ND: no moter PtacM and artificial RBS (GAAAGGACTTGAACG) sequence to replace the native promoter of gene gdhA1 in genome the candidate strain into 4 mL liquid LBHIS media and cultured at 30 °C, 200 rpm for 12–16 h to get pre- culture, 0.5–1 mL of the culture was inoculated into 50 mL media (LBHIS supplemented with Tween-80 1 g/L, l-glycine 25 g/L, l-glycine separately sterilized) and shaking at 30 °C, 200 rpm for 4–5 h until OD562nm reached 0.9–1.0, bath the cells in ice for 15–20 min, centrifugate it at 4000 rpm (~ 3040 g) for 5–10 min to harvest the cells. Wash the cell twice with pre-cooling 10% (vol. / vol.) glycerol, resuspend it in 1 mL 10% glyc- erol, split to 80 μL per tube, and store at –70 °C. Gene gdhA1, glnA1 and glnA2 mutations and l‑Glu fermentation Amplified targeted sgRNA using primers gdhA1sg-F/sgRNA-R, amplified upstream using primers gdhA1-U-F/gdhA1-U-R, amplified PtacM and downstream using primers gdhA1-D-F/gdhA1-D-R (the plasmid pJYW-5-gdhA1 as template), fused three frag- ment by overlap PCR, and ligated the fragment with BamHI-AflII-digested pCCG1 by one-step homologous recombinant cloning kit, transformed the ligated prod- uct into E. coli DH5α to construct plasmid pCCG1- PtacM-gdhA1, and electroporated the purified plasmid into C. glutamicum ATCC 13032. The new strain called CGG001. The next, the plasmids pCCG1-glnA1 and pCCG1-glnA2 was constructed using similar method to delete gene glnA1 and glnA2, the new strains named as CGN004 and CGN005 respectively. To examine the effect, the strains ATCC 13032, CGG001, CGN004 and CGN005 was cultured in the seed media at 30 °C, 200 rpm for 8–10 h until the OD562nm reached 40 ± 3, inocu- late 10% culture in l-glutamic acid fermentation media and fermentation at 30 °C, 200 rpm for 72 h. Supplement urea 4 g/L at 0 h and supplement 2.4 g/L at 10 h, 12 h, 14 h, 17 h and 20 h respectively, the overall additive amount of urea was 16 g/L. The strain CGN004 turns to l-glu- tamine auxotroph after glnA1 been deleted, so, proper l-glutamine must be supplemented in media to make the cells survive, 100 mg/L l-glutamine was added in seed media, fermentation broths add 100 mg/L l-glutamine twice at 0 h and 24 h. HPLC (Shi et al. 2013) was used to assay the concentration of l-glutamic acid and l-Glu- tamine in fermentation broths. arms could be fused to single strand with overlap PCR technology to promote the construction efficiency of recombinant plasmids in E. coli. The sequence of artifi- cial multiple cloning sites (MCS) (Fig. 1b) under PtacM promoter was optimized to ensure every restriction site could be efficiently cut by relevant restriction enzymes. Small sgRNA gene fragment was synthesized and ligated with plasmid pBluescript II SK (the new plasmid named as plasmid pBS-sgRNA, Fig. 1a) to facilitate its conserva- tion and cloning. The full sequence of sgRNA (without N20) in pBS-sgRNA was showed in Fig. 1c. Application of the system for genomic editing in C. glutamicum In order to test our CRISPR/cas9 system, gene ldh, eutD, gabP, glnA1, glnA2, alaT, argR, gabTD, aceAB, poxB were deleted in C. glutamicum ATCC 13032 genome, the results were listed in Table 4. The deletion efficiency of genes was mainly between 26%-90% in C. glutamicum ATCC 13032. gabP was also deleted in C. glutamicum ATCC 14067 and C. glutamicum ssp. lactofermentum genome, the efficiency was 38.4% and 25.0% respectively, lower than 82.6% in C. glutamicum ATCC 13032. Para- doxically, the gene deletion efficiency was great related to gene itself, for example, the deletion efficiency of argR in ATCC 13032 was 60.0% using TCGCGGGCGATGCTA TCTAC sequence, nevertheless, the gene cluster argFR deletion efficiency was 0% when with the same N20 sequence. In this research, four genes were inserted into targeted genes in C. glutamicum ATCC 13032 genome: eutD:: speC, gabP:: gadB, eutD:: gdhA and alaT:: gdhA, Based on the experimental results, the efficiency of gene inser- tion was lower than deletion in most cases, meanwhile, the different gene insertion at same locus might show dif- ferent efficiency even if using the same N20 and homolo- gous repaired arms. In this research, derived from E. coli W3110 gene speC (2261 bp, including promoter) and gdhA (1459 bp, including promoter) were inserted into eutD with same N20 and homologous arms, the effi- ciency were 34.8% and 69.6% respectively, the editing efficiency was contrary to the size of inserted gene, and lower than eutD deletion (86.4%), the efficiency of gabP:: gadB (30.4%) also lower than gabP deletion (82.6%). Gene gdhA1, glnA1 and glnA2 mutations and l‑Glu fermentation Corynebacterium glutamicum is an important indus- trial strain for l-glutamic acid and l-Glutamine produc- tion. Glutamate dehydrogenase (gdhA1) and glutamine Yao et al. AMB Expr (2021) 11:70 Yao et al. AMB Expr (2021) 11:70 Page 8 of 15 Page 8 of 15 synthase (glnA1 and glnA2) were key genes related to both amino acids’ biosynthesis in C. glutamicum ATCC 13032 (Fig. 2a). In this research, the strong constitutive promoter PtacM and artificial RBS was used to substi- tute gdhA1 native promoter in C. glutamicum ATCC 13032 chromosome. Amplified targeted sgRNA using primers gdhA1sg-F/sgRNA-R, amplified upstream using primers gdhA1-U-F/gdhA1-U-R, amplified PtacM and downstream using primers gdhA1-D-F/gdhA1-D-R (the plasmid pJYW-5-gdhA1 as template), fused three frag- ment by overlap PCR, and ligated the fragment with BamHI-AflII-digested pCCG1 by one-step homologous recombinant cloning kit, transformed the ligated prod- uct into E. coli DH5α to construct plasmid pCCG1- PtacM-gdhA1, and electroporated the purified plasmid into C. glutamicum ATCC 13032. The new strain called CGG001. The next, the plasmids pCCG1-glnA1 and pCCG1-glnA2 was constructed using similar method to delete gene glnA1 and glnA2, the new strains named as CGN004 and CGN005 respectively. To examine the effect, the strains ATCC 13032, CGG001, CGN004 and CGN005 was cultured in the seed media at 30 °C, 200 rpm for 8–10 h until the OD562nm reached 40 ± 3, inocu- late 10% culture in l-glutamic acid fermentation media and fermentation at 30 °C, 200 rpm for 72 h. Supplement urea 4 g/L at 0 h and supplement 2.4 g/L at 10 h, 12 h, 14 h, 17 h and 20 h respectively, the overall additive amount of urea was 16 g/L. The strain CGN004 turns to l-glu- tamine auxotroph after glnA1 been deleted, so, proper l-glutamine must be supplemented in media to make the cells survive, 100 mg/L l-glutamine was added in seed media, fermentation broths add 100 mg/L l-glutamine twice at 0 h and 24 h. HPLC (Shi et al. 2013) was used to assay the concentration of l-glutamic acid and l-Glu- tamine in fermentation broths. synthase (glnA1 and glnA2) were key genes related to both amino acids’ biosynthesis in C. glutamicum ATCC 13032 (Fig. 2a). In this research, the strong constitutive promoter PtacM and artificial RBS was used to substi- tute gdhA1 native promoter in C. glutamicum ATCC 13032 chromosome. Construction of CRISPR/Cas9‑assisted system for one‑step genomic editing in C. glutamicum The E. coli—C. glutamicum shuttle plasmid of CRISPR/ Cas9 vector were constructed and named as pCCG1/ pCCG2. The overview diagram of its construction was showed in Fig. 1a. The vector pCCG1 (10077 bp) was first constructed, and then another vector pCCG2 (10147 bp) was following been constructed, both plasmids are functional in genomic editing of C. glutamicum. cas9 expression was designed under the control of inducible promoter Ptrc. sgRNA was under constitutive promoter PtacM to insure its expression level. The construction of recombinant plasmid for targeted gene could utilize the method of restriction enzymes digestion-T4 ligase or homologous recombinant cloning kit. The fragment sgRNA, upstream and downstream homologous repair The vector pCCG1 and pCCG2 were both used to insert gdhA into alaT. Gene gdhA was cloned from E. coli W3110 genome and ligated it between alaT up and down homologous arms. The efficiency was 26.1% and 21.7% for pCCG1 and pCCG2 respectively. Editing of genes gdhA1, glnA1 and glnA2 in C. glutamicum In this research, the plasmid pCCG1-PtacM-gdhA1 was designed to replace the gdhA1 native promoter using Yao et al. AMB Expr (2021) 11:70 Page 9 of 15 the strong promoter PtacM and artificial RBS, the plas- mid profile and agarose gel eletrophoresis was showed in Fig 2b pCCG1 glnA1 and pCCG1 glnA2 were used to In this research, the plasmid pCCG1-PtacM-gdhA1 was used to study the stability of recombinant plasmid pCCG1/pCCG2 in C glutamicum Some cells which har Fig. 1 Construction of CRISPR/Cas9-assisted system. a The overview diagram of vectors pCCG1, pCCG2 and pBS-sgRNA construction, E. coli—C. glutamicum shuttle expression vector pCCG1 and pCCG2 is kanamycin resistance, Cas9 nuclease gene is under the control of the inducible promoter Ptrc, pBL1TS is temperature-sensitive replicon in C. glutamicum, constitutive promoter PtacM and multiple cloning site (MCS) were artificial synthesis, sgRNA were expressed by promoter PtacM. Small sgRNA sequence was ligated with plasmid pBluescript II SK. sgRNA fragment were amplified from the plasmid pBS-sgRNA when needed. b The sequence of multiple cloning sites, including BamHI, NheI, Asc I, AflII and SmaI (XmaI) restriction enzyme sites. c The full nucleotide sequence of sgRNA fragment (171 bp) in plasmid pBS-sgRNA, the red part is Cas9 nuclease recognition site (82 bp) Fig. 1 Construction of CRISPR/Cas9-assisted system. a The overview diagram of vectors pCCG1, pCCG2 and pBS-sgRNA construction, E. coli—C. Construction of CRISPR/Cas9‑assisted system for one‑step genomic editing in C. glutamicum glutamicum shuttle expression vector pCCG1 and pCCG2 is kanamycin resistance, Cas9 nuclease gene is under the control of the inducible promoter Ptrc, pBL1TS is temperature-sensitive replicon in C. glutamicum, constitutive promoter PtacM and multiple cloning site (MCS) were artificial synthesis, sgRNA were expressed by promoter PtacM. Small sgRNA sequence was ligated with plasmid pBluescript II SK. sgRNA fragment were amplified from the plasmid pBS-sgRNA when needed. b The sequence of multiple cloning sites, including BamHI, NheI, Asc I, AflII and SmaI (XmaI) restriction enzyme sites. c The full nucleotide sequence of sgRNA fragment (171 bp) in plasmid pBS-sgRNA, the red part is Cas9 nuclease recognition site (82 bp) In this research, the plasmid pCCG1-PtacM-gdhA1 was used to study the stability of recombinant plasmid pCCG1/pCCG2 in C. glutamicum. Some cells which har- boring plasmid pCCG1-PtacM-gdhA1 was cultured at different temperature, the statistical result was showed the strong promoter PtacM and artificial RBS, the plas- mid profile and agarose gel eletrophoresis was showed in Fig. 2b, pCCG1-glnA1 and pCCG1-glnA2 were used to delete glnA1 and glnA2 from chromosome. The efficiency was 95.7%, 56.5% and 69.6% respectively. Page 10 of 15 Page 10 of 15 Yao et al. AMB Expr (2021) 11:70 Yao et al. AMB Expr (2021) 11:70 Table 4 The results of gene editing a, the mutation length is the length of deleted or/and inserted fragments. 166- bp region has been deleted from genome and 150-bp fragment was inserted when replace native gdhA1 promoter in C. glutamicum ATCC 13032; 877-bp region has been deleted from genome and 1459-bp fragment was inserted when insert gdhA into eutD; 877-bp region has been deleted from genome and 2261-bp fragment was inserted when insert speC into eutD; 930-bp region has been deleted from genome and 1557-bp fragment was inserted when insert gadB into gabP; 930-bp region has been deleted from genome and 3167-bp fragment was inserted when insert gene cluster gadB2B1m into gabP; 614-bp region has been deleted from genome and 1459-bp fragment was inserted when insert gdhA into alaT. The gene speC, gdhA and gadB are all under the control of PtacM promoter; b, T: the total number of colonies for PCR verification, C: the number of correct colonies, I: the number of incorrect colonies. The efficiency was calculated by (C/T) 100% Strains and genes Mutation length (bp)a Results (C/I/T)b Efficiency (%) Gene deletion C. Construction of CRISPR/Cas9‑assisted system for one‑step genomic editing in C. glutamicum glutamicum ATCC 13032 ldh 568 15/5/20 75.0 eutD 877 19/3/22 86.4 gabP 930 19/4/23 82.6 glnA1 730 13/10/23 56.5 glnA2 728 16/7/23 69.6 alaT 614 17/6/23 73.9 argR 317 6/4/10 60.0 gabTD 1949 6/17/23 26.1 aceAB 3450 10/13/23 43.5 poxB 980 15/8/23 65.2 C. glutamicum ATCC 14067 gabP 930 8/15/23 34.8 C. glutamicum ssp. lactofermentum gabP 930 2/6/8 25.0 Gene insertion (deletion/insertion) Φ(PtacM-gdhA1) 166/150 22/1/23 95.7 eutD::speC 877/2261 8/15/23 34.8 gabP::gadB 930/1557 7/16/23 30.4 gabP::gadB2B1m 930/3167 5/18/23 21.7 eutD::gdhA 877/1459 16/7/23 69.6 alaT::gdhA (pCCG1) 614/1459 6/17/23 26.1 alaT::gdhA (pCCG2) 614/1459 5/18/23 21.7 Table 4 The results of gene editing fermentation media, the results were depicted in the Fig. 3. The concentration of the l-Glutamic acid reached maximum at 60 h, the strains CGN004 (22.03 g/L) and CGN005 (20.64 g/L) decreased 20.1% and 25.1% com- pared to ATCC 13032 (27.57 g/L). The l-glutamine concentration in CGG001 (2.06 g/L) and CGN005 (4.73 g/L) broths increased 30.4% and 199.4% compared to ATCC 13032 (1.58 g/L), there is no l-glutamine could be detected in CGN004 broths. The cell density of ATCC 13032, CGG001 and CGN005 reached maximum at 36 h while CGN004 was increasing in 72 h and 1.21-fold than ATCC 13032. The average glucose consumption rate decreased 42.9% in CGN004 compared to ATCC 13032 in 24 h. fermentation media, the results were depicted in the Fig. 3. The concentration of the l-Glutamic acid reached maximum at 60 h, the strains CGN004 (22.03 g/L) and CGN005 (20.64 g/L) decreased 20.1% and 25.1% com- pared to ATCC 13032 (27.57 g/L). The l-glutamine concentration in CGG001 (2.06 g/L) and CGN005 (4.73 g/L) broths increased 30.4% and 199.4% compared to ATCC 13032 (1.58 g/L), there is no l-glutamine could be detected in CGN004 broths. The cell density of ATCC 13032, CGG001 and CGN005 reached maximum at 36 h while CGN004 was increasing in 72 h and 1.21-fold than ATCC 13032. The average glucose consumption rate decreased 42.9% in CGN004 compared to ATCC 13032 in 24 h. Discussion In this research, an improved method of CRISPR/Cas9- assisted system for C. glutamicum was established, all elements for the gene editing were designed in one vec- tor pCCG1/pCCG2. Gene editing could be done by electrotrasformation at one time. the recombinant plas- mid for targeted gene could be one-step constructed in E. coli DH5α, the overall procedure of genomic editing was showed in Fig. 4, this method demonstrates simplest operation and time-saving compared to the reported, 8–9 days can complete the whole work of genomic editing and 5 days for continuous editing (the next plasmid was prepared before electrotransformation), so this method is highly efficient and suitable for abundant gene mutation in C. glutamicum. Corynebacterium glutamicum is an important indus- trial microorganism, the studies of metabolic engineering were due to rationally design and optimize the metabolic pathway, these strategies’ implementation often based on high efficient genomic editing tools. The reported genomic editing methods were mainly multiple-plasmid system (Peng et al. 2017; Zhao et al. 2020), or integrated cas9/recET into chromosome before editing targeted gene with traditional method (Cho et al. 2017), the pro- cesses of plasmid construction, introduced one by one, and plasmid curing were complex. Wang et al. (Wang et al. 2018) introduced cas9 and recET into chromosome using plasmids pIN-cas9 and pIN-recET in advance, the plasmids were SacB-based method and need two rounds of homologous recombination, so these methods are labor-intensive and gene cas9/recET remain on chromo- some after editing, sometimes, the exogenous cas9/recET in chromosome might disturbed certain metabolism of C. glutamicum and not suitable to the studies of meta- bolic engineering. Our method designed cas9 in plasmid and don’t need introduce any genes or plasmid into chro- mosome before genomic editing, meanwhile, there was no residual sequence in chromosome after editing, this is in Fig. 2c. The cells showed maximum stability when cul- tured at 28 °C, while the plasmid will start to loss when the temperature higher than 30 °C, the plasmid curing reached nearly 90% at 37 °C, the stability at 25 °C was similar to 28 °C while the cell growth rate will decrease and editing cycle increase at least one day, so the 28 °C is optimal culture temperature when the cells harboring recombinant plasmid pCCG1/pCCG2. Comparison of l‑Glu and l‑Gln production in the mutant strainsh The new strains CGG001, CGN004, CGN005 and ATCC 13032 (as control) were cultured in l-Glutamic acid Yao et al. AMB Expr (2021) 11:70 Page 11 of 15 Fig. 2 l-Glu and l-Gln biosynthetic pathway and the results of gene editing. a l-Glu and l-Gln biosynthetic pathway. Pyr: pyruvate; Ac-CoA: acetyl-CoA; Cit: Citrate; Aco: cis-aconitate; Icit: isocitate; α-KG: alpha-ketoglutaramate; Suc-CoA: succinyl-CoA; Suc: succinate; Fum: fumarate; Mal: malate; OAA: oxaloacetate. b The first lane is DNA marker and the second lane is the control before editing, the upstream primer of gdhA1 was designed in promoter PtacM, there is no PCR product in negative cells. c The plasmid stability at different temperature. The cells harboring plasmid were cultured at 25 °C, 28 °C, 30 °C and 37 °C respectively, the total number of colonies were counted by cfu per plate Fig. 2 l-Glu and l-Gln biosynthetic pathway and the results of gene editing. a l-Glu and l-Gln biosynthetic pathway. Pyr: pyruvate; Ac-CoA: acetyl-CoA; Cit: Citrate; Aco: cis-aconitate; Icit: isocitate; α-KG: alpha-ketoglutaramate; Suc-CoA: succinyl-CoA; Suc: succinate; Fum: fumarate; Mal: malate; OAA: oxaloacetate. b The first lane is DNA marker and the second lane is the control before editing, the upstream primer of gdhA1 was designed in promoter PtacM, there is no PCR product in negative cells. c The plasmid stability at different temperature. The cells harboring plasmid were cultured at 25 °C, 28 °C, 30 °C and 37 °C respectively, the total number of colonies were counted by cfu per plate 2013; Shen et al. 2014, 2019). In this research, take poxB deletion in ATCC 13032 as example, N20 sequence of AATCAGCAGATCCGCCTCAT cannot target Cas9 to cut off the genome but GGTCACCGGATACTTCACCG sequence did. Large fragments or genes (> 3 kb) insertion were not suggested due to the size of plasmids, the vector pCCG1/pCCG2 were about 10 kb, the editing efficiency will dramatically decline when the recombinant plasmid larger than 14 kb, 1.6-kb and 3.2-kb fragment integration into gabP, the efficiency was 30.4% and 21.7% respec- tively (Table 4). So large gene (> 3.5 kb) integration in chromosome was suggested to break the gene into some fragments and insert it one by one. Both of pCCG1 and pCCG2 were effective in C. glutamicum while there were some differences, pCCG1 might have higher efficiency while pCCG2 have higher concentration extract from E. coli DH5α, pCCG1 was mainly used in this research. Comparison of l‑Glu and l‑Gln production in the mutant strainsh To compare the pCCG1 and pCCG2, gdhA was inserted into alaT by pCCG1 and pCCG2 with same N20 and repair another advantage for metabolic engineering research in C. glutamicum. Peng (Peng et al. 2018) constructed two- plasmid system and improved it all-in-one CRISPR/Cas9 system, the chloramphenicol resistant pFSTC is 11.2-kb- sized vector, larger than pCCG1/pCCG2 (10 kb), so pCCG1/pCCG2 have larger loading capacity than pFSTC theoretically. Furthermore, C. glutamicum had higher resistance to kanamycin than chloramphenicol, the incu- bation time of C. glutamicum harboring pCCG1/pCCG2 on LBHIS plate will decrease 2–3 days than pFSTC, pCCG1/pCCG2 indicated shorter editing cycle and more efficient. fi The genomic editing of C. glutamicum was time-con- suming and low efficiency, the efficiency of Cas9/Cpf1- based editing were often between 20–90%, The plasmid pCCG1/pCCG2 also have some deficiencies as well, it’s hard to reach 100% might because the lack of typical CRISPR RNA sequence in C. glutamicum (Jiang et al. 2017) or Off-Target effect of Cas9 nuclease (Fu et al. Yao et al. AMB Expr (2021) 11:70 Page 12 of 15 Fig. 3 The fermentation results of the strains CGG001, CGN004 and CGN005. C. glutamicum ATCC 13032 was used as control. l-Glu: l-Glutamic acid; l-Gln: l-Glutamine. The results showed the concentration of l-Glu and l-Gln in fermentation broths. Meanwhile, the cell density (OD562nm) and the glucose concentration also been showed in the results. Data was shown as mean ± CI (Confidence Interval), 3 independent experiments for statistics, α = 0.01 Fig. 3 The fermentation results of the strains CGG001, CGN004 and CGN005. C. glutamicum ATCC 13032 was used as control. l-Glu: l-Glutamic acid; l-Gln: l-Glutamine. The results showed the concentration of l-Glu and l-Gln in fermentation broths. Meanwhile, the cell density (OD562nm) and the glucose concentration also been showed in the results. Data was shown as mean ± CI (Confidence Interval), 3 independent experiments for statistics, α = 0.01 cell survived (0.01 mM was suggested). Cas9 limited- expression can be achieved with two methods: first, supplement 0.01–0.02 mM IPTG in LBHIS agar plates, this method can make cas9 little continuous expres- sion; second, supplement 0.1 mM IPTG in liquid recov- ery LBHIS media after electroporation and short-time induce cas9 expression for 1–1.5 h, then centrifugate the culture to collect the cell, discard the supernatant to remove IPTG, resuspend the cells in 0.1 mL LBHIS and plate streaking, the first method maybe more con- venient. Abbreviations l h h E. coli: Escherichia coli; PCR: Polymerase chain reaction; IPTG: Isopropyl β-d- 1-thiogalactopyranoside; LBHIS: LB media containing brain heart infusion solution. Acknowledgements We thank Professor Zhonghu Bai from Jiangnan University for providing the plasmid pFSC in this research. Comparison of l‑Glu and l‑Gln production in the mutant strainsh sgRNA fragment was amplified from plasmid pBS-sgRNA, because sgRNA is 82-bp-sized small frag- ment, we amplified downwards the sequence to obtain about 200-bp-size production (Fig. 1c) to facilitate the later experiment (sgRNA-R was used as univer- sal primer). This CRISPR/Cas9-assisted method needs homology-directed system to repair double-strand breaks (DSBs) (Malyarchuk et al. 2007; Wilson et al. 2003), the proper length of homologous repaired arms arms, the efficiency is 26.1% and 21.7% respectively (Table 4), we lack enough data to compare the efficiency of pCCG1 and pCCG2, but both of them were effective in C. glutamicum.h This method was ideal in C. glutamicum ATCC 13032 and ATCC 14067, other strains of C. glutamicum might show lower effective (Table 4), perhaps the replicon pBL1TS was not suitable for these strains. Some recombi- nant plasmids might impact the growth of E. coli DH5α, the cells’ growth rate and density might lower than regu- lar E. coli DH5α harboring empty vector pCCG1/pCCG2. To solve this problem, E. coli DH5α could be cultured for longer time (16–20 h) or use not less than 5 mL culture to extract recombinant plasmids. Cas9 nuclease is toxic for C. glutamicum. In this strategy, cas9 were expressed under inducible pro- moter Ptrc, low concentration IPTG were supple- mented in media to limit cas9 expression, 0.01–0.05 mM IPTG was proper concentration and can make the Yao et al. AMB Expr (2021) 11:70 Page 13 of 15 Fig. 4 The diagram of genomic editing procedure. a Day 1–3: The recombinant plasmid construction for targeted gene X in E. coli DH5α. Day 4–6, electroporate the plasmid into the electrocompetent cell of C. glutamicum and cultured for 2–3 days at 28 °C, examine the colonies using colony PCR method. N20 sequence at 5′-end of sgRNA was complementary to the targeted gene X, which guide Cas9 to break genome at PAM (NGG) site, the upstream and downstream homologous sequence in plasmid was used to repair the DSBs. Day 7–9, cure the plasmids in positive cells. If continuous editing was needed, make electrocompetent cells of the positive cells, electroporate another plasmid into it for the next editing cycle Fig. 4 The diagram of genomic editing procedure. a Day 1–3: The recombinant plasmid construction for targeted gene X in E. coli DH5α. Day 4–6, electroporate the plasmid into the electrocompetent cell of C. Comparison of l‑Glu and l‑Gln production in the mutant strainsh It showed the maximum number of colonies at 28 °C, so 28 °C was utilized to culture the cells harboring recom- binant plasmid pCCG1/pCCG2 considering with Cas9 nuclease activity, homologous repaired activity and cell growth velocity. Meanwhile, the incubation time could be decreased one day at 28 °C compared to 25 °C on the LBHIS plates, and revealed higher efficiency for large gene editing (> 2 kb). The cells harboring recom- binant plasmid pCCG1/pCCG2 sometimes appeared some extremely big colonies on LBHIS plates after Comparison of l‑Glu and l‑Gln production in the mutant strainsh glutamicum and cultured for 2–3 days at 28 °C, examine the colonies using colony PCR method. N20 sequence at 5′-end of sgRNA was complementary to the targeted gene X, which guide Cas9 to break genome at PAM (NGG) site, the upstream and downstream homologous sequence in plasmid was used to repair the DSBs. Day 7–9, cure the plasmids in positive cells. If continuous editing was needed, make electrocompetent cells of the positive cells, electroporate another plasmid into it for the next editing cycle were suggested between 500–1000 bp. pCCG1/pCCG2 is temperature-sensitive plasmid in C. glutamicum due to the replicon pBL1P47S mutation, plasmid pCCG1- PtacM-gdhA1 was used to test its stability in C. glu- tamicum ATCC 13032 (Fig. 2c), the colonies in plates were counted at 25 °C, 28 °C, 30 °C and 37 °C. It’s stable at 25–28 °C and will lost when the temperature ≥ 30 °C. The plasmid curing efficiency will up to 90% at 37 °C. It showed the maximum number of colonies at 28 °C, so 28 °C was utilized to culture the cells harboring recom- binant plasmid pCCG1/pCCG2 considering with Cas9 nuclease activity, homologous repaired activity and cell growth velocity. Meanwhile, the incubation time could be decreased one day at 28 °C compared to 25 °C on the LBHIS plates, and revealed higher efficiency for large gene editing (> 2 kb). The cells harboring recom- binant plasmid pCCG1/pCCG2 sometimes appeared some extremely big colonies on LBHIS plates after electrotansformation, these colonies were fast-growing (might appeared in one day on plate) compared to reg- ular one on LBHIS plates, it is not correct mutant and not necessary to examine it with colony PCR method, the reasons of this phenomenon are unknown, perhaps related to the replicon itself. were suggested between 500–1000 bp. pCCG1/pCCG2 is temperature-sensitive plasmid in C. glutamicum due to the replicon pBL1P47S mutation, plasmid pCCG1- PtacM-gdhA1 was used to test its stability in C. glu- tamicum ATCC 13032 (Fig. 2c), the colonies in plates were counted at 25 °C, 28 °C, 30 °C and 37 °C. It’s stable at 25–28 °C and will lost when the temperature ≥ 30 °C. The plasmid curing efficiency will up to 90% at 37 °C. References Altenbuchner J (2016) Editing of the Bacillus subtilis genome by the CRISPR– Cas9 system. 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Plasmid 103:25–35. https://doi.org/10.1016/j.plasmid.2019.04.001 Author details 1 1 State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, China. 2 International Joint Laboratory On Food Safety, Jiangnan Univer- sity, Wuxi, Jiangsu, China. 3 Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China. Liu X, Yang Y, Zhang W, Sun Y, Peng F, Jeffrey L, Harvey L, McNeil B, Bai Z (2016) Expression of recombinant protein using Corynebacterium Glutamicum: progress, challenges and applications. Crit Rev Biotechnol 36(4):652–664. https://doi.org/10.3109/07388551.2015.1004519 Received: 12 May 2021 Accepted: 13 May 2021 Liu J, Wang Y, Lu Y, Zheng P, Sun J, Ma Y (2017) Development of a CRISPR/Cas9 genome editing toolbox for Corynebacterium glutamicum. Microb Cell Fact 16(1):205. https://doi.org/10.1186/s12934-017-0815-5 Lv Y, Wu Z, Han S, Lin Y, Zheng S (2012) Construction of recombinant Corynebacterium glutamicum for L-threonine production. Biotechnol Bioproc E 17(1):16–21. https://doi.org/10.1007/s12257-011-0360-8 Availability of data and materials The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request. Hu J, Tan Y, Li Y, Hu X, Xu D, Wang X (2013) Construction and application of an efficient multiple-gene-deletion system in Corynebacterium glutamicum. Plasmid 70(3):303–313. https://doi.org/10.1016/j.plasmid.2013.07.001 Competing interests The authors declare that they have no conflict of interest. Jiang Y, Qian F, Yang J, Liu Y, Dong F, Xu C, Sun B, Chen B, Xu X, Li Y, Wang R, Yang S (2017) CRISPR-Cpf1 assisted genome editing of Corynebacterium glutamicum. Nat Commun 8:15179. https://doi.org/10.1038/ncomm s15179 Authors’ contributions XW and XH proposed the idea, CY designed the experiments and wrote the first manuscript, XW modified and supervised this manuscript. All authors read and approved the final manuscript. Yao et al. AMB Expr (2021) 11:70 Page 14 of 15 Page 14 of 15 Yao et al. AMB Expr (2021) 11:70 Consent for publication Not applicable. Consent for publication Not applicable. Jiang Y, Chen B, Duan C, Sun B, Yang J, Yang S (2015) Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system. Appl Environ Micro- biol 81(7):2506–2514. https://doi.org/10.1128/AEM.04023-14 Declarations Hu J, Li Y, Zhang H, Tan Y, Wang X (2014) Construction of a novel expression system for use in Corynebacterium glutamicum. Plasmid 75:18–26. https:// doi.org/10.1016/j.plasmid.2014.07.005 Hu J, Li Y, Zhang H, Tan Y, Wang X (2014) Construction of a novel expression system for use in Corynebacterium glutamicum. 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Nat Commun 10(1):1053. https://doi.org/10.1038/ s41467-019-09005-3 Tan Y, Xu D, Li Y, Wang X (2012) Construction of a novel sacB-based system for marker-free gene deletion in Corynebacterium glutamicum. Plasmid 67(1):44–52. https://doi.org/10.1016/j.plasmid.2011.11.001 Zhang Y, Zhang X, Xiao S, Qi W, Xu J, Yuan Z, Wang Z (2019b) Engineer- ing Corynebacterium glutamicum Mutants for 3-Methyl-1-butanol Production. References Biochem Genet 57(3):443–454. https://doi.org/10.1007/ s10528-019-09906-4 Wada M, Sawada K, Ogura K, Shimono Y, Hagiwara T, Sugimoto M, Onuki A, Yokota A (2016) Effects of phosphoenolpyruvate carboxylase desensitiza tion on glutamic acid production in Corynebacterium glutamicum ATCC 13032. J Biosci Bioeng 121(2):172–177. https://doi.org/10.1016/j.jbiosc. 2015.06.008 Zhao N, Li L, Luo G, Xie S, Lin Y, Han S, Huang Y, Zheng S (2020) Multiplex gene editing and large DNA fragment deletion by the CRISPR/Cpf1-RecE/T system in Corynebacterium glutamicum. 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https://openalex.org/W3162259975	http://www.odermatol.com/odermatology/2021e/E333.Lipedematous-SarsharF.pdf	English	null	Lipedematous scalp with lipedematous alopecia: A case report	Nasza Dermatologia Online	2,021	cc-by	1,536	How to cite this article: Sarshar F, Amin SS, Adil M, Zahra FT. Lipedematous scalp with lipedematous alopecia: A case report. Our Dermatol Online. 2021;12(e):e46. Submission: 30.02.2021; Acceptance: 23.04.2021 DOI:10.7241/ourd.2021e.46 INTRODUCTION and erythema (Fig. 1). On palpation, scalp had a thick, soft and spongy consistency underneath the alopecia patch (Fig. 2). Hair pull test was negative. Trichoscopic examination revealed follicular plugging, white dots and variable erythema (Fig. 3). Ultrasonography (USG) of scalp showed well defined heterogeneous hyperechoic lesion with mild internal vascularity in subcutaneous plane of Bilateral Fronto-Parieto- Temporal region suggestive of lipomatous lesion. Magnetic resonance imaging (MRI) scalp showed the thickness of 11.4 mm in the vertex region (Fig. 4). Punch biopsy of scalp showed increased subcutaneous tissue thickness with fibro-collagenous dermis showing hair follicle and sebaceous gland and mature adipose tissue encroaching the dermis (Fig. 5). Based on above findings diagnosis of lipedematous alopecia was made. Lipedematous scalp is a rare disorder of unknown etiology characterised by thickening of subcutaneous tissue which presents as thick and boggy scalp usually localised to vertex and parietal area. Lipedematous alopecia refers to the condition where hair growth abnormalities co-exist with lipedematous scalp. Lipedematous alopecia may be confused with other causes of localised hair loss such as alopecia areata [1]. Herein, we report a case of lipedematous scalp and alopecia present at the same site over the scalp. CASE REPORT A 40 years female presented with complaints of headache for 1 year and gradually progressive asymptomatic swelling over vertex of scalp noticed for 5 months. The swelling was insidious in onset and associated with single patch of hair loss for 3 months. Medical and family history was unremarkable. On inspection 2*3 cm solitary patch of non-scarring alopecia was present over vertex area with no scaling Case Report Case Report ABSTRACT Lipedematous scalp and lipedematous alopecia are a rare cutaneous disorders of unknown etiology characterised by thickening of subcutaneous fat layer presenting with thick, boggy scalp of spongy consistency. In addition to changes in texture of skin, varying degree of hair loss are seen in patients of lipedematous alopecia. It was first described by Cornbleet in 1935. This is the report of 40 years female who presented with asymptomatic boggy swelling over affected scalp for 5 months and headache for 1 year. The swelling was associated with patchy hair loss. Exact etiopathogenesis remains unclear. Lipedematous scalp can be a possible cause of dysaesthesia of scalp. Herein, we report a case of lipedematous alopecia due to its rare occurrence and classical presentation with emphasis on trichoscopic, histopathological and radiological findings. Key words: Lipedematous scalp; Lipedematous alopecia; Lipomatosis Lipedematous scalp with lipedematous alopecia: A Lipedematous scalp with lipedematous alopecia: A case report case report Fariz Sarshar, Syed Suhail Amin, Mohammad Adil, Fatima Tuz Zahra Jawaharlal Nehru Medical College (JNMC), Aligarh Muslim University (AMU), Aligarh, India Fariz Sarshar, Syed Suhail Amin, Mohammad Adil, Fatima Tuz Zahra Jawaharlal Nehru Medical College (JNMC), Aligarh Muslim University (AMU), Aligarh, India Corresponding author: Fatima Tuz Zahra, MD, E-mail: ftz0606@gmail.com DISCUSSION Lipedematous scalp was first described by Cornbleet in 1935 and Coskey et al. in 1961 first reported the case of lipedematous alopecia. The disease is more common © Our Dermatol Online e.2021 1 www.odermatol.com The disease presents as a boggy swelling of the scalp with predominant involvement of the vertex and occiput. Patient may also have associated symptoms of pain, paraesthesia, tenderness and pruritus. No definite cause has been established but role of leptin has been proposed which causes adipocytes hyperplasia and displacement of adipocytes into dermis [4]. Hormonal and genetic factors and use of tight head gear have been implicated [5] Martin et al proposed that thickened Figure 2: Boggy scalp underneath the alopecia patch. Figure 1: Patch of non-scarring alopecia present over vertex. Figure 4: Magnetic Resonance Imaging showing increased thickness of scalp. Figure 5: Histopathology showing increased subcutaneous tissue thickness with hair follicle, sebaceous gland and mature adipose tissue encroaching the dermis. www.odermatol.com Figure 1: Patch of non-scarring alopecia present over vertex. Figure 4: Magnetic Resonance Imaging showing increased thickness of scalp. www.odermatol.com Figure 4: Magnetic Resonance Imaging showing increased thickness of scalp. Figure 1: Patch of non-scarring alopecia present over vertex. Figure 1: Patch of non-scarring alopecia present over vertex. Figure 4: Magnetic Resonance Imaging showing increased thickness of scalp. Figure 2: Boggy scalp underneath the alopecia patch. Figure 5: Histopathology showing increased subcutaneous tissue thickness with hair follicle, sebaceous gland and mature adipose tissue encroaching the dermis. Figure 2: Boggy scalp underneath the alopecia patch. Figure 2: Boggy scalp underneath the alopecia patch. Figure 5: Histopathology showing increased subcutaneous tissue thickness with hair follicle, sebaceous gland and mature adipose tissue encroaching the dermis. Figure 3: Dermoscopy showing follicular plugging, white dots and erythema. The disease presents as a boggy swelling of the scalp with predominant involvement of the vertex and occiput. Patient may also have associated symptoms of pain, paraesthesia, tenderness and pruritus. No definite cause has been established but role of leptin has been proposed which causes adipocytes hyperplasia and displacement of adipocytes into dermis [4]. Hormonal and genetic factors and use of tight head gear have been implicated [5]. Martin et al. proposed that thickened adipose tissue may cause pressure effect on hair follicle or impairment of nutrition of the hair matrix leading to decrease in hair growth or shortening of anagen phase [6]. CONCLUSION 6. Martín JM, Monteagudo C, Montesinos E, Guijarro J, Llombart B, Jordá E. Lipedematous scalp and lipedematous alopecia: a clinical and histologic analysis of 3 cases. J Am Acad Dermatol. 2005;52:152-6. Till now no concrete and successful treatment option exists to treat lipedematous scalp. Intralesional steroids, mycophenolate mofetil and surgical debulking and scalp reduction have been tried. Our patient received a trial of intralesional triamcinolone injection. Follow-up showed no clinical signs of improvement. Given the limited cases of successful treatment, further clinical evidence is needed to reach a consensus for treatment of lipedematous alopecia. 7. Sahu P, Sangal B, Dayal S, Kumar S. Lipedematous scalp with varied presentations: A case series of four patients. Indian Dermatol Online J. 2019;10:571-3. 8. Dincy Peter CV, Jennifer A, Raychaudhary T, Chandrashekhar L, Merilyn S, Gowda S, et al. Lipedematous scalp. Indian J Dermatol Venereol Leprol. 2014;80:270-2. Copyright by Fariz Sarshar, 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. REFERENCES 1. Dhurat RS, Daruwalla SB, Ghate SS, Jage MM, Sharma A. Distinguishing lipedematous scalp, lipedematous alopecia and diffuse alopecia areata. Skin Appendage Disord. 2019;5:316-9. A little over 50 cases of lipedematous scalp and lipedematous alopecia have been reported till now. The condition has been reported to co-exist with psoriasis, mucinosis, lupus erythematosus and androgenetic alopecia [2,5]. Sahu et al. reported concomitant alopecia areata and lipedematous scalp over parietotemporal region of scalp in a 29 year old male [7]. Dhurat et al. also described a similar case and emphasized that lipedematous scalp and lipedematous alopecia are not distinct conditions [1]. 2. Müller CS, Niclou M, Vogt T, Pföhler C. Lipedematous diseases of the scalp are not separate entities but part of a spectrum of lipomatous lesions. J Dtsch Dermatol Ges. 2012;10:501-7. 3. Lee HE, Kim SJ, Im M, Kim CD, Seo YJ, and Lee JH, et al. Congenital lipedematous alopecia: Adding to the differential diagnosis of congenital alopecia. Ann Dermatol. 2015;27:87-9. 4. Yasar S, Gunes P, Serdar ZA, Tosun I. Clinical and pathological features of 31 cases of lipedematous scalp and lipedematous alopecia. Eur J Dermatol. 2011;21:520-8. 5. Carrasco-Zuber JE, Alvarez-Veliz S, Cataldo-Cerda K, Gonzalez-Bombardiere S. Lipedematous scalp: a case report and review of the current literature. J Dtsch Dermatol Ges. 2016;14:418-21. DISCUSSION This might be responsible for the broken hair and associated hair loss in areas corresponding to the soft, boggy scalp area. Lymphangiectasia has also been proposed to cause hair loss. Headache may result from pressure on dermal nerves caused by dermal oedema and thickening [7]. Figure 3: Dermoscopy showing follicular plugging, white dots and erythema. in females (90%) and in European, African American and Egyptian people [2]. It can begin at any age and in fact, congenital cases have been reported [3]. © Our Dermatol Online e.2021 2 www.odermatol.com The diagnosis depends on clinical features & histopathological findings. Subcutaneous tissue thickening with encroachment into the dermis, without inflammation is the chief histopathological feature [5]. MRI, CT and USG are used to measure increased scalp thickness. In India, the mean scalp thickness on MRI was found to range from 5.5 to 7.75 mm in lipedematous alopecia and from 9.2 to 16 mm in lipedematous scalp in different regions of the scalp [8]. The authors certify that they have obtained all appropriate patient consent forms, in which the patients gave their consent for images and other clinical information to be included in the journal. The patients understand that their names and initials will not be published and due effort will be made to conceal their identity, but that anonymity cannot be guaranteed. © Our Dermatol Online e.2021 Consent The examination of the patient was conducted according to the principles of the Declaration of Helsinki. 3 © Our Dermatol Online e.2021 3
https://openalex.org/W3105731248	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0242458&type=printable	English	null	Fault diagnosis method of submersible screw pump based on random forest	PloS one	2,020	cc-by	7,072	Editor: Qichun Zhang, University of Bradford, UNITED KINGDOM Received: August 4, 2020 Accepted: November 2, 2020 Published: November 16, 2020 Copyright: © 2020 Jiang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. PLOS ONE RESEARCH ARTICLE a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Minzheng Jiang1, Tiancai ChengID1, Kangxing Dong1, Shufan Xu2, Yulong Geng3 1 School of Mechanics Science & Engineering, Northeast Petroleum University, Daqing, Heilongjiang, China, 2 The Second Oil Production Plant of Daqing Oilfield Co., Ltd., Daqing, Heilongjiang, China, 3 Daqing Oilfield Construction Group Co., Ltd., Daqing, Heilongjiang, China tiancai0926@126.com * tiancai0926@126.com a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Jiang M, Cheng T, Dong K, Xu S, Geng Y (2020) Fault diagnosis method of submersible screw pump based on random forest. PLoS ONE 15(11): e0242458. https://doi.org/10.1371/journal. pone.0242458 Editor: Qichun Zhang, University of Bradford, UNITED KINGDOM Abstract The difficulty in directly determining the failure mode of the submersible screw pump will shorten the life of the system and the normal production of the oil well. This thesis aims to identify the fault forms of submersible screw pump accurately and efficiently, and proposes a fault diagnosis method of the submersible screw pump based on random forest. HDFS storage system and MapReduce processing system are established based on Hadoop big data processing platform; Furthermore, the Bagging algorithm is used to collect the training set data. Also, this thesis adopts the CART method to establish the sample library and the decision trees for a random forest model. Six continuous variables, four categorical vari- ables and fault categories of submersible screw pump oil production system are used for training the decision trees. As several decision trees constitute a random forest model, the parameters to be tested are input into the random forest models, and various types of deci- sion trees are used to determine the failure category in the submersible screw pump. It has been verified that the accuracy rate of fault diagnosis is 92.86%. This thesis can provide some meaningful guidance for timely detection of the causes of downhole unit failures, reducing oil well production losses, and accelerating the promotion and application of sub- mersible screw pumps in oil fields. Introduction Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. Fault diagnosis method of submersible screw pump based on random forest Minzheng Jiang1, Tiancai ChengID1*, Kangxing Dong1, Shufan Xu2, Yulong Geng3 PLOS ONE PLOS ONE Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. At present, there are more than 170,000 pumping wells as part of PetroChina, and pumping- unit lifting technology has always occupied the dominant position of artificial lifting [1]. With the increase in the water content of the produced fluid, a new rod pump oil recovery technol- ogy-the same well injection and production technology began to be applied [2–4]. However, with the emergence of complex well types and oil production conditions, serious problems such as eccentric wear of pipe and rod caused by rod pump oil production systems are still unavoidable. As a rodless pumping equipment, submersible screw pump is applicable to spe- cial situations, such as heavy oil, high sand concentration, places with wax or gas, inclined well or horizontal well. Compared with rod pump, submersible screw pump can avoid the trans- mission loss caused by the expansion and distortion of sucker rod and improves the efficiency; Funding: JIANG Minzheng host the National Key Research and Development Program of China (Grant No. 2018YFE0196000) Cheng Tiancai host the Post-Graduate Innovative Research Project of Northeast Petroleum University (Grant No. JYCX_CX04_2018). Competing interests: No authors have competing interests. PLOS ONE \| https://doi.org/10.1371/journal.pone.0242458 November 16, 2020 1 / 17 PLOS ONE Fault diagnosis method of submersible screw pump based on random forest at the same time, it eliminates the eccentric wear of pipe and rod, reduces the chance of rup- ture and breaking off, and prolongs the pump inspection cycle [5]. Therefore, submersible screw pump as a new rodless lifting technology is very promising. However, the main components of the submersible screw pump are all placed under- ground, such as submersible motor, flexible shaft and screw pump, so manual observation can hardly identify all the problems [6]. Therefore, some failures of submersible screw pumps can- not be discovered in time, and will continue to operate. With the passage of time, the oil wells that only need simple maintenance may have more failures and cause greater economic losses. At present, the commonly used fault diagnosis methods include polished rod force method, electrical parameter method, current method, voltage holding method and neural network- based diagnosis method. The first four methods can only be adopted by experienced workers or experts according to the changes of current, power and other parameters and the working characteristics of screw pump on site. These four methods can hardly be used skillfully or supervised and accuracy rate is relatively low [7]. Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. In order to dispose some faults of screw pump wells widely applied in the oil field exploitation and improve the veracity of fault diagno- sis and the capablility of multi-fault diagnosis, Li established an intelligent integrated fault diagnosis expert system based on the fuzzy neural network, and the construction of the system was given [8]. Xue proposed a fault diagnosis method for screw pump wells based on BP neural network and expert system [9]. The fault diagnosis method based on neural network generally analyzes faults based on the active power parameters of submersible screw pump. The diagno- sis accuracy is relatively low, and the fault category of submersible screw pump cannot be iden- tified accurately. Random forest algorithm uses machine learning for data mining, which is a method based on statistical analysis and is able to mine information and discover knowledge without clear assumptions [10]. The fault information of submersible screw pump is contained in various working parame- ters, including both continuous variables and classified variables. The random forest algorithm can deal with both continuous variables and classified variables at the same time, establish a decision tree and form a random forest to assist in decision-making. Therefore, based on a large amount of data generated in the production process and MapReduce parallel processing system, this paper establishes HDFS distributed storage system and Hadoop big data process- ing platform for diagnosed faults in submersible screw pump. Working principle and fault types of submersible screw pump Structure and working principles of submersible screw pump As shown in Fig 1, the production system of submersible screw pump is mainly composed of two parts: ground device, including transformer, control cabinet, frequency converter, and junction box and downhole device, like submersible motor, flexible coupling, protector, screw pump, submersible cable, drain valve, and single flow valve. The power from the ground power supply is transmitted to the transformer, control cabinet and junction box first, and then the submersible motor through the submersible cable. Under the action of the protector, the submersible motor propels the flexible shaft to drive the screw pump to rotate at a low speed. After being pressurized by the screw pump, the oil is lifted to the ground through the oil pipeline [11]. Fig 1. Schematic diagram of production system of submersible screw pump. Fault types of submersible screw pump The failure of the surface-driven screw pump oil production system is mainly divided into two categories, namely, surface fault and downhole fault. Among them, ground faults include abnormal power supply and reducer failure; while downhole faults include broken sucker rod, 2 / 17 PLOS ONE \| https://doi.org/10.1371/journal.pone.0242458 November 16, 2020 PLOS ONE Fault diagnosis method of submersible screw pump based on random forest Fig 1. Schematic diagram of production system of submersible screw pump. https://doi.org/10.1371/journal.pone.0242458.g001 g p p Fig 1. Schematic diagram of production system of submersible screw pump. 3 / 17 PLOS ONE Fault diagnosis method of submersible screw pump based on random forest eccentric wear of sucker rod, tubing trip, pump empty pumping, pump jamming, pump leak- age, string leakage, and tubing wax deposition [12]. Compared with the surface-driven screw pump, the submersible screw pump is not equipped with the sucker rod and reducer, and puts the motor upside down in the underground. The screw is connected to the motor with a flexi- ble shaft. The fault of the submersible screw pump system can be divided into seven types: pump emptying, wax blockage, pump leakage, flexible shaft fault, oil pipe leakage, abnormal power supply and pump jamming. Hadoop ecosystem Hadoop is an open-source and distributed computing platform with three layers. The first layer is the underlying data source, composing of a large amount of data generated by each user. The middle layer, as the core layer of Hadoop, is also called big data layer, which includes the two cores of the Hadoop ecosystem: Hadoop Distributed File System (HDFS) and MapRe- duce processing system. In addition, the big data layer also contains the distributed column database Hbace for real-time query, and the big data algorithm library Mahout [13]. The third layer is the Inquiry layer which can analyze, query, and mine data. The overall framework of Hadoop platform is shown in Fig 2. Hadoop Distributed File System. As shown in Fig 3, HDFS adopts master/ slave architec- ture. Each cluster contains a name node and several data storage nodes. The name node is responsible for generating data directory and managing users’ access to data files while the data storage node is used to store data files. Fig 2. Overall framework of Hadoop platform. https://doi.org/10.1371/journal.pone.0242458.g002 E \| https://doi.org/10.1371/journal.pone.0242458 November 16, 2020 4 / 1 Fig 2. Overall framework of Hadoop platform. Fig 2. Overall framework of Hadoop platform. https://doi.org/10.1371/journal.pone.0242458.g002 PLOS ONE \| https://doi.org/10.1371/journal.pone.0242458 November 16, 2020 4 / 17 PLOS ONE Fault diagnosis method of submersible screw pump based on random forest Fig 3. HDFS frame structure. https://doi.org/10.1371/journal.pone.0242458.g003 Fig 3. HDFS frame structure. Fig 3. HDFS frame structure. https://doi.org/10.1371/journal.pone.0242458.g003 MapReduce. In Hadoop system, the operation can be divided into two stages, Map and Reduce respectively [14]. During the Map stage, a set of intermediate key-value pairs based on the input key-value is generated. Then, MapReduce framework collects the generated interme- diate key-value pairs and distributes them to Reduce. Reduce is used to process intermediate key-value pairs and related sets. Finally, these key-value pairs are merged to obtain results, as shown in Fig 4. (1) Divide the overall data into training data and test data; (2) Randomly take n sets of sample data from the training data to form a training sample set; (3) Repeat step (1) k times to get k training sample sets. The training sample sets are indepen- dent of each other, and different training sample sets may have duplicate elements; (3) Repeat step (1) k times to get k training sample sets. The training sample sets are indepen- dent of each other, and different training sample sets may have duplicate elements; (4) In terms of K training sample sets, train K models, which are determined by specific problems; (4) In terms of K training sample sets, train K models, which are determined by specific problems; (5) The final result is produced by voting carried by the results of K models. Basic principles and algorithm of decision tree. Decision tree has an inverted tree struc- ture. The nodes include root node, branch node and leaf node. The root node represents a type of test and is located at the top: the test is conducted by using attribute 1. Different branch nodes represent varied test results. Then, attribute 2 is used to get the leaf node. The leaf nodes store the classification label value, representing any possible classification results [17]. The decision tree algorithm is used to classify the unknown samples. The classification process is shown in Fig 5 as follows. Three common methods are usually used for building a decision tree: ID3 algorithm, C4.5 algorithm and cart algorithm. The main difference lies in the evaluation methods used for dif- ferent attributes of original data. Random forest classification algorithm Random forest algorithm is an integrated learning method, the framework of which is a bagging algorithm one. A decision tree classifier is adopted for base classifier, so as to classify both con- tinuous variables and discrete variables [15]. Random forest algorithm is highly capable of very precise generalization, proposes no requirements for training data attributes, and can meet the requirements of high parallel operation of big data processing. It can be well integrated with MapReduce processing system of the Hadoop platform. The electric submersible screw pump data set mainly has two features: numerical variables and category variables. Therefore, random forest algorithm is applied to diagnose and analyze submersible screw pump faults. Bagging algorithm. Bagging sampling algorithm refers to multiple rounds of random sampling with replacement of the overall data. Each round can extract various sets of data. The probability of each group of data selected is line with the uniform probability distribution. After multiple rounds of data sampling, multiple training sets are obtained, and each training set generates a base classifier. All the base classifiers classify and vote on the data to be tested respectively, and finally determine the data to be tested as the highest category of votes [16]. The process of bagging algorithm is shown as follows: Fig 4. MapReduce data flow diagram. https://doi.org/10.1371/journal.pone.0242458.g004 Fig 4. MapReduce data flow diagram. https://doi.org/10.1371/journal.pone.0242458.g004 https://doi.org/10.1371/journal.pone.0242458.g004 5 / 17 PLOS ONE \| https://doi.org/10.1371/journal.pone.0242458 November 16, 2020 oi.org/10.1371/journal.pone.0242458 November 16, 202 PLOS ONE Fault diagnosis method of submersible screw pump based on random forest (1) Divide the overall data into training data and test data; (2) Randomly take n sets of sample data from the training data to form a training sample set; (1) Divide the overall data into training data and test data; (1) ID3 algorithm h l h (1) ID3 algorithm h l h The ID3 algorithm uses the information entropy of different attributes as the judgment cri- terion when selecting data feature attributes. To this end, one specific attribute for node split- ting is selected [18]. Entropy is an indicator for the purity of sample set. The calculation formula of information entropy is shown as follows: EntropðSÞ ¼ X m i¼1 pilog2ðpiÞ ð1Þ ð1Þ Fig 5. Decision tree classification. https://doi.org/10.1371/journal.pone.0242458.g005 Fig 5. Decision tree classification. https://doi.org/10.1371/journal.pone.0242458.g005 https://doi.org/10.1371/journal.pone.0242458.g005 6 / 17 PLOS ONE \| https://doi.org/10.1371/journal.pone.0242458 November 16, 2020 PLOS ONE Fault diagnosis method of submersible screw pump based on random forest Where, m represents the total number of sample attributes. pi means the probability of obtaining NO.i attribute; Entrop(S) refers to the information entropy when attribute is not taken into consideration. If the value of Entrop(S) decreases, the purity of the data sample will become higher. However, ID3 can only discrete feature variable attributes rather than handle continuous variable feature attributes. At the same time, ID3 generally inclines to select feature attributes with more attribute values, which reduces the classification accuracy. (2) C4.5 algorithm (2) C4.5 algorithm (2) C4.5 algorithm Information gain is used to measure the importance of an attribute in the data set [19]. For example, the information gain of attribute A is as follows: GainðS; AÞ ¼ EntropðSÞ X v2ValueðAÞ jSVj S EntropðSVÞ ð2Þ ð2Þ Where, Value(A) refers to the set of values of attribute A. V is a certain attribute value of A. SV represents the sample set with a value of V in S. \|SV\| means the amount of samples con- tained in SV. The larger the value of information gain Gain(S,A) is, the more important attri- bute A in the dataset becomes. Samples should be divided according to attribute A. In feature selection process, C4.5 algorithm uses information gain rate as the standard indi- cator to circumvent the problem arising from ID3 algorithm [20]. The information gain rate of attribute A is expressed as: Gain ratioðAÞ ¼ GainðS; AÞ= X v2ValueðAÞ jSVj S log2 jSVj jSj ! ð3Þ ð3Þ The feature attributes with high information gain rate is influential on the data set, so the feature attributes with high information gain rate are preferred when splitting the decision tree. (1) ID3 algorithm h l h On the one hand, C4.5 algorithm effectively solves the problems of ID3 algorithm, enhances the rationality of decision tree and improves the classification accuracy. However, on the other hand, C4.5 algorithm also has certain defects: the algorithm has to repeatedly calculate and tra- verse the data, and store all the data sets. Hence, more time is needed for calculating the algo- rithm and more space is needed. PLOS ONE \| https://doi.org/10.1371/journal.pone.0242458 November 16, 2020 (3) Cart algorithm l h (3) Cart algorithm l h Cart algorithm uses the minimum Gini index criterion in the node splitting of decision tree and adopts Gini index to reflect the purity of the sample [21]. Gini index is similar to informa- tion entropy, but it requires obviously less calculation than information entropy. Cart tree adopts node dichotomy to remove ID3’s defect which tends to select more variables of catego- ries. The calculation formula is as follows: GiniðSÞ ¼ X m i¼1 pið1 piÞ ¼ 1 X m i¼1 p2 i ð4Þ ð4Þ In nature, Gini index is to randomly take two groups of data from the sample set S. These two groups of data are within the probability of the same category. Therefore, the smaller the Gini coefficient is, the higher the purity of the sample set is. If A is a characteristic attribute of the sample set S, then the Gini coefficient of characteristic attribute A is as follows: GiniðS; AÞ ¼ X v2ValueðAÞ jSVj S GiniðSVÞ ð5Þ ð5Þ 7 / 17 PLOS ONE \| https://doi.org/10.1371/journal.pone.0242458 November 16, 2020 PLOS ONE Fault diagnosis method of submersible screw pump based on random forest Fault diagnosis process of submersible screw pump The fault diagnosis for submersible screw pump based on random forest algorithm depends on big data processing platform. The specific process is as follows: (1) Build a Hadoop big data processing platform and establish a distributed file processing system of HDFS and MapReduce processing system; (2) Cleanse the data collected in the oil field, remove the data noise and data that is not related to mining, and fill in the missing data; (3) Evaluate the importance of characteristic attributes of all relevant data to submersible screw pumps and sort out data based on their rank of importance; (4) Divide data into a training set and a test set. In the training set, the Bagging sampling method is adopted to establish a sample library, and then the CART method is used to build a decision tree to form a random forest. The test set aims to test the accuracy of the random forest model to diagnose faults; (5) Input the data to be logged, call the random forest model to judge, and finally output the diagnosis results. PLOS ONE \| https://doi.org/10.1371/journal.pone.0242458 November 16, 2020 PLOS ONE PLOS ONE Table 1. Working parameters of submersible screw pump. Serial number Characteristic attributes Letter code Unit Variable type Remarks 1 Electric current I A Continuous Real time monitoring 2 Speed n r/min Continuous Real time monitoring 3 Voltage U V Continuous Real time monitoring 4 Active power Pa kW Continuous Calculated 5 Power factor Pf - Continuous Calculated 6 Reactive power Pr Kw Continuous Calculated 7 Submergence Sub m Continuous Capillary test 8 Pump hanging depth Dep m Continuous Measure 9 Moving liquid level Dy m Continuous Calculated 10 Liquid production Yl t/d Continuous Daily output 11 Oil production Yo t/d Continuous Daily output 12 Water content Yw t/d Continuous Daily output 13 Oil pressure Pc MPa Continuous Once a day 14 Casing pressure Pt MPa Continuous Once a day 15 Sand bearing Sand - Categorical Value 0a or 1b 16 Waxy Wax - Categorical Value 0 or 1 17 Pump type Type - Categorical - 18 Well No. Well - Categorical - a0 indicates that the sand and wax contents are relatively low Table 1. Working parameters of submersible screw pump. (3) Finally, six continuous variables, including mean active power, active power variance, sub- mergence, liquid production, oil pressure and casing pressure, and four categorical vari- ables, including sand, wax, pump type, and well number, are chosen to be input in the random forest model, to train a random forest model, and analyze faults on submersible screw pumps. PLOS ONE \| https://doi.org/10.1371/journal.pone.0242458 November 16, 2020 https://doi.org/10.1371/journal.pone.0242458.t001 a0 indicates that the sand and wax contents are relatively low. Data characteristics analysis of submersible screw pump After the field investigation, the following working parameters are mainly collected for sub- mersible screw pump wells in the oilfield: current, speed, voltage, active power, power factor, reactive power, submergence, pump hanging depth, liquid production, oil production, oil pressure and casing pressure. Also, the failure category of screw pump is written down. In addition, well fluid parameters are included, such as sand and wax content. The basic parame- ters of submersible screw pump wells cover pump type and well number. All the parameters are shown in Table 1. The random forest algorithm is used to analyze the faults and the data. The specific process is as follows: (1) Among several sets of feature attributes with high correlation, one set of input data are reserved as a fault diagnosis model. Among current, voltage, active power, power factor and reactive power, active power is selected as the model input data; among submergence, pumping depth and dynamic liquid level, submergence is selected as the model input data; among fluid production, oil production and water content, liquid production is selected as the input data of the model. (2) The active power is calculated based on the current and voltage monitored in real time. To calculate the active power, the researcher takes the data of a period before the failure and analyzes the data of this period of time. The data of this period of time can be regarded as a vector. The data mainly have two statistical characteristics, the central measurement of the data and the measurement of the dispersion degree of the data. These two characteris- tics are used in processing the power vector to characterize this data with two continuous variables of mean and variance. In this way, the power data is processed into two continu- ous variables. The mean value of active power is represented by "Mean" and the variance, by "Sd". PLOS ONE \| https://doi.org/10.1371/journal.pone.0242458 November 16, 2020 8 / 17 Fault diagnosis method of submersible screw pump based on random forest Descriptive files generated The parameters of the random forest model are set according to the input data set, including the size of the training set, the type of data variables in the training set, and the characteristics of the training set. The training data for the fault diagnosis model on submersible screw pump mainly include the following 10 parameters: six continuous variable parameters (active power mean, active power variance, submergence, liquid production, oil pressure, casing pressure) and four cate- gorical variable parameters, including sand, wax, pump type and well number. Part of the data is shown in Table 2. According to the type of input data, a descriptive file is generated. The description file is used to input the category attributes of the training data into the computer and provide the information about whether to participate in modeling, and other information. The first col- umn of the training data of the submersible screw pump fault diagnosis model is the serial number column, which is not used in the modeling, and is represented by "I"; the second col- umn and 9–11 columns of the training data refer to the categorical variables, and are repre- sented by "C"; Column 12 is the category label, namely, the screw pump fault category code, "0–5" represents six working status of "pump emptying, pump leakage, wax plugging, pipe leakage, flexible shaft failure, normal operating conditions" in the submersible screw pump, which are indicated by "L" in the program. Fig 6 shows the procedure and process for generat- ing descriptive files. The generated descriptive file "rf.info" is stored in HDFS and is put aside before the model is built. Data pre-processing As the data are originated from different systems, there are certain differences in data format and integrity. In order to facilitate data mining, it is necessary to clean the data. (1) Standardization of names Hashtag information is unified. There are two formats of the original well number, one is the combination of Chinese characters and numbers, the other is the combination of letters and numbers. Hashtag information is unified. There are two formats of the original well number, one is the combination of Chinese characters and numbers, the other is the combination of letters and numbers. (2) Time field normalization The recording intervals are different for each working parameter data of submersible screw pump in the oil field. Some parameters are monitored in real time, such as, current, active power, voltage, power factor, and reactive power, while some parameters are measured once a day, such as oil pressure, casing pressure, and dynamic liquid level. The time format is normal- ized during data cleansing. (3) Normalization of relevant data units The units for the data from different oil fields are unified and labelled. (4) Data missing value processing 9 / 17 PLOS ONE \| https://doi.org/10.1371/journal.pone.0242458 November 16, 2020 PLOS ONE Fault diagnosis method of submersible screw pump based on random forest Due to machine or human reasons, the recorded data may have missing values. Combined with the actual situation of the monitoring parameters of submersible screw pump wells, the missing values will be filled or deleted. After data cleaning, the data from different oil fields and departments are merged. Based on the fault category, the current, voltage, active power, power factor, reactive power, oil pressure, casing pressure, dynamic liquid level and other working parameter data of the submersible screw pump well are combined into one list when different faults occur, a total of 132 groups of submersible screw pump failure data are sorted out, 90 groups of which are randomly selected as training data, 42 groups are selected as test data, and stored in the HDFS distributed storage system. PLOS ONE \| https://doi.org/10.1371/journal.pone.0242458 November 16, 2020 Fault diagnosis model of submersible screw pump Construction of random forest model. The original training set is collected using bag- ging sampling. The new training set D_t is sampled, and the sampling is performed for T times; and m feature attributes are selected from the training set D_t to split the decision tree for establishing a decision tree. Each training set generates a decision tree, and finally T deci- sion trees are generated and converted into a random forest model. The modeling process and the modeling results are shown in in Figs 7 and 8 respectively. Based on the results, there are 90 input records, more specifically, 90 sets of training data; and 5 output records, a random forest built based on 5 decision trees. The decision tree is split up for 4 times, and the generated random forest model is stored in the forest.seq file in the out- put-forest folder. Evaluation of the random forest model. The test data are used to evaluate the random forest model generated above and analyze the accuracy rate. The process is shown in Fig 9. Evaluation of the random forest model. The test data are used to evaluate the random forest model generated above and analyze the accuracy rate. The process is shown in Fig 9. The test data is input into the random forest fault diagnosis model, to obtain the result shown forest model generated above and analyze the accuracy rate. The process is shown in Fig 9. The test data is input into the random forest fault diagnosis model, to obtain the result shown The test data is input into the random forest fault diagnosis model, to obtain the result shown PLOS ONE \| https://doi.org/10.1371/journal.pone.0242458 November 16, 2020 10 / 17 PLOS ONE Fault diagnosis method of submersible screw pump based on random forest Table 2. Part of the data of the fault diagnosis model. Serial number Well No. Active power mean/kW Active power variance Sub/m Pc/MPa Pt/MPa Yl/(t/d) Pump type Wax Sand Working condition category 1 B2—312-53 1.9685 0.1376 13 0.06 3.30 10 200 0 1 0 2 B3-D5-P51 1.5417 0.0413 20 0.21 1.20 7 200 1 0 0 3 B3-D3-P51 1.0728 0.1151 515 0.12 1.20 2 200 0 1 0 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Fault diagnosis model of submersible screw pump 23 B2—D3-55 1.1106 0.0874 876 0.32 1.20 61 200 1 0 1 24 B3—D5-P32 0.8512 0.4888 495 0.21 3.10 79 200 1 1 1 25 B2—D3-425 1.3079 0.1384 480 0.11 0.80 63 200 0 1 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 B2—D3-455 1.3998 0.3178 926 0.12 1.52 54 200 1 0 2 46 B3—D5-P59 1.5638 0.0374 1048 0.31 3.91 44 200 1 0 2 47 B3—D5-P57 1.5598 0.0102 441 0.34 2.22 57 200 1 1 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67 B2—D3-P69 1.6729 0.0089 515 0.11 1.71 69 200 0 0 3 68 B2—20-455 1.4321 0.0243 783 0.17 2.60 48 100 0 1 3 69 B2—D4-P56 2.0112 0.1442 352 0.21 2.71 75 200 0 1 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 B2—342-P36 2.0206 0.0896 467 0.02 2.70 2 100 0 0 4 90 B3—20-P51 1.7439 0.0317 777 0.05 3.20 3 200 1 0 4 91 B2—D3-SP65 2.0365 0.0333 1038 0.01 2.30 1 200 0 0 4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130 ZF90-52 2.0943 0.0329 343 0.11 3.70 48 100 0 0 5 131 ZF81-47 2.4628 0.5735 325 0.30 1.60 56 200 0 1 5 132 B2-342-P37 5.1565 7.3457 798 0.30 1.40 66 100 0 0 5 https://doi.org/10.1371/journal.pone.0242458.t002 Table 2. Part of the data of the fault diagnosis model. in Fig 10. The result includes the test accuracy rate and the confusion matrix, which is obtained by using the test set in the random forest model. in Fig 10. The result includes the test accuracy rate and the confusion matrix, which is obtained by using the test set in the random forest model. in Fig 10. PLOS ONE \| https://doi.org/10.1371/journal.pone.0242458 November 16, 2020 Fault diagnosis model of submersible screw pump The result includes the test accuracy rate and the confusion matrix, which is obtained by using the test set in the random forest model. From the results, it can be seen that the random forest model categorizes 42 sets of test data. Based on the reference to the fault category code of submersible screw pump, wax plugs and flexible shaft faults are easier to distinguish, and other fault categories are all misjudged. The submersible screw pump fault diagnosis model correctly classifies 39 sets of data, while errors occur when the model classifies 3 sets of data classification errors. The accuracy rate is about 92.86%, indicating that the random forest-based submersible screw pump fault diagnosis model works well and can be applied to reality. In order to verify the random forest fault diagnosis method, the researchers used both BP neural network and probabilistic neural network for fault diagnosis based on the above data. The fault diagnosis method based on BP neural network and probabilistic neural network is Fig 6. Descriptive documents. https://doi.org/10.1371/journal.pone.0242458.g006 11 / 17 PLOS ONE \| https://doi.org/10.1371/journal.pone.0242458 November 16, 2020 PLOS ONE Fault diagnosis method of submersible screw pump based on random forest Fig 7. Flow chart of generating random forest model. https://doi.org/10.1371/journal.pone.0242458.g007 Fig 7. Flow chart of generating random forest model. Fig 7. Flow chart of generating random forest mod https://doi.org/10.1371/journal.pone.0242458.g007 12 / 17 PLOS ONE Fault diagnosis method of submersible screw pump based on random forest Fig 8. Random forest model. https://doi.org/10.1371/journal.pone.0242458.g008 compared with the random forest fault diagnosis method. The test accuracy rates of the latter two diagnostic methods are 86% and 90.5%, respectively. The accuracy of the random forest- based algorithm is improved by 6.86 and 2.36 percentage compared with the other two methods. PLOS ONE \| https://doi.org/10.1371/journal.pone.0242458 November 16, 2020 PLOS ONE \| https://doi.org/10.1371/journal.pone.0242458 November 16, 2020 Comparative analysis of fault diagnosis methods Previous scholars mainly studied the fault diagnosis method of submersible screw pump based on neural network. However, this paper proposes the fault diagnosis method of submersible screw pump based on random forest algorithm. Therefore, for these two methods, a compara- tive analysis is carried out on the parameters, algorithms and scope of application. Comparative analysis of parameters. The two fault diagnosis methods are based on the operating data and various parameters of the submersible screw pump to identify the working conditions, but the specific parameters are different. The diagnosis method based on neural network uses five parameters of active power, liquid production, oil pressure, casing pressure, and dynamic liquid surface depth as the basis for fault diagnosis; The diagnosis method based on random forest includes the following 10 parameters: six continuous variable parameters (active power mean, active power variance, submergence, liquid production, oil pressure, cas- ing pressure) and four categorical variable parameters, including sand, wax, pump type and well number. Comparative analysis of algorithm. The neural network-based diagnosis method takes active power as the core parameter, performs wavelet packet decomposition and reconstruc- tion, and obtains the energy information contained in each frequency band. Under different PLOS ONE \| https://doi.org/10.1371/journal.pone.0242458 November 16, 2020 13 / 17 PLOS ONE Fault diagnosis method of submersible screw pump based on random forest Fig 9. Flow of testing random forest model. https://doi.org/10.1371/journal.pone.0242458.g009 Fig 9. Flow of testing random forest model. https://doi.org/10.1371/journal.pone.0242458.g009 https://doi.org/10.1371/journal.pone.0242458.g009 14 / 17 PLOS ONE Fault diagnosis method of submersible screw pump based on random forest Fig 10. Test results of random forest model. https://doi.org/10.1371/journal.pone.0242458.g010 https://doi.org/10.1371/journal.pone.0242458.g010 working conditions, the energy contained in the same frequency band is different, so as to determine the fault category of the screw pump. The fault diagnosis method based on random forest processes the data statistically, analyzes the potential correlation between parameter data and fault categories, uses bagging algorithm to sample the original data, and establishes a deci- sion tree classifier to form a random forest model. working conditions, the energy contained in the same frequency band is different, so as to determine the fault category of the screw pump. The fault diagnosis method based on random forest processes the data statistically, analyzes the potential correlation between parameter data and fault categories, uses bagging algorithm to sample the original data, and establishes a deci- sion tree classifier to form a random forest model. Comparative analysis of fault diagnosis methods Comparative analysis of scope of application. At present, the long-term monitoring parameters that can be directly obtained in screw pump wells mainly include voltage, current, production, oil pressure, casing pressure, dynamic liquid surface depth, etc. The fault diagnosis method based on neural network is suitable for this situation. However, with the continuous development of smart oil fields and big data, the database will be more robust, and the use of big data platforms to process data will be faster and more efficient. In this case, the fault diag- nosis method of submersible screw pump based on random forest is more applicable and the analysis result is more accurate. PLOS ONE \| https://doi.org/10.1371/journal.pone.0242458 November 16, 2020 Author Contributions Author Contributions Conceptualization: Minzheng Jiang. Data curation: Kangxing Dong, Shufan Xu. Formal analysis: Shufan Xu. Funding acquisition: Minzheng Jiang, Tiancai Cheng. Investigation: Yulong Geng. Methodology: Minzheng Jiang. Resources: Kangxing Dong. Writing – original draft: Tiancai Cheng. Writing – review & editing: Tiancai Cheng. Author Contributions Conceptualization: Minzheng Jiang. Data curation: Kangxing Dong, Shufan Xu. Formal analysis: Shufan Xu. Funding acquisition: Minzheng Jiang, Tiancai Cheng. Investigation: Yulong Geng. Methodology: Minzheng Jiang. Resources: Kangxing Dong. Writing – original draft: Tiancai Cheng. W iti i & diti Ti i Ch Methodology: Minzheng Jiang. Resources: Kangxing Dong. Writing – original draft: Tiancai Cheng. Writing – review & editing: Tiancai Cheng. Conclusion (1) Given the difficulty in diagnosing faults and low accuracy of current fault diagnosis meth- ods of submersible screw pumps, this paper proposes a fault diagnosis method of submers- ible screw pump based on random forest. Six continuous variables and four classified variables of the production system of submersible screw pump are used to analyze and judge the fault in the wells. The parameters are fully used and the accuracy is high. (2) Based on the establishment of Hadoop big data processing platform, HDFS distributed file storage system and MapReduce parallel processing system are established to store and pro- cess the data for submersible screw pump. (3) Bagging algorithm is used to take samples from the training set data and establish the sam- ple database. The cart method is used to establish the decision tree and forms the random forest model. The accuracy rate of the model is 92.86%, which proves the fault diagnosis methods can be applied to the fault diagnosis of submersible screw pumps. PLOS ONE \| https://doi.org/10.1371/journal.pone.0242458 November 16, 2020 15 / 17 PLOS ONE Fault diagnosis method of submersible screw pump based on random forest Supporting information S1 File. (ZIP) S1 Table. Working parameters of submersible screw pump. (XLS) S2 Table. Part of the data of the fault diagnosis model. (XLS) References 1. Liu H, Hao ZX, Wang LG, Cao G. Current Situation and Development Trend of Artificial Lift Technology. Acta Petrolei Sinica, 2015; 36(11):1441–1448. 1. Liu H, Hao ZX, Wang LG, Cao G. Current Situation and Development Trend of Artificial Lift Technology. Acta Petrolei Sinica, 2015; 36(11):1441–1448. 2. Jiang MZ, Cheng TC, Dong KX, Liu JT, Zhang HY. An Efficient Downhole Oil/Water-Separation System with Sucker-Rod Pump. SPE Production & Operations. 2020; 35(3):522–536. https://doi.org/10.2118/ 201234-pa 2. Jiang MZ, Cheng TC, Dong KX, Liu JT, Zhang HY. An Efficient Downhole Oil/Water-Separation System with Sucker-Rod Pump. SPE Production & Operations. 2020; 35(3):522–536. https://doi.org/10.2118/ 201234-pa 3. Dong KX, Jiang MZ, Cheng TC, Zhang HY. Rational Matching Injection and Production Pump for Rod Pump Injection-Production Systems. 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https://openalex.org/W2767091204	https://researchonline.lshtm.ac.uk/id/eprint/4651189/1/After%20the%20epidemic%20Zika%20virus%20projections%20for%20Latin%20America%20and%20the%20Caribbean.pdf	English	null	After the epidemic: Zika virus projections for Latin America and the Caribbean	PLoS neglected tropical diseases	2,017	cc-by	11,331	RESEARCH ARTICLE Editor: Uwem Friday Ekpo, Federal University of Agriculture, NIGERIA Editor: Uwem Friday Ekpo, Federal University of Agriculture, NIGERIA Felipe J. Colo´n-Gonza´lez1, Carlos A. Peres1☯, Christine Steiner São Bernardo2☯, Paul R. Hunter3‡, Iain R. Lake1‡ 1 School of Environmental Sciences, University of East Anglia, Norwich, Norfolk, United Kingdom, 2 Universidade do Estado de Mato Grosso, Rua São Pedro s/n, Cavalhada, Ca´ceres, Mato Grosso, Brazil, 3 Norwich Medical School, University of East Anglia, Norwich, Norfolk, United Kingdom ☯These authors contributed equally to this work. ‡ PRH and IRL also contributed equally to this work. F.Colon@uea.ac.uk ☯These authors contributed equally to this work. ‡ PRH and IRL also contributed equally to this work. C @ a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Methodology/Principal findings Received: May 22, 2017 We produced high-resolution spatially-explicit projections of Zika cases, associated congen- ital syndromes and monetary costs for Latin America and the Caribbean now that the epi- demic phase of the disease appears to be over. In contrast to previous studies which have adopted a modelling approach to map Zika potential, we project case numbers using a sta- tistical approach based upon reported dengue case data as a Zika surrogate. Our results indicate that 12.3 (0.7–162.3) million Zika cases could be expected across Latin America and the Caribbean every year, leading to 64.4 (0.2–5159.3) thousand cases of Guillain- Barre´ syndrome and 4.7 (0.0–116.3) thousand cases of microcephaly. The economic bur- den of these neurological sequelae are estimated to be USD 2.3 (USD 0–159.3) billion per annum. Copyright: © 2017 Colo´n-Gonza´lez 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: Epidemiological data are available from the Brazilian Ministry of Health (http://www2.datasus.gov.br/DATASUS/index.php? area=0203&id=30009960&VObj=http://tabnet. datasus.gov.br/cgi/deftohtm.exe?sinanwin/cnv/ dengue), the Mexican Health Secretariat (http:// www.epidemiologia.salud.gob.mx/anuario/html/ anuarios.html). Climatic data are available from the Climatic Research Unit Archive (https://crudata. uea.ac.uk/cru/data/hrg/). Population data are available from the Socioeconomic Data and Applications Center (http://sedac.ciesin.columbia. OPEN ACCESS Zika is one of the most challenging emergent vector-borne diseases, yet its future public health impact remains unclear. Zika was of little public health concern until recent reports of its association with congenital syndromes. By 3 August 2017 217,000 Zika cases and 3,400 cases of associated congenital syndrome were reported in Latin America and the Caribbean. Some modelling exercises suggest that Zika virus infection could become endemic in agreement with recent declarations from the The World Health Organisation. Citation: Colo´n-Gonza´lez FJ, Peres CA, Steiner São Bernardo C, Hunter PR, Lake IR (2017) After the epidemic: Zika virus projections for Latin America and the Caribbean. PLoS Negl Trop Dis 11(11): e0006007. https://doi.org/10.1371/journal. pntd.0006007 Introduction Zika virus (ZIKV) is a vector-borne disease that is transmitted among humans through the bite of infectious Aedes mosquitoes. ZIKV is a member of the Flaviviridae family, and genus Flavivirus. The symptoms of ZIKV infection are usually mild and similar to those of other arboviral infections such as dengue including fever, macopapular rash, conjunctivitis, myalgia, and headache [1]. In most infected people the disease is benign. However, in some cases ZIKV infection may result in serious complications such as Guillain–Barre´ syndrome, microcephaly and maculopathy [2–4]. To the date of this study, there are no vaccines or antiviral therapy readily available for ZIKV infection [5]. However, this could be feasible in the future [6]. In April 2015, a ZIKV outbreak was reported in Brazil, and subsequently in several Latin American and Caribbean countries. By 3 August 2017 217,000 confirmed ZIKV cases, and 3,400 cases of associated congenital syndrome had been reported to the Pan-American Health Organization [7]. Current research on ZIKV activity has rightly focused upon the disease epidemic stage [8] and its consequences. The next big question is whether Zika will become endemic in Latin America and the Caribbean (LATAM), and what are the potential health and economic burdens. Although it is impossible to ascertain whether ZIKV will become endemic in LATAM, a recent study based on a numerical epidemic model predicts that the virus will eventually Although it is impossible to ascertain whether ZIKV will become endemic in LATAM, a recent study based on a numerical epidemic model predicts that the virus will eventually become endemic [9]. The lack of vaccines for ZIKV [5], the environmental suitability of the region [10], and the endemic status of other arboviruses that share the same vector (e.g. den- gue fever) also suggest that such an endemic state is plausible. become endemic [9]. The lack of vaccines for ZIKV [5], the environmental suitability of the region [10], and the endemic status of other arboviruses that share the same vector (e.g. den- gue fever) also suggest that such an endemic state is plausible. One key aspect for the control of mosquito-borne diseases is vector control. Past experience indicates that aggressive control of Aedes mosquitoes using traditional insecticide-based mea- sures is effective only if implemented in a comprehensive and sustained manner [11]. This may be difficult due to public resistance, lack of expertise, and finance [11, 12]. After the epidemic: Zika virus projections for Latin America and the Caribbean edu/data/collection/grump-v1). Birth rate data are available from the World Population Prospects (https://esa.un.org/unpd/wpp/). Conclusions/Significance Zika is likely to have significant public health consequences across Latin America and the Caribbean in years to come. Our projections inform regional and federal health authorities, offering an opportunity to adapt to this public health challenge. 1 / 19 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0006007 November 1, 2017 Author summary In February 2016 the World Health Organisation (WHO) declared Zika virus infection in the Americas as a Public Health Emergency of International Concern (PHEIC). By November 2016, Zika was declared a long-term public health challenge. This change of status implies that Zika is likely to become an endemic problem in the region. Due to the PHEIC status of Zika, most current research has rightly focused on the epidemic stage of the disease; however, it is timely and critical to consider the public health consequences after such epidemic phase. We used one of the largest and most spatially diverse panels of epidemiological surveillance data comprising 12 years of dengue case observations from Brazil and Mexico, and covering an area of over ten million km2. State-of-the-art statistical models, and high-resolution (0.5 × 0.5 degrees) climate and demographic data were used to produce spatially-explicit projections of Zika infection for Latin America and the Caribbean. Model projections were then used to estimate the number of cases with neurological sequelae and their economic cost. Our findings indicate that the poten- tial health and economic burden of Zika could be considerably large for the region should it become endemic. The estimated burden of Zika under an endemic state high- lights the need for health authorities in the countries at risk to promote preventive and control measures. Funding: FJCG, PRH and IRL were funded by the National Institute for Health Research Health Protection Research Unit (NIHR HPRU) in Emergency Preparedness and Response at King’s College London in partnership with Public Health England (PHE), and the University of East Anglia (Grant number HPRU-2012-10141, www.nihr.ac. uk). The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. After the epidemic: Zika virus projections for Latin America and the Caribbean Physical control measures against the vector such as house screens, and the environmental modification or sanitation of larval sites may also be effective [14]; however, these measures may be unavailable to poor residents in crowded urban areas where the impacts of ZIKV are greatest [5]. Recent studies have mapped the potential global scale range of ZIKV based on combina- tions of environmental, vector abundance, and socioeconomic factors [10, 15]. One limitation of the method used by these studies is that it maps the environmental suitability which does not necessarily imply that the disease will occur in that area [10]. This issue is critical because experience from similar diseases indicates that such modelling approaches tend to overesti- mate the geographical areas where disease could occur as they cannot take into account the complex local factors that determine whether potential risk actually translates into disease [16]. An alternative approach is a statistical analysis based on spatially-explicit monthly reports of confirmed ZIKV cases; a difficult task due to the limited time period for which reliable human spatially-explicit case reports are available for LATAM [17–19]. We overcome these limitations by using human dengue case data across LATAM as a sur- rogate for ZIKV. The advantage of this approach is that it is based upon knowledge on where disease transmission from mosquito to humans occurs in reality. One challenge, however, is that reported disease counts are a fraction of the true incidence (it has been estimated that for each official dengue report 10–27 cases go unreported [20]). We argue that this approach is valid because the dengue virus shows remarkable similarities to ZIKV. For example, both viruses have single positive stranded RNA genome encoding three structural proteins (C, prM/M and E), and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5); are vectored by Aedes mosquitoes; and seem to have similar infectious and viral replica- tion mechanisms [21]. Moreover, phylogenetic analyses have shown that ZIKV is closer to dengue virus than to any other flavivirus [22]. As a consequence of such similarities, there is cross-reaction of anti- bodies to dengue with ZIKV [23]. Not surprisingly, previous ZIKV modelling studies are largely based upon dengue parameters [8, 24]. We acknowledge, however, that whilst there is only one ZIKV serotype, there are four different dengue serotypes which do not confer protec- tive immunity against all serotypes [6]. Thus, while Zika could infect an individual only once, dengue could cause disease repeatedly which poses a key difference in the ecology and epide- miology of these two diseases. The aims of our work are three-fold. Focusing upon the post-epidemic period of ZIKV, we first examine the likely incidence of ZIKV in childbearing women across LATAM and the potential number of microcephaly and Guillain-Barre´ syndrome (GBS) cases. Sec- ond, we identify areas where ZIKV transmission may be sporadic and hence remains epi- demic re-emerging every few years. Finally, we quantify how case numbers are likely to fluctuate in affected areas due to seasonal and meteorological effects such as an El Niño (ENSO) event. Introduction A recent meta- review on the effectiveness of Aedes control strategies has found that this type of vector control does not seem to be associated with long-term reductions of mosquito populations [13]. 2 / 19 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0006007 November 1, 2017 Meteorological data High-resolution gridded datasets of monthly global mean temperature, total precipitation, and potential evapotranspiration (PET) data [31] were obtained from the CRU TS3.24 Climatic Research Unit climate archives at a 0.5 × 0.5 degree resolution for land cells only, and for the period January 1991 to December 2015. Moving averages were computed for the current and previous two months to account for the delayed effects of temperature, precipitation, and PET on incidence [32]. Mean temperature, mean PET, and total precipitation estimates for each administrative unit in the study were calculated using the extract method included in the R [29] raster package [33]. After the epidemic: Zika virus projections for Latin America and the Caribbean whilst the Brazilian dataset was obtained at the municipal county level (n = 5,566, mean popu- lation = 0.47 million people). Missing counts were imputed for municipalities with less than 20% missing entries using a singular value decomposition-based method [27], included in the bcv package [28] for R [29]. Areas with over 20% missing counts (n = 4,177) were removed from the dataset. Brazilian municipal counties are considerably smaller in area and population than the Mexican States. Such small areas were typically characterized by low counts of cases. We aggregated the Brazilian municipal counties into larger geographical units by dividing the centroid coordinates into 286 latitude-longitude intervals, and merging all counties with cen- troid coordinates within each latitude-longitude bin together. The merged areas (n = 286, mean population = 0.45 million people) were used for analysis. Whilst the presence of four dengue serotypes is an important difference with ZIKV that we acknowledge, it would be impossible to disentangle the dengue epidemiological sur- veillance data to obtain four different time series, one for each serotype. The ratio of dengue to ZIKV cases is hard to estimate due to the limited period for which ZIKV data are avail- able. Given that the transmission dynamics of ZIKV and dengue are similar when observed in the same setting [30], for simplicity, we initially assumed that each confirmed dengue report is equivalent to a ZIKV case (1:1 ratio). To account for uncertainties in this assump- tion, we also considered scenarios where the ZIKV to dengue ratio varied between 0.1:1 and 10:1. Demographic data Global gridded total population count estimates were retrieved at a 2.5 arc minutes resolution from the Gridded Population of the World project [34] at five year intervals for the period 2000–2010. For consistency with the meteorological data, demographic data were aggregated at a 0.5 × 0.5 degree resolution using the Climate Data Operators software [35]. Total popula- tion estimates were scaled to agree with the United Nations World Population Prospects yearly population estimates [36]. Monthly estimates for each grid-box were derived using linear interpolation [32, 37]. The estimated population for each geographical unit included in the study was then calculated using the extract method included in the R [29] raster package [33]. Crude birth rates per country were also retrieved from the United Nations World Population Prospects [36]. Epidemiological surveillance data Epidemiological surveillance data Monthly counts of laboratory confirmed dengue cases were obtained from the Mexican [25], and Brazilian [26] Ministries of Health for the period January 2001 to December 2012 (144 months). Together, these two countries cover a latitudinal range between 30˚N and 30˚S, and account for over 60% of the reported dengue cases and 53% of the LATAM population. Our dataset consists of nearly 4 million dengue reports (Brazil 88%, Mexico 12%). The Mexi- can dataset was obtained at the State level (n = 32, mean population = 3.2 million people), 3 / 19 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0006007 November 1, 2017 Model specification The expected number of Zika virus infections E(Yit) for area i = 1, . . ., I at time t = 1, . . ., T was modelled using a generalized additive mixed model (GAMM) approach. To account for possi- ble over-dispersion in the data, we fitted Negative Binomial and quasi-maximum likelihood 4 / 19 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0006007 November 1, 2017 After the epidemic: Zika virus projections for Latin America and the Caribbean Poisson models. We selected the model specification with the lowest mean absolute error (MAE). The general algebraic definition of both the Negative Binomial and quasi-maximum likelihood Poisson models is given by: Poisson models. We selected the model specification with the lowest mean absolute error (MAE). The general algebraic definition of both the Negative Binomial and quasi-maximum likelihood Poisson models is given by: logðmitÞ ¼ Zit ð1Þ ð1Þ logðmitÞ ¼ Zit Zit ¼ a þ LogðxitÞ þ t0 þ s0 þ X P p¼1 f ðxitÞ þ di ð2Þ ð2Þ where ηit is a logarithmic link function of the expectation E(Yit μit), with Yit as the time series of monthly dengue reports. The term α corresponds to the intercept; Log(ξit) denotes the loga- rithm of the population at risk for area i and time t included as an offset to adjust the epidemi- ological data by population. Here, t0 is a cubic regression spline function of the time variable with 1 degree of freedom (df) for every M years of data to control for possible long-term trends. Seasonal trends are modelled using Fourier terms (s0) with N sine/cosine pairs. Long- term and seasonal trends in all variables in the model are controlled for because they may be related to factors other than climate [38] such as changes in reporting or coverage, holidays or seasonal water storage. The term f(xit) corresponds to smoothed relationships between the cli- matic predictors and the crude incidence rate defined by the cubic regression splines. Area- specific random effects (di) were included to account for the effects of unknown or unobserved variables in the model such as diagnostic performance variability, immunity, and intervention measures. PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0006007 November 1, 2017 After the epidemic: Zika virus projections for Latin America and the Caribbean the following matrix: MAEk;h ¼ MAE1;1 MAE1;2 MAE1;H MAE2;1 MAE2;2 MAE2;H .. . .. . .. . .. . MAEK;1 MAEK;2 MAEK;H 2 666666664 3 777777775 the following matrix: MAEk;h ¼ MAE1;1 MAE1;2 MAE1;H MAE2;1 MAE2;2 MAE2;H .. . .. . .. . .. . MAEK;1 MAEK;2 MAEK;H 2 666666664 3 777777775 the following matrix: MAEk;h ¼ MAE1;1 MAE1;2 MAE1;H MAE2;1 MAE2;2 MAE2;H .. . .. . .. . .. . MAEK;1 MAEK;2 MAEK;H 2 666666664 3 777777775 The MAE for each modelled subset (henceforth MAEk,h) was calculated by averaging the subset-specific values (h) across all time steps (k). With this process, we aimed to identify the most accurate model or group of models. Model predictions for Latin America Cross-validated model outputs were used to predict the total number of ZIKV infections for an average month, a typical ENSO month, a strong El Niño (based on the 1997–1998 and 2015–2016 events) [42], and a typical non-El-Niño month across LATAM under the assump- tion of a 0.1:1, 1:1 and 10:1 ZIKV to dengue ratios. To account for uncertainties in the under- reporting of the health data, we multiplied the predicted number of cases for a given geograph- ical area by a factor of 10, 18.5 or 27 [20]. Model predictions were computed using mean monthly gridded climatic and population data at a 0.5 × 0.5 degree resolution for each of the aforementioned periods. ENSO events are defined here as periods where the 3-month running mean of the Oceanic Niño Index is greater than 0.5˚C. The length of an ENSO event was the length indicated by the USA National Weather Service, Climate Prediction Center [43] plus three months to account for potential delayed effects on the local climate. Country-wide totals were retrieved using standard routines within the raster [33] R package. Specification of the long-term and seasonal trends TSCV was used to identify the specification of long-term and seasonal trends with the lowest MAEk,h. Specifically, we modified the number of df per year (ranging from 1 df for every two years of data to 1 df for every four years) for the cubic spline function of time, as well as the number of sine/cosine pairs for the Fourier terms (ranging from three to six). All possible combinations of long-term and seasonal trends were explored. Selection of climatic predictors A time series cross-validation (TSCV) algorithm [39] was implemented to select the set of cli- mate predictors producing the lowest prediction error. TSCV was preferred over k-fold or leave-one-out cross-validation algorithms because epidemiological surveillance time series are typically serially correlated [40] violating the assumptions that data are independent and iden- tically distributed. Models were fitted using all climatic predictors (i.e. mean monthly tempera- ture, mean monthly PET and total monthly precipitation) in isolation, as well as in all possible combinations. Therefore, we successively fitted all possible models containing one climatic predictor at a time, then two predictors at a time, and so on, until all predictors were included altogether in a single model. We measured the accuracy of each model calculating their MAE. The MAE was selected as the measure for model accuracy because it is a natural and unambig- uous measure of average error magnitude [41]. TSCV was implemented dividing the dataset into a training and a test sets. The initial training set comprised 90% of the total number of months (n = 144). Each time step (k), a further month of data was added to the training set. Thus, at time step k = 1, the training set comprised observations for month t = 1, . . ., 130; at k = 2 it comprised observations for t = 2, . . ., 131, and so on. The test set comprised the first observation for each geographical area immediately after the last observation in the training set. Consequently, at time step k = 1, the test set contained all area-specific observations for t = 131; at k = 2, it contained all observations for t = 132, and so on until the test set contained the observations for month t = n; where n is the total number of months in the dataset. The MAE was calculated at each time step k = 1, . . ., K, and for each subset of climatic predictors h = 1, . . ., H as in 5 / 19 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0006007 November 1, 2017 Total Costi ¼ Microcephalyi þ GBSi ð5Þ Identification of epidemic-prone regions Epidemic-prone areas were defined as areas where the month-to-month relative standard deviation (RSD) of the model estimates is greater than the mean for a given grid box. The RSD is defined here as the ratio of the standard deviation (σ) to the mean (μ). We defined epidemic areas as those where the RSD of the estimated number of cases was larger than one, and highly epidemic areas where this ratio was greater than 1.5 [47]. RSD ¼ s=m ð6Þ ð6Þ RSD ¼ s=m Results/Discussion Previous studies have used combinations of environmental, vector abundance, and socioeco- nomic factors to map the environmental suitability for ZIKV [10, 15]. However, the fact that a region is environmentally suitable for transmission does not necessarily imply autochthonous transmission will necessarily occur in that area [10]. Recent research suggests that such model- ling approaches overestimate the geographical areas where disease is likely to occur [19]. The lack of long-term spatially-explicit monthly reports of confirmed ZIKV cases for LATAM [17– 19] poses serious difficulties for the development of alternative approaches based on ZIKV epi- demiological surveillance. To overcome these limitations, we used one of the largest panels of epidemiological surveillance dengue case time series for LATAM as a surrogate for ZIKV. Compared to models aimed to predict the environmental suitability for ZIKV, our approach has the advantage of being based upon knowledge of where disease transmission really occurs. Estimating the risk of neurological sequelae and their economic impact Model estimates of mean monthly cases were downscaled by the proportion of cases occurring in childbearing women (i.e. 15–44 years of age) based on the proportion of cases per gender and age reported to the Mexican Ministry of Health over the period 2010–2012 [25]. The num- ber of cases in childbearing women was then used to estimate the potential number of ZIKV- affected pregnancies by multiplying them by the corresponding country-specific crude birth rates [36]. The risk of microcephaly due to ZIKV infection during the first trimester of preg- nancy was calculated using the 0%, 50% and 100% percentiles of the distribution of the range of values estimated for the risk of microcephaly due to infection in women aged 15–44 (i.e. 0.88–14.4) [44] to account for uncertainties on our estimates. Similarly, the potential number of GBS cases in both males and females was estimated using the 0%, 50% and 100% percentiles of the distribution of risk estimates of GBS per 1000 ZIKV infections based on previous research conducted in French Polynesia and LATAM [3, 45]. 6 / 19 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0006007 November 1, 2017 After the epidemic: Zika virus projections for Latin America and the Caribbean The economic impact of the estimated number of cases with neurological sequelae was esti- mated based on the direct medical cost of each microcephaly and GBS case [46]. Thus, the esti- mated mean annual number of microcephaly (X) and GBS cases (Y) for each administrative unit (i) was multiplied by the estimated medical cost per case based on previous research [46]. The direct medical cost of each microcephaly case (δ) was assumed to be USD 91,102 whilst that of each GBS case (γ) was assumed in USD 28,818 [46]. The total economic impact of the neurological sequelae was estimated as follows: Microcephalyi ¼ Xi d Microcephalyi ¼ Xi d ð3Þ ð3Þ GBSi ¼ Yi g ð4Þ Total Costi ¼ Microcephalyi þ GBSi ð5Þ After the epidemic: Zika virus projections for Latin America and the Caribbean spline with three df, and the seasonality was specified with a Fourier term with three sine and cosine functions of time. The final model explained 79.5% of the deviance in the health data. The structure of the final model was then used to compute estimates based on both 0.1:1 and 10:1 ZIKV-dengue ratios. S1 Fig compares the observed and predicted temporal trends in the number of cases for each country. We noted that the final model’s predictions (and their corresponding error esti- mates) capture quite closely the temporal variations observed in the observed data with some underestimations in both countries related to major outbreaks that could be related to loca- tion-specific non-climatic factors (e.g. human behaviour and interventions) not explicitly accounted for in the model [16]. GAMMs are essentially a nonparametric method; therefore, it is difficult to express their results using mathematical equations. Instead, the GAMM-estimated smoothed relationships between ZIKV incidence, T0:2 and PET0:2 are presented in S2 Fig. The solid lines in the figure represent the estimated functional form of the relationship between ZIKV incidence and each predictor. S2A Fig shows an almost null response of ZIKV to T0:2 below 20˚C, with rapid increases in ZIKV cases as T0:2 surpasses this threshold. The estimated effect is consistent with the biology of both the vector and ZIKV because rising temperatures shorten the development time and gonotrophic cycle of the vector, and increase its biting rate; also, they reduce the time required for viral development inside the vector all of which results in an increased risk of transmission [42, 48]. S2B Fig indicates that there is a log-negative relationship between ZIKV incidence and PET0:2 with the risk of infection drastically decreasing between one and three mm per month and remaining low after that threshold. We were unable to identify studies investigating the effects of PET on ZIKV or Aedes mosquitoes. However, previous research using anopheline data [49] has shown similar relationships between PET and vector abun- dance with low levels being more conducive of vectorial activity than high PET levels, and so increasing the risk of disease transmission. High temperatures and low humidity levels have been found to reduce the oviposition rate and life span of Aedes mosquitoes [50]. Model output We fitted 23 different model specifications to test all possible combinations of climatic pre- dictors long-term and seasonal trends whilst assuming a ZIKV-dengue ratio of 1:1. The TSCV algorithm applied to the dengue-derived ZIKV data (henceforth ZIKV data) favoured a Negative Binomial GAMM with a MAEk,h of 105 cases per month that included tempera- ture lagged zero to two months (T0:2), and PET lagged zero to two months (PET0:2) as cli- matic covariates. Precipitation lagged zero to two months was not included in the final model. The incorporation of an interaction term between T0:2 and PET0:2 did not increase the predictive ability of the model, and so it was not included in the final model. After per- forming a sensitivity analysis testing different specifications for the df of long-term and sea- sonal trends, the long-term trends in the final model were specified with a cubic regression PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0006007 November 1, 2017 7 / 19 After the epidemic: Zika virus projections for Latin America and the Caribbean Table 1. Country-level projections of Zika virus (ZIKV) infection under the assumption of endemicity for the total population and women on child- bearing age, and estimated health and economic burden of Guillain-Barre´ syndrome, and microcephaly. Values in brackets represent the mean esti- mates under the assumption of a 0.1:1 and 10:1 ZIKV-dengue ratios. Country Cases (Thousand) Cases women 15- 44 (Thousand) Guillain-Barre´ (Individuals) Affected pregnancies (Individuals) Microcephaly (Individuals) Economic impact (Million USD) Brazil 7344 (426–95899) 2300 (59–50096) 38335 (102– 3047671) 34499 (2001–450544) 2636 (18–64878) 1345 (5–93738) Colombia 1153 (67–15209) 361 (9–7914) 6020 (16–483352) 5655 (327–74642) 432 (3–10748) 213 (1–14908) Mexico 1094 (62–14785) 343 (9–7745) 5708 (15–469855) 6207 (355–83840) 474 (3–12073) 208 (1–14640) Venezuela 841 (48–11453) 263 (7–6014) 4391 (12–363991) 4952 (284–67556) 378 (2–9728) 161 (1–11376) Cuba 284 (17–3708) 89 (2–1927) 1484 (4–117833) 935 (55–12193) 71 (0–1756) 49 (0–3556) Peru 212 (12–2722) 66 (2–1420) 1104 (3–86512) 1228 (72–15837) 94 (1–2281) 40 (0–2701) Dominican Republic 176 (10–2305) 55 (1–1190) 918 (2–73253) 1179 (69–15449) 90 (1–2225) 35 (0–2314) Guatemala 168 (9–2291) 53 (1–1189) 878 (2–72803) 1013 (57–13870) 77 (1–1997) 32 (0–2280) Haiti 108 (6–1403) 34 (1–724) 562 (2–44582) 824 (49–10726) 63 (0–1545) 22 (0–1425) Ecuador 105 (6–1409) 33 (1–725) 546 (1–44779) 644 (36–8692) 49 (0–1252) 20 (0–1404) Panama 102 (6–1311) 32 (1–679) 531 (1–41653) 612 (37–7862) 47 (0–1132) 20 (0–1304) Bolivia 96 (6–1280) 30 (1–666) 503 (1–40691) 660 (38–8762) 50 (0–1262) 19 (0–1288) Argentina 87 (5–1201) 27 (1–634) 453 (1–38161) 332 (18–4563) 25 (0–657) 15 (0–1160) Costa Rica 81 (5–1076) 26 (1–555) 425 (1–34198) 281 (16–3724) 21 (0–536) 14 (0–1034) Puerto Rico 72 (4–925) 23 (1–480) 376 (1–29397) 273 (16–3506) 21 (0–505) 13 (0–893) El Salvador 69 (4–909) 22 (1–467) 361 (1–28883) 184 (11–2416) 14 (0–348) 12 (0–864) Nicaragua 68 (4–912) 21 (1–473) 355 (1–28973) 392 (22–5264) 30 (0–758) 13 (0–904) Paraguay 61 (3–843) 19 (0–436) 317 (1–26790) 268 (14–3737) 20 (0–538) 11 (0–821) Jamaica 56 (3–709) 17 (0–366) 291 (1–22544) 308 (18–3910) 24 (0–563) 11 (0–701) Guyana 52 (3–668) 16 (0–346) 273 (1–21214) 287 (17–3667) 22 (0–528) 10 (0–659) Honduras 43 (2–584) 14 (0–301) 225 (1–18551) 218 (12–2962) 17 (0–427) 8 (0–573) French Guiana 17 (1–221) 5 (0–115) 90 (0–7035) 108 (6–1389) 8 (0–200) 3 (0–221) Trinidad and Tobago 14 (1–180) 4 (0–94) 72 (0–5729) 64 (4–830) 5 (0–120) 3 (0–176) Suriname 10 (1–126) 3 (0–65) 51 (0–3989) 44 (3–572) 3 (0–82) 2 (0–122) Belize 9 (1–122) 3 (0–64) 49 (0–3888) 36 (2–472) 3 (0–68) 2 (0–118) Uruguay 4 (0–51) 1 (0–27) 19 (0–1607) 14 (1–193) 1 (0–28) 1 (0–49) Chile 3 (0–41) 1 (0–36) 17 (0–1294) 14 (1–172) 1 (0–25) 1 (0–40) Total 12329 (713– 162343) 3861 (99–84746) 64354 (170- 5159256) 61231 (3541–807354) 4676 (29– 116261) 2281 (8–159271) The economic impact is measured in thousand USD. Model predictions for Latin America and the Caribbean We then used the model output to predict the mean monthly number of cases across LATAM for a typical year at a 0.5 × 0.5 degree resolution. A sensitivity analysis was performed to com- pute predictions under the assumption of a 0.1:1, 1:1, and 10:1 ZIKV to dengue ratio to explore the uncertainties in our assumptions of the relationship between the estimated number of ZIKV and dengue virus infections. Based on such sensitivity analysis, we estimate that should ZIKV become endemic 12 million (range: 713 thousand to 162 million) ZIKV cases could occur across LATAM every year (Table 1). About 4 million of those cases (range: 99 thousand to 85 million) are expected to occur in childbearing women (15–44 years of age) annually. The country-level estimates suggest that Brazil will experience the largest disease burden (60%); more than six times the estimated burden for Mexico (7%) or any other LATAM country (Table 1). Other countries such as Colombia, Mexico, Venezuela, Cuba and Peru are also expected to experience large numbers of ZIKV infection. The risk of ZIKV infection has been estimated to be larger in South America than in any other part of the world [42]. Brazil will experience the largest disease and economic burden particularly in the south-east and north-east where ZIKV infections are currently the highest in the country [1, 51]. This is not surprising given that Brazil has the largest population in LATAM (205 million), and its climate is conducive for year-round transmission across large urban and rural areas. Other countries with large ZIKV values are Colombia, Mexico, Venezu- ela and Cuba where high risk of infection has been previously estimated [8, 19, 42]. Some PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0006007 November 1, 2017 8 / 19 https://doi.org/10.1371/journal.pntd.0006007.t001 The economic impact is measured in thousand USD. The following countries were considered in the analysis but the model estimated a risk lower than 0.01 cases per month and a negligible economic burden: Anguilla, Antigua and Barbuda, Aruba, Barbados, Bahamas, Bermuda, Bonaire, Saint Eustatius and Saba, British Virgin Islands, Cayman Islands, Clipperton Island, Curacao, Dominica, Grenada, Guadeloupe, Martinique, Montserrat, Saint Kitts and Nevis, Saint Lucia, Saint Martin, Saint Vincent and the Grenadines, Saint-Barthelemy, Sint Maarten, Turks and Caicos Islands, US Minor Outlying Islands, US Virgin Islands. The following countries were considered in the analysis but the model estimated a risk lower than 0.01 cases per month and a negligible economic burden: Anguilla, Antigua and Barbuda, Aruba, Barbados, Bahamas, Bermuda, Bonaire, Saint Eustatius and Saba British Virgin Islands Cayman Islands Clipperton Island Curacao Dominica Grenada Guadeloupe Martinique Montserrat Saint Kitts and Nevis Table 1. Country-level projections of Zika virus (ZIKV) infection under the assumption of endemicity for bearing age, and estimated health and economic burden of Guillain-Barre´ syndrome, and microcephaly mates under the assumption of a 0.1:1 and 10:1 ZIKV-dengue ratios. The economic impact is measured in thousand USD. The following countries were considered in the analysis but the model estimated a risk lower than 0.01 cases per month and a negligible economic burden: Anguilla, Antigua and Barbuda, Aruba, Barbados, Bahamas, Bermuda, Bonaire, Saint Eustatius and Saba, British Virgin Islands, Cayman Islands, Clipperton Island, Curacao, Dominica, Grenada, Guadeloupe, Martinique, Montserrat, Saint Kitts and Nevis, Saint Lucia, Saint Martin, Saint Vincent and the Grenadines, Saint-Barthelemy, Sint Maarten, Turks and Caicos Islands, US Minor Outlying Islands, US Virgin Islands. The economic impact is measured in thousand USD. The following countries were considered in the analysis but the model estimated a risk lower than 0.01 cases per month and a negligible economic burden: Anguilla, Antigua and Barbuda, Aruba, Barbados, Bahamas, Bermuda, Bonaire, Saint Eustatius and Saba, British Virgin Islands, Cayman Islands, Clipperton Island, Curacao, Dominica, Grenada, Guadeloupe, Martinique, Montserrat, Saint Kitts and Nevis, Saint Lucia, Saint Martin, Saint Vincent and the Grenadines, Saint-Barthelemy, Sint Maarten, Turks and Caicos Islands, US Minor Outlying Islands, US Virgin Islands. https://doi.org/10.1371/journal.pntd.0006007.t001 differences were observed in the ranking of the countries most affected by ZIKV when we compared the estimated the number of cases and the number of affected pregnancies. One notable feature of these results was Cuba which was fifth in terms of overall infections in child- bearing women but ninth in terms of pregnancies due to low crude birth rate [8]. h h l d d d h h h h differences were observed in the ranking of the countries most affected by ZIKV when we compared the estimated the number of cases and the number of affected pregnancies. One notable feature of these results was Cuba which was fifth in terms of overall infections in child- bearing women but ninth in terms of pregnancies due to low crude birth rate [8]. Fig 1 shows the mean annual case predictions and indicates that the highest case estimates correspond to low elevation coastal areas of Brazil, Southern Mexico, the Caribbean, the Pacific coast of Central America, Ecuador, Colombia and Venezuela likely because the differences were observed in the ranking of the countries most affected by ZIKV when we compared the estimated the number of cases and the number of affected pregnancies. One notable feature of these results was Cuba which was fifth in terms of overall infections in child- bearing women but ninth in terms of pregnancies due to low crude birth rate [8]. Fig 1 shows the mean annual case predictions and indicates that the highest case estimates correspond to low elevation coastal areas of Brazil, Southern Mexico, the Caribbean, the Pacific coast of Central America, Ecuador, Colombia and Venezuela likely because the Fig 1 shows the mean annual case predictions and indicates that the highest case estimates correspond to low elevation coastal areas of Brazil, Southern Mexico, the Caribbean, the Pacific coast of Central America, Ecuador, Colombia and Venezuela likely because the PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0006007 November 1, 2017 9 / 19 After the epidemic: Zika virus projections for Latin America and the Caribbean Fig 1. Mean monthly estimates. Estimated mean monthly Zika virus (ZIKV) cases the total population for an average year at a 0.5 × 0.5 degree resolution. Blank cells indicate risk-free areas. This Figure was created using ArcGIS Desktop 10.3 based on the model outputs projected onto a 0.5 × 0.5 grid. The shapefile for the countries was obtained using the wrld_simpl layer of the maptools R package. https://doi.org/10.1371/journal.pntd.0006007.g001 Fig 1. Mean monthly estimates. Estimated mean monthly Zika virus (ZIKV) cases the total population for an average year at a 0.5 × 0.5 degree resolution. Blank cells indicate risk-free areas. This Figure was created using ArcGIS Desktop 10.3 based on the model outputs projected onto a 0.5 × 0.5 grid. The shapefile for the countries was obtained using the wrld_simpl layer of the maptools R package. https://doi.org/10.1371/journal.pntd.0006007.g001 Fig 1. Mean monthly estimates. Estimated mean monthly Zika virus (ZIKV) cases the total population for an average year at a 0.5 × 0.5 degree resolution. Blank cells indicate risk-free areas. This Figure was created using ArcGIS Desktop 10.3 based on the model outputs projected onto a 0.5 × 0.5 grid. PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0006007 November 1, 2017 The shapefile for the countries was obtained using the wrld_simpl layer of the maptools R package. abundance of Aedes spp. mosquitoes declines sharply at elevations above 1,700 metres above sea level [52] due to the effect of low temperatures on the biology of the virus and the mos- quito. Particularly large case numbers are expected in south-eastern and north-eastern Brazil, the Mexican Isthmus, Cuba, Puerto Rico, northern Colombia, and northern Venezuela. The estimated spatial distribution of cases agrees with observations of ZIKV infection in the region [1], with previous studies estimating the environmental suitability [10, 19, 53, 54] and risk of arboviral infection [8, 42], and with records of other arboviral diseases in LATAM [55–59]. Although not explicitly accounted for, urbanisation plays a major role in the occurrence of Aedes-related diseases as it increases its larval habitats [60]. Recent studies indicate that the presence of Aedes mosquitoes and ZIKV incidence is larger in urban than in rural areas [60, 61]. Our predicted geographical distribution of cases agrees well with such studies as the higher number of cases are predicted to occur in areas where population densities are high. Two distinctive seasonal cycles are observed in the predicted ZIKV cases. Fig 2 shows the estimated seasonal cycles for the six countries with the highest predicted ZIKV burden. In the southern hemisphere the high transmission season is between April and June (e.g. Brazil, and Peru), whilst in the northern hemisphere it peaks between September and November (see PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0006007 November 1, 2017 10 / 19 After the epidemic: Zika virus projections for Latin America and the Caribbean Fig 2. Mean annual cycle. Estimated mean annual cycle of ZIKV infections in the six countries with the highest predicted health burden. https://doi.org/10.1371/journal.pntd.0006007.g002 Fig 2. Mean annual cycle. Estimated mean annual cycle of ZIKV infections in the six countries with the highest predicted health burden. https://doi.org/10.1371/journal.pntd.0006007.g002 Mexico, Colombia, Venezuela and Cuba) in agreement with previous studies [62]. A bimodal seasonal cycle is observed in Colombia which may be related to a bimodal annual cycle of pre- cipitation observed in the central and western regions [63]. Although precipitation was not included in the final model, by including PET0:2, we have accounted for some of its effects on ZIKV incidence. It is reminded that through evapotranspiration, atmospheric moisture returns to land as rainfall [64]. Factors such as intervention measures could also play a role in defining the seasonal trends of ZIKV transmission, yet have not been explicitly accounted for in the model. Our modelling framework also allowed us to investigate spatio-temporal changes in ZIKV occurrence. We estimate that in the Southern hemisphere, ZIKV transmission could extend to 35˚S between February and May, contracting thereafter to 30˚S (see S1 Video in Supple- mentary material). In the Northern hemisphere, transmission remains stable up to 20˚N most of the year, expanding to 30˚N between July and November. Epidemic-prone regions Under the assumption of endemicity, there are areas that will likely remain epidemic due to intermittent or short transmission seasons. Our model identified the Mexican Plateau, the Andean foothills, and parts of northern Paraguay as highly epidemic (Fig 3). Some areas with regular transmission also showed a high RSD. Cold regions (< 20˚C) are marginally permissive for vector development and viral transmission [48]. Populations in these areas are likely to have low herd immunity due to low transmission intensity and viral density [68] increasing their likelihood of succumbing to epidemics. Childbearing women would therefore be more at risk in epidemic-prone areas due to low herd immunity. Areas with reg- ular transmission may also be epidemic-prone due to outbreaks occurring earlier or later than usual with unusual high peaks in seasonal transmission as a consequence [47]. These changes may be related to variability in environmental, socioeconomic or meteorological factors [69]. After the epidemic: Zika virus projections for Latin America and the Caribbean Given that the Asian lineage is related to brain developmental abnormalities [66], and that it is the lineage present in LATAM, we also estimated the potential number of microcephaly cases. Based on the crude birth rates per country [36], we estimate that 61 (3–807) thousand pregnancies (Table 1) could be affected by prenatal ZIKV transmission (i.e. ZIKV infection of the mother at some point during pregnancy). Assuming that only first trimester ZIKV infec- tions may cause microcephaly, and a risk of microcephaly due to infection of between 0.88% and 14.4% [44], we estimate that 5 (0–116) thousand children could develop microcephaly yearly in LATAM. With an estimated direct medical cost of USD 28,818 per GBS and of USD 91,102 per microcephaly case per lifetime [46], the ZIKV-related neurological sequelae would add an economic burden of USD 2.3 (USD 0–159.3) billion each year. The large confidence intervals indicate that the economic impact is largely sensitive to selected zika to dengue ratio. This sensitivity has major implications for surveillance systems and public health preparedness to adequately respond to the presence of neurological sequelae. There are uncertainties in our estimates of neurological risk. First, available data on the risk of GBS and microcephaly due to ZIKV infection are limited, especially in areas where the infection rates are unknown [44] posing problems for the use of country-specific risk factors. Second, the risk of microcephaly has dramatically increased in some locations over the past year [67] suggesting that the risk estimates should be revised relatively often. Third, the intro- duction of a vaccine and its combination with effective control measures could reduce the risk of infection and hence the risk of neurological sequelae. Risk of neurological sequelae GBS is an acute immune-mediated muscle weakness that affects the peripheral nervous system leading to paralysis that has been attributed to ZIKV infections [3, 65]. Assuming a risk of GBS between 0.24 and 31.78 cases per 1000 ZIKV infections [3, 45], and a ZIKV-dengue ratio of 0.1:1, 1:1, and 10:1, we estimate 64 thousand GBS cases per annum (range: 0.2–51596 thou- sand) across the LATAM region. 11 / 19 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0006007 November 1, 2017 After the epidemic: Zika virus projections for Latin America and the Caribbean Fig 3. Epidemic-prone regions. Location of areas of high variability (i.e. epidemic) in Zika incidence. Blank cells indicate a relative standard deviation lower than one. This Figure was created using ArcGIS Desktop 10.3 based on the model outputs projected onto a 0.5 × 0.5 grid. The shapefile for the countries was obtained using the wrld_simpl layer of the maptools R package. https://doi org/10 1371/journal pntd 0006007 g003 Fig 3. Epidemic-prone regions. Location of areas of high variability (i.e. epidemic) in Zika incidence. Blank cells indicate a relative standard deviation lower than one. This Figure was created using ArcGIS Desktop 10.3 based on the model outputs projected onto a 0.5 × 0.5 grid. The shapefile for the countries was obtained using the wrld_simpl layer of the maptools R package. https://doi.org/10.1371/journal.pntd.0006007.g003 Fig 3. Epidemic-prone regions. Location of areas of high variability (i.e. epidemic) in Zika incidence. Blank cells indicate a relative standard deviation lower than one. This Figure was created using ArcGIS Desktop 10.3 based on the model outputs projected onto a 0.5 × 0.5 grid. The shapefile for the countries was obtained using the wrld_simpl layer of the maptools R package. https://doi.org/10.1371/journal.pntd.0006007.g003 ENSO effects A typical ENSO event is likely to increase the monthly case load across most of LATAM. Fig 4 shows the areas where increases in transmission are expected during a typical ENSO. Epidemic areas such the Andean foothills in Ecuador and Peru may show increases between 1.2 and 2.5 times the average case load of a typical non-ENSO period. During a strong ENSO event (e.g. 1997–1998 or 2015–2016), many more regions are expected to experience large increases (1.2– 2.5 times the average case load during a non-ENSO period) in ZIKV cases including south- eastern Mexico, Honduras, Nicaragua and the western lowlands of South America. Previous studies indicate that ENSO could increase the risk of ZIKV infection due to an amplification effect by providing conducive conditions for transmission [42] particularly during strong events, and so ENSO may be an important driver of inter-annual variation in ZIKV transmis- sion [70]. PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0006007 November 1, 2017 12 / 19 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0006007 November 1, 2017 Limitations Our spatially explicit projections of ZIKV risk for LATAM provide useful information for pub- lic health preparedness. However, there are several caveats that ought to be mentioned. First, our estimates are based on the occurrence of a different organism (i.e. dengue virus) which, despite its remarkable similarities with ZIKV, has important differences [21] that may affect our results. One key difference is that there are four dengue serotypes while there is only one ZIKV serotype (subdivided into two lineages and three genotypes) [71]. None of the dengue serotypes confers protective neutralizing antibody responses against all four serotypes [6]. Thus, a single person may succumb to dengue more than once in a lifetime. ZIKV, however, induces a humoural antibody response that seems to confer lifelong immunity against reinfec- tion, although this assumption still needs to be confirmed [72]. The assumption of a lifelong immunity to ZIKV indicates that once individuals (succumbing to the disease only once in a lifetime) become immune they also become unavailable for future infections. This situation means that recurring outbreaks would necessarily be related to the remaining susceptible individuals in a population, in addition to newly born hosts. Another important difference PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0006007 November 1, 2017 13 / 19 After the epidemic: Zika virus projections for Latin America and the Caribbean Fig 4. Effects of El Niño. (A) Ratio of an average El Niño month to an average non-El-Niño month. (B) Ratio of an average strong El Niño month to an average non-El-Niño month. Blank cells indicate a ratio lower than one. This Figure was created using QGIS Desktop 2.0 based on the model outputs projected onto a 0.5 × 0.5 grid. The shapefile for the countries was obtained using the wrld_simpl layer of the maptools R package. htt //d i /10 1371/j l td 0006007 004 Fig 4. Effects of El Niño. (A) Ratio of an average El Niño month to an average non-El-Niño month. (B) Ratio of an average strong El Niño month to an average non-El-Niño month. Blank cells indicate a ratio lower than one. This Figure was created using QGIS Desktop 2.0 based on the model outputs projected onto a 0.5 × 0.5 grid. The shapefile for the countries was obtained using the wrld_simpl layer of the maptools R package. https://doi.org/10.1371/journal.pntd.0006007.g004 between the two viruses is the presence of a sexual transmission mode in ZIKV [73]. Supporting information S1 Fig. Model estimates. GAMM-estimated monthly ZIKV cases for the period January 2001 to December 2012 for Brazil and Mexico. The shaded area indicates the 95% confidence inter- vals. S2 Fig. Smoothed relations. GAMM-estimated relationships between average monthly inci- dence and (A) temperature lagged zero to two months, and (B) potential evapotranspiration (PET) lagged zero to two months. The solid lines represent the functional form of the relation- ship between the incidence rate and the predictor. The shaded area indicate the estimated 95% confidence intervals. The “X” axis represents variations on each predictor. The “Y” axis is labelled s(cov, edf) where cov is the name of the predictor, and edf are the estimated degrees of freedom of the smoother. S1 Video. Estimated Zika cases per month. Spatially explicit estimates of mean Zika infec- tions per month across Latin America and the Caribbean under the assumption of an endemic state. The Figures displayed on this video were created using ArcGIS Desktop 10.3 based on the model outputs projected onto a 0.5 × 0.5 grid. The shapefile for the countries was obtained using the wrld_simpl layer of the maptools R package. The video was created using ImageMa- gick and converted to mp4 using Convertio online. (MP4) After the epidemic: Zika virus projections for Latin America and the Caribbean economic burden across LATAM under the assumption of endemicity. The geographic distri- bution of other vector-borne diseases sharing the same vector [53, 54], the lack of a vaccine [5], the absence of effective vector control measures [11], and the environmental suitability of the region [10] suggest that ZIKV will likely become endemic throughout LATAM in the near future. This hypothesis concurs with a recent study that on the basis of a numerical model pre- dicts that the virus will eventually become endemic [9]. Recent declarations from the WHO also suggest that ZIKV infection will become endemic [76]. We produced to our knowledge, the first high-resolution spatially-explicit projections of future ZIKV cases under the assump- tion of endemicity. Across LATAM, our projections suggest that ZIKV may impose a health burden of 12 (1–162) million cases per year, 69 (0–5276) thousand of which are likely to have major neurological sequelae. The economic burden imposed across the LATAM region amounts to USD 2 (0–159) billion per year, and this may increase up to *2 times in the aftermath of a strong ENSO event particularly in epidemic areas where public health systems are unprepared for major outbreaks. These projections can inform public health preparedness and response, and offer an opportunity to enhance capabilities in LATAM. Acknowledgments The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health. Limitations Sexual transmission could occur from asymptomatic or symptomatic individuals through genital, oral, or anal intercourse; and from male-male, male-female, and female-male contact [72, 73]. Not only sexual transmission does not occur in dengue, but also it is not driven by temperature and PET which are the main transmission drivers in our disease model. The extent to which sexual transmission can modify disease occurrence across time and space is unclear and requires further investigation. Second, recent research has shown that the ecological niches of dengue and ZIKV are sig- nificantly different, with the niche of ZIKV expanding more than that of dengue [19]. There- fore, the potential distribution of ZIKV could expand a greater geographical area than that predicted by our model [19]. Third, our results do not account for the potential deployment of a vaccine which would significantly reduce the risk of ZIKV infection. Recent studies suggest that two ZIKV vaccine prototypes recently entered a phase-1 human-safety testing [74] and an epitope-focused vaccine for viruses in the so-called ZIKV-dengue super serogroup could be developed soon [22]. However, large-scale efficacy trials and the mass production of such a vaccine may still be years away [75]. Fourth, in the absence of long-term datasets for compareable viruses, we have based our estimates of ZIKV on one of the largest and more spatially diverse dengue datasets (accounting for over 60% of the reported cases across LATAM); however, local socioeconomic determi- nants of disease (e.g. access to protective measures, intervention deployment, urbanisation indices, and international travel data) in countries not included in our dataset may signifi- cantly alter disease occurrence [16]. This issue is important because socioeconomic factors vary at fine scales for political or administrative reasons and so our model could over or under estimate the risk of infection in some regions. This fact highlights the need for spatially explicit, high-resolution, publicly available epidemiological and socioeconomic time series data for LATAM. There are remarkable genomic and epidemiological similarities between dengue virus and ZIKV [21, 22]. Based on such similarities, we have used a detailed panel of time series of con- trywide dengue reports as a surrogate for ZIKV infection to estimate the potential health and PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0006007 November 1, 2017 14 / 19 Funding acquisition: Iain R. Lake. Investigation: Felipe J. Colo´n-Gonza´lez, Carlos A. Peres, Christine Steiner São Bernardo, Paul R. Hunter, Iain R. Lake. Methodology: Felipe J. Colo´n-Gonza´lez, Paul R. Hunter, Iain R. Lake. Project administration: Felipe J. Colo´n-Gonza´lez. Resources: Felipe J. Colo´n-Gonza´lez, Carlos A. Peres, Christine Steiner São Bernardo. Software: Felipe J. Colo´n-Gonza´lez. Supervision: Carlos A. Peres, Paul R. Hunter, Iain R. Lake. Supervision: Carlos A. Peres, Paul R. Hunter, Iain R. Lake. Validation: Felipe J. Colo´n-Gonza´lez. Visualization: Felipe J. Colo´n-Gonza´lez. Visualization: Felipe J. Colo´n-Gonza´lez. Writing – original draft: Felipe J. Colo´n-Gonza´lez. Writing – original draft: Felipe J. Colo´n-Gonza´lez. Writing – review & editing: Felipe J. Colo´n-Gonza´lez, Carlos A. Peres, Christine Steiner São Bernardo, Paul R. Hunter, Iain R. Lake. Author Contributions Conceptualization: Felipe J. Colo´n-Gonza´lez, Paul R. Hunter, Iain R. Lake. Data curation: Felipe J. Colo´n-Gonza´lez, Carlos A. Peres, Christine Steiner São Bernardo. Formal analysis: Felipe J. Colo´n-Gonza´lez. Conceptualization: Felipe J. Colo´n-Gonza´lez, Paul R. Hunter, Iain R. Lake. Data curation: Felipe J. Colo´n-Gonza´lez, Carlos A. Peres, Christine Steiner São Bernardo. Formal analysis: Felipe J. Colo´n-Gonza´lez. Data curation: Felipe J. Colo´n-Gonza´lez, Carlos A. Peres, Christine Steiner São Bernardo. 15 / 19 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0006007 November 1, 2017 After the epidemic: Zika virus projections for Latin America and the Caribbean After the epidemic: Zika virus projections for Latin America and the Caribbean 14. 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https://openalex.org/W4380853752	https://alustath.uobaghdad.edu.iq/index.php/UJIRCO/article/download/2199/1520	Arabic	null	حـركـــــيَّة الـنسيجِ النصيِّ في الشعرِ الأندلسيِّ (امتدادُ التأثرِ وارتدادِ الأثر )	Al-ustād̲	2,023	cc-by	7,881	جملة األستاذ للعلوم اإلنسانية واالجتماعية جملد (62) العدد (2) حزيران لسنة 2023 حـركـــــيَّة الِّـنسيجِ النصيِّ يف الشعرِ األندلسي ) (امتدادُ التأثرِ وارتدادِ األثر م.د. أسيل حممد ناصر جامعة الكرخ للعلوم - العراق aseel.m@kus.edu.iq :التقديم28 / 1 / 2023 :القبول1 / 3 / 2023 :النشر15 / 6 / 2023 Doi: https://doi.org/10.36473/ujhss.v62i2.2199 This work is licensed under a Creative Commons Attribution 4.0 International Licenses ال ملخص َّتأتي مشكلة البحث محاولة تجديديَّة إلعادة استقراء الشعرِ األندلسي ِ وظاهرة على وفق متطلبات النقد المُعاصر، وإن الدراسة هنا جاءت محاولةً تجديدية في إعادة النظر بتراثِ الشعرِ العربي ِ ، وقراءته برؤيةٍ معاصرة تستجيبُ لمريدات النقد العربي ِ المُعاصر، وتحديث.القراءات التراثية على وفق متطلباته العصري ِة الهدف من البحث هو الوقوف على مدى استجابة الشعرِ األندلسي ِ لنظريات النقد المعاصرة، والتماهي مع منطلقاتها، ومدى التأثُّر والتأثير الذي اتضحت معالمه .في نصوص الشعراء األندلسيين احتكمت الدراسة إلجراء تطبيقي ٍ على عدد من النصوص الشعريَّة التي اتضحت فيها مديات التغيير الذي طرأ على الشعرِ العربي ِ ؛ نتيجةً للتحول المكاني ِ الذي طرأ على الشاعر األندلسي ِ ؛ من حيثُ األلفاظ ٍ والمعاني والصور، ومدى انعكاس ذلك على الشعرِ في األندلس لغيرِ العرب، عبرَ منهج بحثي ٍ تطبيقي ٍ تحليلي ُيحتكم . للرؤى النقديَّة المعاصرة في تفكيك النصوصِ وإعادة استقرائها مجددًا تبينَ أنَّ النصَّ الشعريَّ األندلسيَّ قادرٌ على ،االستجابة لمتطلبات النقد المعاصر، وأثبت األثر الذي تركهُ الشعراء العربُ في األندلس في مضمون الحياة األندلسية وانعكاس ذلك على شعرا.) ء أوروبا في شعر ( التروبادور الكلمات المفتاحيَّة : األندلسي، الشع ر، النسيج النصيُّ، النقد المعاصر . The Movement of Textual Texture in Andalusian Poetry جملة األستاذ للعلوم اإلنسانية واالجتماعية جملد (62) العدد (2) حزيران لسنة 2023 حـركـــــيَّة الِّـنسيجِ النصيِّ يف الشعرِ األندلسي ) (امتدادُ التأثرِ وارتدادِ األثر م.د. أسيل حممد ناصر جامعة الكرخ للعلوم - العراق aseel.m@kus.edu.iq :التقديم28 / 1 / 2023 :القبول1 / 3 / 2023 :النشر15 / 6 / 2023 Doi: https://doi.org/10.36473/ujhss.v62i2.2199 This work is licensed under a Creative Commons Attribution 4.0 International Licenses ال ملخص َّتأتي مشكلة البحث محاولة تجديديَّة إلعادة استقراء الشعرِ األندلسي ِ وظاهرة على وفق متطلبات النقد المُعاصر، وإن الدراسة هنا جاءت محاولةً تجديدية في إعادة النظر بتراثِ الشعرِ العربي ِ ، وقراءته برؤيةٍ معاصرة تستجيبُ لمريدات النقد العربي ِ المُعاصر، وتحديث.القراءات التراثية على وفق متطلباته العصري ِة الهدف من البحث هو الوقوف على مدى استجابة الشعرِ األندلسي ِ لنظريات النقد المعاصرة، والتماهي مع منطلقاتها، ومدى التأثُّر والتأثير الذي اتضحت معالمه .في نصوص الشعراء األندلسيين احتكمت الدراسة إلجراء تطبيقي ٍ على عدد من النصوص الشعريَّة التي اتضحت فيها مديات التغيير الذي طرأ على الشعرِ العربي ِ ؛ نتيجةً للتحول المكاني ِ الذي طرأ على الشاعر األندلسي ِ ؛ من حيثُ األلفاظ ٍ والمعاني والصور، ومدى انعكاس ذلك على الشعرِ في األندلس لغيرِ العرب، عبرَ منهج بحثي ٍ تطبيقي ٍ تحليلي ُيحتكم . للرؤى النقديَّة المعاصرة في تفكيك النصوصِ وإعادة استقرائها مجددًا تبينَ أنَّ النصَّ الشعريَّ األندلسيَّ قادرٌ على ،االستجابة لمتطلبات النقد المعاصر، وأثبت األثر الذي تركهُ الشعراء العربُ في األندلس في مضمون الحياة األندلسية وانعكاس ذلك على شعرا.) ء أوروبا في شعر ( التروبادور الكلمات المفتاحيَّة : األندلسي، الشع ر، النسيج النصيُّ، النقد المعاصر . The Movement of Textual Texture in Andalusian Poetry جملد (62) العدد (2) حزيران لسنة 2023 جملة األستاذ للعلوم اإلنسانية واالجتماعية حـركـــــيَّة الِّـنسيجِ النصيِّ يف الشعرِ األندلسي ) (امتدادُ التأثرِ وارتدادِ األثر م.د. جملة األستاذ للعلوم اإلنسانية واالجتماعية جملد (62) العدد (2) حزيران لسنة 2023 حـركـــــيَّة الِّـنسيجِ النصيِّ يف الشعرِ األندلسي ) (امتدادُ التأثرِ وارتدادِ األثر م.د. أسيل حممد ناصر جامعة الكرخ للعلوم - العراق aseel.m@kus.edu.iq :التقديم28 / 1 / 2023 :القبول1 / 3 / 2023 :النشر15 / 6 / 2023 Doi: https://doi.org/10.36473/ujhss.v62i2.2199 This work is licensed under a Creative Commons Attribution 4.0 International Licenses ال ملخص َّتأتي مشكلة البحث محاولة تجديديَّة إلعادة استقراء الشعرِ األندلسي ِ وظاهرة على وفق متطلبات النقد المُعاصر، وإن الدراسة هنا جاءت محاولةً تجديدية في إعادة النظر بتراثِ الشعرِ العربي ِ ، وقراءته برؤيةٍ معاصرة تستجيبُ لمريدات النقد العربي ِ المُعاصر، وتحديث.القراءات التراثية على وفق متطلباته العصري ِة الهدف من البحث هو الوقوف على مدى استجابة الشعرِ األندلسي ِ لنظريات النقد المعاصرة، والتماهي مع منطلقاتها، ومدى التأثُّر والتأثير الذي اتضحت معالمه .في نصوص الشعراء األندلسيين احتكمت الدراسة إلجراء تطبيقي ٍ على عدد من النصوص الشعريَّة التي اتضحت فيها مديات التغيير الذي طرأ على الشعرِ العربي ِ ؛ نتيجةً للتحول المكاني ِ الذي طرأ على الشاعر األندلسي ِ ؛ من حيثُ األلفاظ ٍ والمعاني والصور، ومدى انعكاس ذلك على الشعرِ في األندلس لغيرِ العرب، عبرَ منهج بحثي ٍ تطبيقي ٍ تحليلي ُيحتكم . للرؤى النقديَّة المعاصرة في تفكيك النصوصِ وإعادة استقرائها مجددًا تبينَ أنَّ النصَّ الشعريَّ األندلسيَّ قادرٌ على ،االستجابة لمتطلبات النقد المعاصر، وأثبت األثر الذي تركهُ الشعراء العربُ في األندلس في مضمون الحياة األندلسية وانعكاس ذلك على شعرا.) ء أوروبا في شعر ( التروبادور الكلمات المفتاحيَّة : األندلسي، الشع ر، النسيج النصيُّ، النقد المعاصر . The Movement of Textual Texture in Andalusian Poetry أسيل حممد ناصر جامعة الكرخ للعلوم - العراق aseel.m@kus.edu.iq :التقديم28 / 1 / 2023 :القبول1 / 3 / 2023 :النشر15 / 6 / 2023 :التقديم28 / 1 / 2023 :التقديم28 / 1 / 2023 This work is licensed under a Creative Commons Attribution 4.0 International Licenses ال ملخص َّتأتي مشكلة البحث محاولة تجديديَّة إلعادة استقراء الشعرِ األندلسي ِ وظاهرة على وفق متطلبات النقد المُعاصر، وإن الدراسة هنا جاءت محاولةً تجديدية في إعادة النظر بتراثِ الشعرِ العربي ِ ، وقراءته برؤيةٍ معاصرة تستجيبُ لمريدات النقد العربي ِ المُعاصر، وتحديث.القراءات التراثية على وفق متطلباته العصري ِة الهدف من البحث هو الوقوف على مدى استجابة الشعرِ األندلسي ِ لنظريات النقد المعاصرة، والتماهي مع منطلقاتها، ومدى التأثُّر والتأثير الذي اتضحت معالمه .في نصوص الشعراء األندلسيين احتكمت الدراسة إلجراء تطبيقي ٍ على عدد من النصوص الشعريَّة التي اتضحت فيها مديات التغيير الذي طرأ على الشعرِ العربي ِ ؛ نتيجةً للتحول المكاني ِ الذي طرأ على الشاعر األندلسي ِ ؛ من حيثُ األلفاظ ٍ والمعاني والصور، ومدى انعكاس ذلك على الشعرِ في األندلس لغيرِ العرب، عبرَ منهج بحثي ٍ تطبيقي ٍ تحليلي ُيحتكم . للرؤى النقديَّة المعاصرة في تفكيك النصوصِ وإعادة استقرائها مجددًا تبينَ أنَّ النصَّ الشعريَّ األندلسيَّ قادرٌ على ،االستجابة لمتطلبات النقد المعاصر، وأثبت األثر الذي تركهُ الشعراء العربُ في األندلس في مضمون الحياة األندلسية وانعكاس ذلك على شعرا.) ء أوروبا في شعر ( التروبادور الكلمات المفتاحيَّة : األندلسي، الشع ر، النسيج النصيُّ، النقد المعاصر . The Movement of Textual Texture in Andalusian Poetry (An Extended Influence and Rebound Effect) Inst. Dr. Aseel Mohammed Nalir Al-Karkh University of Science, Iraq aseel.m@kus.edu.iq :َالمقد ِّمة مثَّل العصرُ األندلسيُّ نقطة انطالقٍ جديدة في حياة الشاعر العربي، ورؤية تجديدية في مسيرة الشعر العربي ِ ؛ بحكم ما اتسمت به هذه الحقبة التاريخية من حيا ة الشعر العربي ِ من تغييرٍ في منظومة التفكير لدى الشاعر العربي ِ الذي راح يبحثُ ويتأملُ في صور الطبيعة، وتمازج األقوام وألسنتهم، والحياة الجديدة التي َتختلفُ عن بيئة الشرق وتفصيالتها؛ األمر الذي جعل من الشاعر العربي ِ مُحاطًا في مراحلَ ثالث؛ ليكتمل عندهُ ا لمشهد، فالمرحلة األولى هي ( مرحلة الخيال ) والمرحلة الثانية هي ( مرحلة التخييل ) والمرحلةُ الثالثة هي ( مرحلة اإلخراج الفني ِ )، وهذه المراحل تمث ِلُ مجتمعةً األداء الحركيَّ لنسيج النصَّ ِِ الذي تحولَّ عقبى ذلك إلى حالة جديدة انعكست وأخذت مداها في على الثقاف ات والشعر األوروبي بنحو عام فانعكست الجذور ،العربيَّة كالزجل الموشَّ ح على الثقافات األوروبيَّة كافة ( ينظر: الطاهر مكي1987 ، ص119 ) THE Taher) Makki,1987,p:119 ).، فهذه المالمحُ والتفاصيل أعطت مساحة فنيَّة للشاعر األندلسي ألن يؤثر ويتأثر، وهذه الرفقة التبادليَّة تحتاجً لوقفةٍ على وفق النقد المعاصر؛ لتتضح معالمها الفنيَّة في تفاصيل النقد الحديث وناقديه عبر الدراسة للمضمون وجوهره ومراحله التي مرَّ عبرها البناء الفن ي للقصيدة األندلسيَّة بوساطة مباحث ثالث: . المبحث األول: مرحلة الخيال .المبحث الثاني: مرحلة التخييل : المبحثِ الثالث . مرحلة امتداد األثر ورجع الصدى وهذا ما ستوضحه تفصيالت هذه الدراسة عبر اتجاهاتها النقديَّة .المُعاصرة Abstract The research problem comes as a renewal attempt to extrapolate Andalusian poetry and its phenomenon according to the requirements of contemporary criticism.The aim of the research is to determine the extent of the response of Andalusian poetry to contemporary theories of criticism, identification with its premises, and the extent of influence and influence whose features became evident in the texts of Andalusian poets .The study resorted to an applied procedure on a number of poetic texts, in which the extents of change that occurred in Arabic poetry became clear. As a result of the spatial shift that occurred to the Andalusian poet; In terms of words, meanings, and images, and the extent of their reflection on poetry in Andalusia for non-Arabs, through an applied and analytical research approach that appeals to contemporary critical visions in deconstructing texts and re-extrapolating them again. It turned out that the Andalusian poetic text is able to respond to the requirements of contemporary criticism, and it proved the impact left by the Arab poets in Andalusia on the content of Andalusian life, and its reflection on European poets in the poetry of the troubadours. Keywords: Andalusian, poetry, textual fabric, contemporary criticism 357 جملد (62) العدد (2) حزيران لسنة 2023 جملة األستاذ للعلوم اإلنسانية واالجتماعية : أالمبحث األول : مرحلة الخيال 13 )، ولغرض استيضاح ذلك بنحو 358 جملد (62) العدد (2) حزيران لسنة 2023 جملة األستاذ للعلوم اإلنسانية واالجتماعية واضح يمكن الوقوف عند قول شاعر األندلس األشهر ابن زيدون وهو يكابدُ ألم الحُب ِ والهجران؛ ليعبر عن : ذلك بقوله واضح يمكن الوقوف عند قول شاعر األندلس األشهر ابن زيدون وهو يكابدُ ألم الحُب ِ والهجران؛ ليعبر عن : ذلك بقوله ،( من البسيط ) : ( ابن زيدون، الديوان2005،ص إِن ي ُذَكَرت ــ ِك بِالزَّهْراءَ مُشت ــ ًاقا وَلِلنَسي ــــــ ِمِ اِعتِاللٌ في أَصائِلِه َكـَ ٌوَالرَ وضُ عَن مائِهِ الفِضِ ي ِ مُبتَسِ م َما ـــــ ك يَومٌ كَأَي امِ لَذ اتٍ لَنا انصَ رَمَت نَلهو بِما يَستَميلُ العَينَ مِن ٍزَهَر كَأَنَّ أَعيُنَهُ إِذ عايَنَت أَرَقي ِوَردٌ تَأَلَّقَ في ضاحي مَنابِتِه ِسَ رى يُنافِحُهُ نَيلوفَرٌ عَب ـــ ٌق كُل َيَهيجُ لَنا ذِكرى تَش ـــ وُّقِنا ال سَ كَّنَ َللاَُ قَلباً عَقَّ ذِك ـ ُرَكُم لَو شاءَ حَملي نَسيمُ الصُ بحِ حينَ سَ رى ،( من البسيط ) : ( ابن زيدون، الديوان2005 ،ص51 ( ) Ibn Zaydun, Al Diwan, 2005, p. : أالمبحث األول : مرحلة الخيال 51 :) إِن ي ُذَكَرت ــ ِك بِالزَّهْراءَ مُشت ــ ًاقا وَاألُفقُ طَلقٌ وَمَرأى األَرضِ قَد راقا وَلِلنَسي ــــــ ِمِ اِعتِاللٌ في أَصائِلِه َك ـــــــــــــ َّـــ أَن هُ رَقَّ ل ـ ي فَاع ــ َّتَل اقا ـ إِشف ٌوَالرَ وضُ عَن مائِهِ الفِضِ ي ِ مُبتَسِ م َـــــ ك َما شَ ق ـقـ َتَ عَنِ الل ـــــــ ب اتِ أَط واقا ـ يَومٌ كَأَي امِ لَذ اتٍ لَنا انصَ رَمَت ـنـ بِت ا لَها حي ـ نَ ن ــــــــــ امَ الدَهرُ سُ ر اقا نَلهو بِما يَستَميلُ العَينَ مِن ٍزَهَر ــــ ج َالَ الن ــ دى فيهِ حَت ى مالَ أَعناقا كَأَنَّ أَعيُنَهُ إِذ عايَنَت أَرَقي َـ بَك ت لِما بي فَج ـ الَ الدَم ــ عُ رَقراقا ِوَردٌ تَأَلَّقَ في ضاحي مَنابِتِه َــف ازدادَ مِنهُ الضُ حى في العَينِ إِشر اقا ِسَ رى يُنافِحُهُ نَيلوفَرٌ عَب ـــ ٌق وَسن ــــ َّانُ نَب ـ هَ مِنهُ الصُ بحُ أَح ـــ داقا كُل َيَهيجُ لَنا ذِكرى تَش ـــ وُّقِنا ـــ إِلَي كِ لَم يَعدُ عَنها الصَ درُ أَن ضاقا ال سَ كَّنَ َللاَُ قَلباً عَقَّ ذِك ـ ُرَكُم َــــ ف ِلَم يَط ـ ر بِجَن ــ َاحِ الش ــ وقِ خَف اقا ـ لَو شاءَ حَملي نَسيمُ الصُ بحِ حينَ سَ رى ــ واف َاكُمُ بِف ـــ تىً أَضن ــ اهُ م ــــ ا القى ال شكَّ أنَّ طريقة تفكير الشاعر ،ذهبت باتجاه جعل الطبيعة نظيرهُ في المعاناة فهي تعتلُّ( تمرض ) العتالله تنقل أشواقهُ، وترسلُ معاناته، وتُخبر عمَّا آل إليهِ مصيره، والشاعر بذلك ينبئ عن امتالكهِ لناصيةِ الخيالِ التي جمَّعها وخزنَّها في ذاكرته؛ ليطلقَ عنان قلمهِ ليترجمها على الورق صورًا مست قطبةً من بؤرة مكانيَّة جعلها صنوًا له في الحركة، واألداء، لتعمل هندسة الدماغ على إعادة ترتيب المخزونات، وترسل مجسَّ اتها؛ موعِزةً للعقل البشري بأن يبثَّها طبقًا لما يراهُ مناسبًا بإزاء هذه اإلدراكات، والحركة النصيَّة، وقيادة الشاعر للنسيج النص ِ ي، وفي هذا ا لشأن يذهب الدكتور محمد مفتاح بالقول إلى أنَّ ( الخطابُ الشعريُّ وليدُ هذه المكونات جميعها؛ يُدرك بنسقي السمع والبصر، ثمَّ يخزنُ ما أُدرِكَ ونُظِ مَ وعُلِمَ في الذاكرة؛ ليُستثارَ بعضٌ منهُ عندما تدعو الحاجة إلى ذلك بقياس المجهول على المعلوم؛ إنَّ الخزنَ في ا ًلذاكرة يتشكَّلُ في بنيات نموذجيَّة تُتيحُ التكهُّنَ بما سيتلو بناء ،على ما ذُكرَ من مؤشرات ) ( مفتاح، محمد2010 ، الجزء3 ، ص342 ( ) Moftah, Muhammad, 2010, Part 3, p. : أالمبحث األول : مرحلة الخيال أ وهي المرحلة األولى من مراحل التكوين اإلبداعي ِ التي يبحث عنها الشاعر؛ لغرض اإلعداد للمرحلة التي تليها، وهي مرتكزة على أساس تفكيري ِ يستندُ للرصد، والمالحظة، والتأمل، فالخيالُ هو( صورة للتجربة تعمل على تقديم الحقيقي وغير الحقيقي في خليط أو مزاج غير مح دد العوالم يقوم الفهم بعد ذلك بترسيب ،الحقيقة منه ) (الربيعي2012 ، ص131 ( ) Al-Rubaie, 2012, p. 131 ُ)، ومعنى ذلك أنَّ ارتباطه األولى متعلقٌ بمجهود الشاعر، وقدرته الذاتيَّة على التقاط الصور والوقائع من تفاصيل الواقع، وخزنها في ذاكرته؛ ليعمل بع ذلك على استرج اعها بالطرائق التي تمكنَّهُ في أن يجعلها مؤثرةً ومقبولة بل ألبعد من ذلك ِتصلُ إلى حالة من االدهاش عبر آلية نقديَّة يُصطلح عليها ( التداعي )؛ بوصفها ( وسيلة خلقٍ فني ٍ لفعل التصوير الذي يستمدهُ الشاعر عبر تداعيات ذاكرته الخياليَّة المحف ِزة لذلك الفعل ـ فعل التصوير ـ للدخول إلى ،بيئة النسيج النص ِ ي ) ( ناصر2016 ، ص133 ( ) Nasser, 2016, p. 133 )؛ ليحقق عبر تلك البيئة نتاجًا شعريًا مختلفًا عمَّا رآهُ في رصدياتهِ السابقة المقتبسة من الطبيعة المشرقيَّة التي تختلفُ في نظيرتها ًاألندلسيَّة ظرفيًا، وثقافيًا، ومكاني ا؛ لتتحول إلى بؤرة نصيَّة يتحركُ خيال الشاعر عندها، ويطرحُ رؤاهُ بإزائها؛ ألن البؤرة المكانيَّة بإمكانها توسيع وجودها، تخلقُ صورها المستمدة من وجودها، ليتحركَ خيال الشاعر في ِ إطار حدودها المرسومة، ويبثُّ أفكارهُ، ويتجهُ صوب ما يحققُ عملية الخلقِ اإلبداعي ،( يُنظر: النصي ِر ،ياسين2011 ، ص13 ( ) See: Al-Naseer, Yassin, 2011, p. : أالمبحث األول : مرحلة الخيال 342 ،ُ) معنى ذلك أنَّ اإليعازات الحسيَّة التي تتحول في منظور الشاعر إلى خيالٍ يرصد ُويستقطب ؛ ليقودَ نسيجهُ النصي ِ إلى حالةٍ من القلقِ الذي تتأرجحُ فيه حركيَّة النص ِ بين الثبات والسكونُ ، فصنع الشاعر ذلك بموهبته فما هو ثابتٌ أنَّهُ يعاني من ألمِ الهوى، وشدَّةِ االشتياق، بيدَ أنَّ ما هو متحرِكٌ فهو يعمل على إرسال هذا االستشعار عبرَ نقاط متحركة ي ،قودها الخيالُ إلى التمظُّهر بصورٍ رمزيَّة، والتفاتاتٍ طبيعيَّة ِعمل الشاعر على تحشيدها ذهنيًا ومن ثمَّ قام بإطالقِها؛ لتكونَ معب ِرًا رسميًا لمن نطقَ بهِ إحساسهِ، وقادت إليه .أشواقهُ؛ ليجعلَ من مكامن الطبيعة وسيلةً إلبالغ ماهيته الخطابيَّة 359 جملة األستاذ للعلوم اإلنسانية واالجتماعية جملد (62) العدد (2) حزيران لسنة 2023 َإنَّ الخيال صفةً مالزمةً للشعرِ وال تبارِحهُ بأي شكلٍ من األشكال؛ بيدَ أنَّها مع الشاعرِ األندلسي ِ أخذتَ مأخذًا آخر، يُجاز لهُ أن يكون تحوالً رياديًا من حيثُ الرؤى، واألنساقُ، وطرائقُ التفكير في التعاطي مع بيئتها وأنموذجها المتفرَّد؛ ألنَّ االستبطانات الشاعرة ينبغي أ ن تكون قادرة على استكشافِ الخفايا الكامنة وراء هذه ُالعوالم، وتلتقطُ مجرياتِ أحداثها، وتعمل بعيدَ ذلك على تثوير مرصودات تلك العوالم، وتصييرها إلى فعلٍ يعكس ،طريقة تعامل الشاعر األندلسي ِ مع حيثياتها عبر ثقافة الخلق الخيالي الممكنة ( يُنظر: عليمات2004، ص 15 ( ) See: Alimat, 2004, p. 15 ) وما دام الحديثُ لمَّا يزل مستمرًا في مساحة االشتغال الخيالي ِ التي ) عمل عبرَها ابن زيدون فيمكن الوقوف عند نونيتهِ الشهيرة في قوله: ( من البسيط ،( ابن زيدون، الديوان 2005 ،ص51 ( ) Ibn Zaydun, Al Diwan, 2005, p. : أالمبحث األول : مرحلة الخيال 51 ) : كانَت ُلَهُ الشَ مس ًظِ ئرا في أَكِلَّتِه بَل ما تَجَل ى لَها إِال أَحايينا كَأَنَّما أُثبِتَت في ِصَ حنِ وَجنَتِه زُهرُ الكَواكِبِ تَعويذاً وَتَزيينا ًما ضَ رَّ أَن لَم نَكُن أَكفاءَهُ شَ رَفا وَفي المَوَدَّةِ كافٍ مِن تَكافينا يا رَ وضَ ةً طالَما أَجنَت لَواحِظَنا ًوَرداً جَالهُ الصِ با غَض ا وَنَسرينا َوَيا ح ــي ـــــــــــــ ًاة تَمَلَّينا بِزَهرَتِها ُــم ًنى ُــــــ ض ًروبا وَلَذ اتٍ أَفانينا َو يا نَعي ــــ ًما خَطَرنا ِمِن غَضارَتِه في وَشيِ نُعمى سَ حَبنا ذَيلَهُ حينا لَسنا نُسَ م ــــ ًيكِ إِجالالً وَتَكرِمَة ِوَقَدرُك َالمُعت ي ــــ لــ عَن ذاكَ يُغنينا َإنَّ الخيال صفةً مالزمةً للشعرِ وال تبارِحهُ بأي شكلٍ من األشكال؛ بيدَ أنَّها مع الشاعرِ األندلسي ِ أخذتَ مأخذًا آخر، يُجاز لهُ أن يكون تحوالً رياديًا من حيثُ الرؤى، واألنساقُ، وطرائقُ التفكير في التعاطي مع بيئتها وأنموذجها المتفرَّد؛ ألنَّ االستبطانات الشاعرة ينبغي أ ن تكون قادرة على استكشافِ الخفايا الكامنة وراء هذه ُالعوالم، وتلتقطُ مجرياتِ أحداثها، وتعمل بعيدَ ذلك على تثوير مرصودات تلك العوالم، وتصييرها إلى فعلٍ يعكس ،طريقة تعامل الشاعر األندلسي ِ مع حيثياتها عبر ثقافة الخلق الخيالي الممكنة ( يُنظر: عليمات2004 ، ص 15 ( ) See: Alimat, 2004, p. 15 ) وما دام الحديثُ لمَّا يزل مستمرًا في مساحة االشتغال الخيالي ِ التي ) عمل عبرَها ابن زيدون فيمكن الوقوف عند نونيتهِ الشهيرة في قوله: ( من البسيط ،( ابن زيدون، الديوان 2005 ،ص51 ( ) Ibn Zaydun, Al Diwan, 2005, p. : أالمبحث األول : مرحلة الخيال 51 ) :َ كانَت ُلَهُ الشَ مس ًظِ ئرا في أَكِلَّتِه كَأَنَّما أُثبِتَت في ِصَ حنِ وَجنَتِه ًما ضَ رَّ أَن لَم نَكُن أَكفاءَهُ شَ رَفا يا رَ وضَ ةً طالَما أَجنَت لَواحِظَنا َوَيا ح ــي ـــــــــــــ ًاة تَمَلَّينا بِزَهرَتِها َو يا نَعي ــــ ًما خَطَرنا ِمِن غَضارَتِه لَسنا نُسَ م ــــ ًيكِ إِجالالً وَتَكرِمَة بَل ما تَجَل ى لَها إِال أَحايينا زُهرُ الكَواكِبِ تَعويذاً وَتَزيينا وَفي المَوَدَّةِ كافٍ مِن تَكافينا ًوَرداً جَالهُ الصِ با غَض ا وَنَسرينا ُــم ًنى ُــــــ ض ًروبا وَلَذ اتٍ أَفانينا في وَشيِ نُعمى سَ حَبنا ذَيلَهُ حينا ِوَقَدرُك َالمُعت ي ــــ لــ عَن ذاكَ يُغنينا بَل ما تَجَل ى لَها إِال أَحايينا زُهرُ الكَواكِبِ تَعويذاً وَتَزيينا وَفي المَوَدَّةِ كافٍ مِن تَكافينا ًوَرداً جَالهُ الصِ با غَض ا وَنَسرينا ُــم ًنى ُــــــ ض ًروبا وَلَذ اتٍ أَفانينا في وَشيِ نُعمى سَ حَبنا ذَيلَهُ حينا ِوَقَدرُك َالمُعت ي ــــ لــ عَن ذاكَ يُغنينا اتخذ الشاعرُ من طريقةَ االستعطافِ واإلجالل ،لحبيبتهِ ( والَّدة بنت المستكفي ) وسيلةً إلبالغ رسالتهِ الخطابيَّة ويعلنُها بأنهُ ليس بمنزلتها شرفًا فهي ربيبة الملوك، بل ذهبَ ألبعدَ من ذلك وجعلَ من الشمسِ ( ضئرًا أي .مُرضِ عةً ) لها وهي مدللة تحت ظالل كِلتها سلكَ الشاعرُ سبيالً فنيًا لملم عبرهُ أشتات ا لمخزونات التي يحتفظ بها ذاكراتيًا افضت إلى ما يصطلح عليه في النقد المُعاصر بعملية (التفكيك ) هذا المصطلح الذي عُني به جاك دريدا، ومن العرب الدكتور عبد الملك مرتاض وأسماهُ ( التقويضيَّة) والدكتور عبدهللا الغذامي وأسماهُ التشريحيَّة، ويُقصد به إجماالً ( تفكي َّك النص ِ ثم ،إعادة بنائهِ، هذه سيلةٌ تفتحُ لإلبداعِ القرائي كي يتفاعل مع النص ِ ( وغليسي2008 ، ص345 ( ) Waglisi , 2008, p. 345 )، وبذلك ذهبَ الشاعرُ األندلسيُّ صوب التفاعل مع الرؤى قُبيل انطالقِها، واألفكار قُبيل عصفها، والصور قُبيل إشاعتها؛ ولذلكَ أكثر ، ِ ما يُلحظ على الشاعر األندلسي ِ بأنَّهُ كثيرٌ العناية بحركيَّة النص ِوتحديد مساراتِهِ، وتأثيثِ مفرداتِهِ؛ ومردُّ ذلكَ كلُّهُ يعودُ إلى طبيعة التحوُّل الفكري ِ والثقافي ِ التي طرأت على حياته وجددتَها بوعي ٍ مختلفٍ ، ورؤى انبثاقيَّة جديدة، ومسارات رياديَّة ،في إعادة صياغة الخطاب الشعري ِ واستقرائه وهذا العمل من شأنهِ إدامةِ الزخمِ لدى الشاعر األندلسي ِ في أن يوجَّهَ خيالَهُ نحو تحريكِ النص ِ الشعري ِ بنحو يحافظ فيهِ على ديناميَّة النص ِ، وتجديدهِ، وتنشيط بناهُ األسلوبيَّة على نحو الذي يكون فيهِ النصُّ األند ُّلسي . : أالمبحث األول : مرحلة الخيال ِ متشبعًا بالحركيَّة واألداء والثِقل الرمزي 360 جملد (62) العدد (2) حزيران لسنة 2023 جملة األستاذ للعلوم اإلنسانية واالجتماعية : المبحث الثاني : مرحلة التخييل تمثلَّـت مرحلة التخييل في مسارِ العمليَّة اإلبداعيَّة على أنَّها تأخذُ بُعدًا جديدًا يتمثُّل بمشاركة القارئ للمبدع في تحليل الخطاب وتوجيه أُطرهِ المعرفيَّة؛ ليقودَ بالنتيج ة إلى الهدفِ الذي تتضحُ فيه معالم اكتمال تجربة المبدع ُونضجها، ومديدات ارتداد أثرها عبرَ تأثيرها بنتاج الثقافات المناظرة لها والمختلفة عنها، ويُعرَّف التخييل بأنَّه (نشاطٌ ذهني يعب ِرُ به عن تفاعلهِ النفسي ِ مع العالم، انفعالهِ الوجداني بمواضيعه وأشيائه ، وهو فعلٌ غير مقصور على فئةٍ دون أخرى أو على جنسٍ دون سواهُ بل يشتركُ فيهِ كلُّ الناس، وال يختلفون إالَّ في نوعية توظيفهِ ودرجتهِ، بيد أنَّ تأمُّل ذلك النشاط والتفكير فيه هو من طبيعة ذهنية أخرى ،ويقتضي حركة إدراكية مغايرة؛ ُألنَّه ًليس أمر ًا غريزي ًا وطبيعي ،ا وإنَّما هو فكري ونظري ،وال يتم إالَّ في اللحظة التي تبتعد فيها الذات ًنفسي ًا وإدراكي ا عن موضوع ، ُّتخيلها ) ( اإلدريسي2015 ، ص25 ) ( Al-Idrisi, 2015, p. 25 ) ومعنى ذلكَ أنها تتعدى حدود ذاتيَّة الشاعر؛ لتتعدى إلى المتلقي الذي يستكمل اشتراطات التكامل ا إلبداعي ِ واستقرائه؛ ِ ليعمل على البحث في مكامن ما يرسلهُ الشاعر، ويستقصي آثاره، ويعمل على استمرار ديناميَّة النص ،الشعري، وربط أواصرهِ، ويفتشُ في مآالت ما يُفضي إليه عبرَ بروزهِ عامالً في إثراء مشهديَّة القراءة النصيَّة وتحديد أبعادِها الصوريَّة، عبر أدوا تهِا المختلفة داخل منطقة التلقي من صور، ورموز، وتعابير مجازيَّة، وما ،إلى ذلك ( يُنظر: عُبيد2010 ، ص32 ) ( See: Obeid, 2010, p. 32 ) ُ، وبحكم ما اتصفَ به النسيج الشعريُّ األندلسيُّ من اتجاهات فنيَّة متنوعة قاد لها فعلُ الخيالِ ؛ ليصيرَها للتخييل فقد كانَ لت ،نوُّع الصور واختالفِ الرموز، وتعدُّدِ اإلشارات واإليماءات والقرائن التي استدلت عليها القراءة عبر مآالت التخييل الذي رسم أبعادَ عمقِ التجربة، وكشفَ خبايا النسيج النصي ِ ، وأضاء مناطق العُتمة التي اكتنفتهُ، ولغرض استيضاح ذلكَ جليًا يمكنُ الوقوف عند مرثية الش اعر أبي البقاء الرندي ِ األندلسي الشهيرة بعد سقوط األندلس التي : مطلعها ِ لكُل ٍشَ يء إِذا ما تَم نُقصانُ فَال َّيُغَر ِبِطيب ِالعَيش ُإِنسان إذ يقول : ( من البسيط ): ( الديوان، ص57 ) ( Al Diwan, p. 57 ) ِ لكُل ٍشَ يء إِذا ما تَم نُقصانُ فَال َّيُغَر ِبِطيب ِالعَيش ُإِنسان فَال َّيُغَر ِبِطيب ِالعَيش ُإِنسان إذ يقول : ( من البسيط ): ( الديوان، ص57 ) ( Al Diwan, p. : أالمبحث األول : مرحلة الخيال 57 ) ُوأين منهم أكاليلٌ وتيــــــــــــــــــجان ُوأين ما ساسه ِّفي الفُرْس ساسان ُوأين عادٌ وشـــــــــــــــــد اد وقحــــــــطان حتى قضوا فكأن القوم ما كانــــــــــوا ُكما حكى عن خيال الط يْفِّ وسْ نان وأم كســــــــــــــــــــرى فما آواه إيوان يوماً وال ملك الد نيا سلي ـــــان م أين المُلوك ذوو ٍالت يجانِّ من يَمن ٍوأين ما شاده شـــــــــــد اد في إرم ُوأين ما حازه ٍقارُ ون من ذهب أتى على الكل أمرٌ ال مـــــــــــــرد له وصار ما كان من مُلكٍ ومن مَلك دار الز مــــــــــــــــــــان على دارا وقاتله كأ ٌن ما الصعب لم يسهل له سبب أين المُلوك ذوو ٍالت يجانِّ من يَمن ُوأين منهم أكاليلٌ وتيــــــــــــــــــجان ٍوأين ما شاده شـــــــــــد اد في إرم ُوأين ما ساسه ِّفي الفُرْس ساسان ُوأين ما حازه ٍقارُ ون من ذهب ُوأين عادٌ وشـــــــــــــــــد اد وقحــــــــطان أتى على الكل أمرٌ ال مـــــــــــــرد له حتى قضوا فكأن القوم ما كانــــــــــوا وصار ما كان من مُلكٍ ومن مَلك ُكما حكى عن خيال الط يْفِّ وسْ نان دار الز مــــــــــــــــــــان على دارا وقاتله وأم كســــــــــــــــــــرى فما آواه إيوان كأ ٌن ما الصعب لم يسهل له سبب يوماً وال ملك الد نيا سلي ـــــان م ... 361 جملة األستاذ للعلوم اإلنسانية واالجتماعية جملد (62) العدد (2) حزيران لسنة 2023 يا مَن ِلِذلَّة قَوم َبَعد َعِز تهِم أَحال حالَهُم ٌكفر ُوَطُغيان ِبِاألَمس كانُوا ًمُلُوكا فِي مَنازِلهِم َوَاليَوم هُم في ِبِالد ِالكُفر ُعُبدان فَلَو تَراهُم حَيارى ال َدَلِيل لَهُم عَلَيهِم من ِثياب ِ الذُل ُأَلوان وَلَو رَأَيت بُكاهُم َعِند َبَيعهمُ لَهالَك ُاألَمر َوَاِستَهوَتك ُأَحزان يا َّرُب ٍ أم ٍوَطِ فل َحيل بينهُما كَما ُتُفَرَّق ٌأَرواح ُوَأَبدان وَطفلَة َمِثل ِحُسن ِالشَ مس إِذ برزت كَأَنَّما َهي ٌياقُوت ُوَمُرجان يَقُودُها ُالعِلج ِلِلمَكروه ًمُكرَهَة ُوَالعَين ٌباكِيَة ُوَالقَلب ُحَيران ِلِمثل هَذا يَبكِي ُالقَلب مِن كَمَدٍ إِن َكان في ِالقَلب ٌإِسالم ُوَإِيمان عُدت هذه القصيدة من عيون الشعرِ العربي في األندلس رثى فيها الشاعر األندلس وما حولها من المدن بعد ضعف حكم المسلمين، وقد دفع بقوى ( الخيال) على استكمال المشهد؛ ليسلمَّهُ إلى ( التخييل ) لدى القارئ الذي راح يستقرأ صرخات الشاعر وبكاءهُ على المُلك العربي ِ واإلسالمي الزائل؛ إلى الحد ِ الذي أصبحَ فيه ( العلج غير العربي ِ ) هو من يتحكم باألمور، ويسوق النسوة واألطفال للعبودية والذل ِ ؛ ليستصرخ الضمير . : أالمبحث األول : مرحلة الخيال لدى قومهِ؛ ولكنَّه يصلُ إلى نتيجةٍ حتميَّة إلى أنَّ كلَّ شيء صائرٌ إلى الزوال وال محيص عن ذلك َإنَّ فعل التخييل خلقَ واقعًا افتراضيًا مماثالً للواقع العيني ِ وكان استنباط ذلك عبر حركة الصورة التي صيغ ُمنها النسيج النصيُّ في لغتها، وموسيقاها، وتراكيبها وكأنَّهُ جعلَ منها واقعًا عينيًا ال غبارَ عليهِ؛ متخذًا من السردِ الحدثي ِ وسيلةَ إبالغٍ نصي ِ تتماهى وتتماشى مع حركة النص ِ وتقلباتهِ في تمهيده، وأحداثهِ، وخاتمته؛ ُألنَّ ( النصَّ المرئيَّ تمثيلٌ للواقع، إالَّ أنَّه ُفي حقيقة األمر خلقٌ لواقعٍ جديدٍ من الزمانِ والمكانِ ، ومن ثمَّ فإنَّه ،يتميز بالحركيَّةِ والتوتُّرِ وامتالك إيقاعهُ الخاص) ( فضل2014 ، ص16 ) ( Fadl, 2014, p. 16 ) والمقصود هُنا في اإليقاع الخاص ليس إيقاع الوزن وحده، وإنمَّا إيقاع توجيه النص ِ وحركيته ا لقائمة على ثنائية تضاديَّة بين مجدٍ سابقٍ رائع، ونكبةٍ حاليةٍ مؤلمة؛ مستتبعًا هذه الحركة بإيقاع الوزن القائم على تفعيلتي ُ( مُسْ تَفْعِلُن / فَاْعِلُن )؛ ليعملَ التخييل على إفراد مساحة وافية لنبرة الحُزن التي استظلت بحركيَّة نصيَّة تقود إلى وقائع وأحداثٍ ود ،الالت ال يمكن استيضاح كنهها إالَّ التعمُّق في التخييل، وفك ِ شفرات النص ِ تباعًا ٍواستيضاح مدلوالتِها، وفهم مغزاها؛ ألنَّ النصَّ ( هو فعاليَّة لغويَّة مواضعات العادة والتقليد، وتلبست بروح ) متمردة رفعتها عن سياقها االصطالحي إلى سياقٍ جديدٍ يخصُّ ها ويمثلها ،( الغذامي2012 ، ص10 ) ( Al-Ghadami, 2012, pg. 10 ) وهذا يعني أنَّ من قاد إلى هذه الفعالية هو الحركيَّة النصيَّة التي أرسلها . الخيال، وفسرَّها التخييل، ووضع لها الحدود المناسبة لفضاء القول والتفسير إنَّ حركيَّة النص ِ تسيرُ في اتجاهاتٍ عدَّة، المهارةُ في لملمة شتات هذه االتجاهات وجعلها تتناغم في عملية إبداعيَّة واحدة هو السبيل الوحيد لصناعة الجمال وتنميته؛ ألنَّ الذات اإلبداعيَّة تميلُ إلى تنامي الحدث وصيرورته إلى حالةٍ يمكن استجالء أثرها، والوصول إلى مفاداتها ( فالذاتُ ال تحصلُ على موضوعها إال إنَّ حركيَّة النص ِ تسيرُ في اتجاهاتٍ عدَّة، المهارةُ في لملمة شتات هذه االتجاهات وجعلها تتناغم في عملية إبداعيَّة واحدة هو السبيل الوحيد لصناعة الجمال وتنميته؛ ألنَّ الذات اإلبداعيَّة تميلُ إلى تنامي الحدث وصيرورته إلى حالةٍ يمكن استجالء أثرها، والوصول إلى مفاداتها ( فالذاتُ ال تحصلُ على موضوعها إال بحركة ما قد تكون عسيرة أو يسيرة ... ومهما يكن األمر فإنَّ هناك تفاعالً يجري في فضاء وزمان معينين ،ويتحقق فيهما عبر العالمات اللغويَّة ) ( مفتاح1990 ،ص8 ) Moftah1990, p. : أالمبحث األول : مرحلة الخيال 8) َّ)، ومعنى ذلك أن 362 جملد (62) العدد (2) حزيران لسنة 2023 جملة األستاذ للعلوم اإلنسانية واالجتماعية حركيَّة النص ِ تتنامى وتستمرُ ديمومة تناميها بعد أن تنطلق من فضاء الخيال، لتص ل إلى فضاء التخييل ،فتصنعُ حدثًا يتخذُ من سمة العمقِ مكانهُ األرحب في االستمرار على حركيته وتصاعد وتيرة أحداثِها ومآالتِها وما دام الحديثُ لمَّا يزل داخل منظومة الحُزن التي التقطتْ صورها، واستمدت أجواءها من فضاء الرؤيا الحُلميَّة لدى الشاعر األندلسي ِ فيمك ن الوقوف عند نص ِ ابن خفاجة األندلسي ِ الذي يتحسُّ ر على (بلنسية) التي يقولُ فيها ( المقر ِي، الجزء6 ، ص199 ) ( Al-Muqri, Part 6, p. 199 ) : ْعاثت ِبساحتِك العدى يا ُدار ومحا ِمحاسنَك البلى ُوالنار فإذا َتردَّد في بِك جنا ناظر ٌِ ... َطال ٌاعتبار فيك ُواستعبار ٌأرض ِتقاذفت ُالخطوب بأهلِها ... ْوتمخضَّ ت بخرابِها ُاألقدار ْكتبت ُيد ِالحدثان في عرصاتِها " ...لا ِأنت ِأنت وال " ُالديارُ ديار ْعاثت ِبساحتِك العدى يا ُدار ومحا ِمحاسنَك البلى ُوالنار فإذا َتردَّد في بِك جنا ناظر ٌِ ... َطال ٌاعتبار فيك ُواستعبار ٌأرض ِتقاذفت ُالخطوب بأهلِها ... ْوتمخضَّ ت بخرابِها ُاألقدار ْكتبت ُيد ِالحدثان في عرصاتِها " ...لا ِأنت ِأنت وال " ُالديارُ ديار انساقَ النصُّ وراء تداعيات ما حدث لمدينة بلنسية األندلسيَّة وما حلَّ فيها من أمورٍ دفعت بأثرِ الحُزن أن يأخذَ مكانهُ من انبثاقات الشاعر التي سطرَّها نصيًا، فكانَ توثيقُ ذلكَ ينبئ باألثر الذي يقدرهُ التخييل في مساحة الرؤية التي صدرَّها إليه الشاعر بطريقة اح ترافية جعلت من عنصرِ التخييل أن يكون في كامل َّجاهزيتهِ، لتحليل متبنيات النص ِ ، وتفكيك ما اعتراهُ من غموضٍ خفي ٍ ، وكما يقول أدونيس ( والواقع أن ،اإليصال ليسَ بذاتهِ مهمًا، فقد يستطيعُ الشاعر أن يبس ِ طَ قصائدهُ بحيث يتلقاها السامعُ أو القارئ بسهولة لكن األك ،ثر أهميةً هُنا هو أن يقدر السامعُ أو القارئ أن يحو ِلَ تلقيه إلى وعي نقدي ٍ فعَّال ) ( أدونيس 2005، ص23 ) ( Adonis, 2005, p. 23 ) ، وهذا ما يفعلهُ عنصر التخييل الذي يعملُ على أن يجعل ِ مراحل اإلبداع تتكامل، وتنصهر في بوتقة واحدة تظهرُ عبرها حركة النسيج النصي ،سائرةً باتجاه التنامي واستمراريَّة الحدث، وإدامة التواصل بين أطراف العمليَّة اإلبداعيَّة، فالبعدان الخيالي والتخييلي متداخالن في ،جوهر النص ِ وديناميته إلى الحد ِ الذي يلتقيان به عند بوابة التأويل ( ينظر:العمري2005 ، ص164 ) ( See: Al-Omari, 2005, p. 164 ) ؛ليستحيل العملُ الشعريُّ وحدة موضوعيَّة تتكامل فيه عناصر . التشكيل واإلنتاج والفهم المتنامي : المبحث الثالث : مرحلة امتداد األثرِّ ورجع الصدى : المبحث الثالث : مرحلة امتداد األثرِّ ورجع الصدى َّإن َّطبيعة التحوالت الزمانيَّة والمكانيَّة من شأنها إحداثِ تغييرات وارتدادات على القيمة النصي ة للمتون اإلبداعيَّة سواءً أكانت شعريَّة أم نثريَّة وهذا األمر ال جدالَ فيه، فخلقَ ذلكَ حركةً نصيَّة اختلفت عن قيمها المضمونيَّة األولى أحيانًا بالمضمون وأحيانًا بالشكل والمضمون، وهذا ما يُفس ِ رُ ظهور أنماط شعريَّة جديدة في األندلس نتيجة عملية امتداد األث َّر ورجع صدى هذا األثر، وقد عزا هيكل سبب ذلك إلى أن ( األندلسيين أحسُّ وا بتخلفِ القصيدة الموحدة إزاء األلحان المنوعة، وشعروا بجمود الشعرِ التقليدي أمام النغم ) في حاضره التجديدي المرن، وأصبحت الحاجة الماسة إلى لون من الشعرِ الجديد يواكب الموسيقى والغناء 363 جملة األستاذ للعلوم اإلنسانية واالجتماعية جملد (62) العدد (2) حزيران لسنة 2023 جملد (62) العدد (2) حزيران لسنة 2023 ( ،هيكل1979 ، ص138 ) ( Heikal, 1979, p. 138 )، وهذه األنماط وثَّق آصرة التواصل بين تأثر العرب بغيرهم والعكس؛ بحكم جنوح هذه األ إلى االقتراب من التبسيط، والتداول المحلي ِ ، وتنوع القوافي، وتعدد رنين األ حركيَّة نصيَّة متجددة بعثت طاقاتٍ إبداعيَّة مضافة لشعر العرب في األند ،على موشح الشاعر ابن سناء الملك الذي يقول فيه ( الملك ابن سناء49 Sana’, 1949, pg70 ) سر ِح جفوني ...في روض وجنتيكا هذي ديوني ...قد بليَت لديكا حسبي مَنوني ...إن كان ْمِن يديكا َّيا كل ٍطيب له ُالجمال نَعْت ...ما بال ذنبي في ِ حب من أحببت يا من تجن ى ...لا َذُقْت ما ُأذوق ٌقلب مُعَنَّى ...ومدمع ُطليق أفديك غصنًا ...وجدي به ُخليق ٌغصن كثيب ُلَدْن التثني ُشَ خْت ...قضيت نحبي مُذْ بان أو مذ ُبِنْت ُالحسن يعلم ... َأنك منه أحسن وأنت ُأكرم ... ُوالموت فيك أَهون يفديك مُغرم ... َّأسَ ر حتى أعلن أنت نصيبي من كل ما ُاقترحت ... َحسبي حسبي ما َشِ يت ال ُما شِ يت أنا َوأنت ...إسوة هذا الهجر بالصبر بِنْتا ...مع انصداع الفجر ومذ رحلتا ...غَن ي الجوى في صدري سافر حبيبي سَ حَر وما ود عتو ...يا وحش قلبي في الليل إذا افتكرتو ( ،هيكل1979 ، ص138 ) ( Heikal, 1979, p. : المبحث الثالث : مرحلة امتداد األثرِّ ورجع الصدى 138 ) ٍ ، وهذه األنماط من الشعر قادت إلى حراكٍ إبداعي ُوثَّق آصرة التواصل بين تأثر العرب بغيرهم والعكس؛ بحكم جنوح هذه األنماط الشعريَّة إلى الغناء فهي تميل إلى االقتراب من التبسيط، والتداول المحلي ِ ، وتنوع القوافي، وتعدد رنين األجراس، األمر الذي قاد إلى ابتكار حركيَّة نصيَّة متجددة بعثت طاقاتٍ إبداعيَّة مضافة لشعر العرب في األندلس، والستجالء ذلك يمكن الوقوف ،على موشح الشاعر ابن سناء الملك الذي يقول فيه ( الملك ابن سناء1949 ، ص70 ) ( Al-Malik Ibn Sana’, 1949, pg70 ) سر ِح جفوني ...في روض وجنتيكا هذي ديوني ...قد بليَت لديكا حسبي مَنوني ...إن كان ْمِن يديكا َّيا كل ٍطيب له ُالجمال نَعْت ...ما بال ذنبي في ِ حب من أحببت يا من تجن ى ...لا َذُقْت ما ُأذوق ٌقلب مُعَنَّى ...ومدمع ُطليق أفديك غصنًا ...وجدي به ُخليق ٌغصن كثيب ُلَدْن التثني ُشَ خْت ...قضيت نحبي مُذْ بان أو مذ ُبِنْت ُالحسن يعلم ... َأنك منه أحسن وأنت ُأكرم ... ُوالموت فيك أَهون يفديك مُغرم ... َّأسَ ر حتى أعلن أنت نصيبي من كل ما ُاقترحت ... َحسبي حسبي ما َشِ يت ال ُما شِ يت أنا َوأنت ...إسوة هذا الهجر بالصبر بِنْتا ...مع انصداع الفجر ومذ رحلتا ...غَن ي الجوى في صدري سافر حبيبي سَ حَر وما ود عتو ...يا وحش قلبي في الليل إذا افتكرتو ،بال ريبٍ أنَّ النصَّ يشي بمقدار التحوُّل في المسارِ الشعري ِ على صعيد القيمة الشكليَّة والموضوعيَّة والفنيَّة عبر االقتراب إلى الذهنيَّة البشريَّة في األندلس؛ بحكم تعدد األجناس والبيئات التي وفدوا منها، فكان من الطبيعي أن تأخذَ مأخذها من ذهنيَّة الشاعر ا لعربي ِ ؛ ليحتكم إلى رؤية جديدة في إيصال نتاج يقتربُ فيه من ُالثقافة الجديدة، وطبيعة التحوالت التي طرأت على فكره وشخصهِ؛ ليعمل خطَّ شروعٍ جديدٍ تلتقي فيه األهواء ِوالمشاربُ بمسمياتِها المختلفة؛ ( ذلك أنَّ العربيَّ الذي كانَ يحد ِدُ هويتهُ انطالقًا من تاريخ جماعة معينة ومن ،دين هذه الجماعة بات يحد ِدُ هويتهُ في ضوءِ عالقة هذه الجماعة بالمكان وتفاعُلهِ معهُ ) (سعيد1982 ، ص 33 ) ( Saeed, 1982, p. : المبحث الثالث : مرحلة امتداد األثرِّ ورجع الصدى ِ عن تنامي النص ِ الشعري ِ األندلسي ِ واستمرار رفد حركيتهِ لبنى النص ِ الشعري لا َنزاه اال في ِالواد والنشْ م ِوالخضر ْوالذل وأنا مع المليحة نشرب والطير ْتولول ُفالعروس اليوم ُتراه لس يخاف ُيصفه ْواصِ ف ْمُـر ترى الوادي مجلل بالصَّ بايا و الوصايف عملوا ثياب من الما ومن ِالشعور ْمالحِف ثم برقعُوها األقمــار َفرأيت ْأجمل ْوأجمل الخليـــج أكثر نزاه ََِ وأطم عاد ْوأطبع َ(إنْ ) دخلت َوأنت مهموم ُفيزول َّالهم ْأجمع فـــــذا َاردت ذاب أن ترى َالغاب ْفاطلع ْوارتبط ْفي الفُحش واشرب وانطرب ِ وغن ْواصهل إَّنَّها لغةٌ خاصة، ورؤيةٌ خاصة، وتوجهاتٌ خاصة في بث ِ نمطٍ شعري ٍ من الواضح أنهُ مستوحى من طبيعة المجتمع األندلسي وهو يقتربُ من لُغةٍ هي للعامية أقربُ منها للفصحى، فالشاعر يتحدثُ هنا عن نزهة تبدَّى َّبها جمال الطبيعة، وأصوات الطيور، والوجوهُ الحِسان؛ ليشكل ُنمطا الموشَّ ح والزجل حركةً ديناميَّة تتواصل مع حركيَّة النص ِ العربي ِ ، وتمدُّه بثقافة جديدة ال تلتزم بحدود االلتزام الصارم التي تقوم عليه تلك الثقافة ومتبنياتها؛ بيد أنَّها تمتحُ من لغتها، ورؤيتها ما تراهُ متماهيًا مع سيرورة الحدثِ المتنامي إلى تبدو ظا هرة ما يُصطلح عليه جوازًا ال وجوبًا بـ( رجعِ الصدى )؛ لتتضح ضرورات هذا االمتداد متواصلة من طرفي الشرق والغرب، وهذا ما اتضح جليًا لدى شعراء ( التروبادور ) وهو شعر أوروبا القديم، وقد أثرى الدكتور محمد ( عباسة في كتابه الموشحات واألزجال وأثارها في شعر التروب ر ادو ) هذا الجانب، وبيَّن مديات تأثُّر شعراء التروبادور بالموشحات واألزجال األندلسيَّة، وهذا يعني أنَّ سلسلة التأثُّر والتأثير شغلت حيزًا وافيًا من عمليَّة اإلنتاج اإلبداعي ِ التي تصدرتها وتميزت بها مشهديَّة األندلس األدبيَّة؛ لتكون دليالً لهُ من القوة بمك ان أن يُعلن . ِ عن تنامي النص ِ الشعري ِ األندلسي ِ واستمرار رفد حركيتهِ لبنى النص ِ الشعري : المبحث الثالث : مرحلة امتداد األثرِّ ورجع الصدى 33 )؛ األمر الذي قاد الشاعر األندلسيَّ إلى تجديد مفاهيمه ورؤاهُ صوب مستجداتِ فرضتها عليه التحوالت القائمة في بيئته الجديدة وثقافتها، وال تخفى هذه التوجهات في حركيَّة 364 جملة األستاذ للعلوم اإلنسانية واالجتماعية جملد (62) العدد (2) حزيران لسنة 2023 النص ِ الشعري ِ األندلسي ِ وتجديده وابتكاراتهِ؛ ولغرض استجالء ذلك بنحو أوضح يمكن اإلشارة إلى أح د ( زجليات ابن قزمان الذي يعدُّ الشاعر األكثر شهرة في هذا اللون من الشعر كورينطي ، 1980 ، ص200 ) Corinti, 1980, p. 200) ) :، إذ يقول Corinti, 1980, p. 200) ) :، إذ يقول لا َنزاه اال في ِالواد والنشْ م ِوالخضر ْوالذل وأنا مع المليحة نشرب والطير ْتولول ُفالعروس اليوم ُتراه لس يخاف ُيصفه ْواصِ ف ْمُـر ترى الوادي مجلل بالصَّ بايا و الوصايف عملوا ثياب من الما ومن ِالشعور ْمالحِف ثم برقعُوها األقمــار َفرأيت ْأجمل ْوأجمل الخليـــج أكثر نزاه ََِ وأطم عاد ْوأطبع َ(إنْ ) دخلت َوأنت مهموم ُفيزول َّالهم ْأجمع فـــــذا َاردت ذاب أن ترى َالغاب ْفاطلع ْوارتبط ْفي الفُحش واشرب وانطرب ِ وغن ْواصهل إَّنَّها لغةٌ خاصة، ورؤيةٌ خاصة، وتوجهاتٌ خاصة في بث ِ نمطٍ شعري ٍ من الواضح أنهُ مستوحى من طبيعة المجتمع األندلسي وهو يقتربُ من لُغةٍ هي للعامية أقربُ منها للفصحى، فالشاعر يتحدثُ هنا عن نزهة تبدَّى َّبها جمال الطبيعة، وأصوات الطيور، والوجوهُ الحِسان؛ ليشكل ُنمطا الموشَّ ح والزجل حركةً ديناميَّة تتواصل مع حركيَّة النص ِ العربي ِ ، وتمدُّه بثقافة جديدة ال تلتزم بحدود االلتزام الصارم التي تقوم عليه تلك الثقافة ومتبنياتها؛ بيد أنَّها تمتحُ من لغتها، ورؤيتها ما تراهُ متماهيًا مع سيرورة الحدثِ المتنامي إلى تبدو ظا هرة ما يُصطلح عليه جوازًا ال وجوبًا بـ( رجعِ الصدى )؛ لتتضح ضرورات هذا االمتداد متواصلة من طرفي الشرق والغرب، وهذا ما اتضح جليًا لدى شعراء ( التروبادور ) وهو شعر أوروبا القديم، وقد أثرى الدكتور محمد ( عباسة في كتابه الموشحات واألزجال وأثارها في شعر التروب ر ادو ) هذا الجانب، وبيَّن مديات تأثُّر شعراء التروبادور بالموشحات واألزجال األندلسيَّة، وهذا يعني أنَّ سلسلة التأثُّر والتأثير شغلت حيزًا وافيًا من عمليَّة اإلنتاج اإلبداعي ِ التي تصدرتها وتميزت بها مشهديَّة األندلس األدبيَّة؛ لتكون دليالً لهُ من القوة بمك ان أن يُعلن . نتائج البحث وخاتمته إاأا  ابن زيدون 2005 ، ،الديوان تحقيق :عبدهللا ،سنده دار ،المعرفة ،بيروت ط1 ،.  ، ُّاإلدريسي يوسف2015 . ، مفهوم التخييل في النقد والبالغة العربيين (األصول واالمتدادات )، دار وجوه للنشر ،التوزيع المملكة العربيَّة ،السعودية ط1 ، أ  ، ُّاإلدريسي يوسف2015 . ، مفهوم التخييل في النقد والبالغة العربيين (األصول واالمتدادات )، دار وجوه للنشر ،التوزيع المملكة العربيَّة ،السعودية ط1 ، َ  ،الربيعي علي محمد ،هادي 2012 ، دار صفاء للنشر ،والتوزيع عمَّان / ،األردن ط1 . أ  ،الربيعي علي محمد ،هادي 2012 ، دار صفاء للنشر ،والتوزيع عمَّان / ،األردن ط1 .  الرندي ،األندلسي أبو ،البقاء ،الديوان تحقيق رفعت برهام ،حسن، القاهرة ط1 (د.ت. ) أإَ  ،الربيعي علي محمد ،هادي 2012 ، دار صفاء للنشر ،والتوزيع عمَان / ،األردن ط1 .  الرندي ،األندلسي أبو ،البقاء ،الديوان تحقيق رفعت برهام ،حسن، القاهرة ط1 (د.ت. ) أإَ  سعيد، د. خالدة ، 1980 ،، حركيَّة اإلبداع دراسة في األدبِ العربي ِ الحديث، دار العودة، بيروت/ لبنان ط2 .  سعيد، د. خالدة ، 1980 ،، حركيَّة اإلبداع دراسة في األدبِ العربي ِ الحديث، دار العودة، بيروت/ لبنان ط2 .  الطاهر مكي ، 1987 ،دراسات أندلسية في األدب والتأريخ والفلسفة ، دار المعارف، القاهرة ،ط3 .  ،عُبيد محمد ،صا 2010 ، بر مرايا التخييل ، ِ الشعري دار مجدالوي للنشر ،والتوزيع عمَّان / ،األردن ط1 .  الطاهر مكي ، 1987 ،دراسات أندلسية في األدب والتأريخ والفلسفة ، دار المعارف، القاهرة ،ط3 .  ،عُبيد محمد ،صا 2010 ، بر مرايا التخييل ، ِ الشعري دار مجدالوي للنشر ،والتوزيع عمَّان / ،األردن ط1 .  الطاهر مكي ، 1987 ،دراسات أندلسية في األدب والتأريخ والفلسفة ، دار المعارف، القاهرة ،ط3 .  ،عُبيد محمد ،صا 2010 ، بر مرايا التخييل ، ِ الشعري دار مجدالوي للنشر ،والتوزيع عمَّان / ،األردن ط1 .  عليمات، د. ،يوسف 2004 ، جماليات التحليل الثقافي (الشعر ُّالجاهلي نموذجًا)،المؤسسة العربيَّة للدراسات ،والنشر عمَّان / ،األردن ط1 .  عليمات، د. ،يوسف 2004 ، جماليات التحليل الثقافي (الشعر ُّالجاهلي نموذجًا)،المؤسسة العربيَّة للدراسات ،والنشر عمَّان / ،األردن ط1 .  ،العمري2005 ، ،محمد البالغة الجديدة بين التخييل ،والتداول أفريقيا ،الشرق المغرب،ط1 .  الغذام،ي عبدهللا ،محمد 2012 ، الخطيئة والتكفير (من البنيوية إلى التشريحيَّة نظرية ،وتطبيق المركز الثقافي ، ُّالعربي بيروت / ،،لبنان ط7 .  الغذام،ي عبدهللا ،محمد 2012 ، الخطيئة والتكفير (من البنيوية إلى التشريحيَّة نظرية ،وتطبيق المركز الثقافي ، ُّالعربي بيروت / ،،لبنان ط7 . نتائج البحث وخاتمته نتائج البحث وخاتمته بُعيد هذا االستقراء االستقصائيُّ في متن الشعرِ األندلسي ِ ، والتطواف في مساحات مضامينهِ يمكن الخلوص :إلى النتائج اآلتية 1 . صالحية النص ِ الشعري ِ األ ندلسي ِ ومديات مرونتهِ في إعادة استقراء ذلك على وفق آليات النقد . المُعاصرة 2 . . وضوح الرؤية النقديَّة وثباتها في أنَّ النصَّ الشعريَّ األندلسيَّ نص متحركٌ وفعَّال ومتنامي بُعيد هذا االستقراء االستقصائيُّ في متن الشعرِ األندلسي ِ ، والتطواف في مساحات مضامينهِ يمكن الخلوص :إلى النتائج اآلتية يآ ج إ ى 1 . صالحية النص ِ الشعري ِ األ ندلسي ِ ومديات مرونتهِ في إعادة استقراء ذلك على وفق آليات النقد . المُعاصرة 2تنا كٌ فَّال ت الش يَّ األندل َّ ن أنَّ الن َّ ال ؤية النقديَّة ثباتها ف ض 1 . صالحية النص ِ الشعري ِ األ ندلسي ِ ومديات مرونتهِ في إعادة استقراء ذلك على وفق آليات النقد . المُعاصرة 2 . . وضوح الرؤية النقديَّة وثباتها في أنَّ النصَّ الشعريَّ األندلسيَّ نص متحركٌ وفعَّال ومتنامي 365 جملة األستاذ للعلوم اإلنسانية واالجتماعية جملد (62) العدد (2) حزيران لسنة 2023 3 . رسوخ األثر لدى الشاعر األندلسي ِ قاد القول إلى أنَّ فعل ( الخيال ) مكَّن الشاعرَ األ ندلسيَّ من . التطبيع مع مشاهداتهِ وضخها لمتنِ النص ِ الشعري ِ ؛ ليبقى على قيد الحركة دائبا 3 . رسوخ األثر لدى الشاعر األندلسي ِ قاد القول إلى أنَّ فعل ( الخيال ) مكَّن الشاعرَ األ ندلسيَّ من . التطبيع مع مشاهداتهِ وضخها لمتنِ النص ِ الشعري ِ ؛ ليبقى على قيد الحركة دائبا 4 . إسهام عنصر ( التخييل ) بنحو فاعلٍ في استكمال عمليةِ الرصد، وتفكيك شفراتِها، وإضاءة ما كان معتمًا من متبنياتها؛ ليكونَ رافدًا أساسيًا في استمرار ديناميَّة حركة النصوص األندلسيَّة، وبلوغها .أقصى نقطة في األثرِ والتأثير 4 . إسهام عنصر ( التخييل ) بنحو فاعلٍ في استكمال عمليةِ الرصد، وتفكيك شفراتِها، وإضاءة ما كان معتمًا من متبنياتها؛ ليكونَ رافدًا أساسيًا في استمرار ديناميَّة حركة النصوص األندلسيَّة، وبلوغها .أقصى نقطة في األثرِ والتأثير أ 5 . ِبلوغ الشعرِ األندلسي ِ مكانةً مهمة تؤكدُ عليها القراءة النقديَّة المُعاصرة في أن الشاعرَ العربيَّ بمقدار ما تأثَّر أحدثَ فنونًا شعريةً جديدة، تمتلك ثقافة خاصة اكتسبها من البيئة الجديدة التي انتقل إليها؛ ليعود هذا التأثرُ ويرجع صداهُ ليكون ذلكَ مصداقًا أكده مسار الشعرِ لدى أألدب األوروبي القديم .) الكامن في شعر ( التروبادور  ابن زيدون 2005 ، ،الديوان تحقيق :عبدهللا ،سنده دار ،المعرفة ،بيروت ط1 ،. 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نفح الطيب من على غصن األندلس الرطيب، تحقيق: محمد محي الدين عبد الحميد، دار الكتاب العربي، بيروت.  الملك، ابن سناء ، 1949 ، دار الطراز في عمل الموشحات ، تحقيق جودة الركابي، مطابع المطبعة .الكاثوليكيَّة، بيروت َ  ،ناصر سيل أ ، محمد 2016 ، أثر الترميز ِ الفني في شعر الغزل ،العذري دار ،الرضوان عمَّان / ،األردن ط1 . َ  ،ناصر سيل أ ، محمد 2016 ، أثر الترميز ِ الفني في شعر الغزل ،العذري دار ،الرضوان عمَّان / ،األردن ط1 .  ،النصي ِر ،ياسين 2011 ، شحنات المكان (جدلية التشكيل والتأثير )، دار الشؤون ،الثقافية بغداد،ط1 .  ، هيكل، أحمد1979 ، األدب األندلسيُّ من الفتح حتى سقوط الخالفة، دار.المعارف، مصر  وغليسي، د. ، يوسف 2008 ، إشكالية المصطلح في الخطاب ِ النقدي ِ العربي ،الجديد منشورات االختالف، بيروت /لبنان،ط1 .  ،النصي ِر ،ياسين 2011 ، شحنات المكان (جدلية التشكيل والتأثير )، دار الشؤون ،الثقافية بغداد،ط1 .  ، هيكل، أحمد1979 ، األدب األندلسيُّ من الفتح حتى سقوط الخالفة، دار.المعارف، مصر References  Moftah, Muhammad, 1990, The Dynamics of the Text (theorizing and brevity), the Arab 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Youssef, 2008, The Problematic Term in the New Arab Critical Discourse, Al-Ikhtif Publications, Beirut / Lebanon, 1st Edition.  Woglisi, d. Youssef, 2008, The Problematic Term in the New Arab Critical Discourse, Al-Ikhtif Publications, Beirut / Lebanon, 1st Edition. 368
https://openalex.org/W2765453854	https://europepmc.org/articles/pmc5654376?pdf=render	English	null	Experimental formation enthalpies for intermetallic phases and other inorganic compounds	Scientific data	2,017	cc-by	9,637	www.nature.com/scientificdata www.nature.com/scientificdata Background & Summary The standard enthalpy of formation of a compound is the energy associated with the reaction to form the compound from its component elements (at the pressure of 1 atm. and the temperature of 298 K). The standard enthalpy of formation plays a pivotal role in the deﬁnition of Gibbs energy of formation of a compound1,2 and is widely used as input in thermodynamic models such as those used in the CALPHAD approach to calculate phase diagrams3–10. For example, Reichmann et al.4 measured standard enthalpies of formation of compounds in the Cd-Pr system and used the data to improve the agreement between the calculated Cd-Pr phase diagram and experimental results. New phases identiﬁed in calorimetry experiments can also help amend existing experimental phase diagrams. For instance, Bittner et al.11 discovered a new phase (Ge4Ti5, space group Pnma) while performing calorimetry experiments in the Ge-Ti system for which there was little literature prior to their work. In addition, enthalpy of formation data of known compounds are valuable resources for empirical prediction of phase stability of new phases in materials discovery. Recently, Miracle et al.2 estimated the Gibbs energies of formation of over 1,000 binary intermetallic compounds from their respective enthalpies of formation. Using this approximation, they predicted the stability region of novel high entropy alloys at 1,500 K2. y p y g g py y Calorimetry provides the only direct method by which the enthalpy of formation is experimentally measured. High-temperature calorimetric methods were used for the measurements of enthalpy of formation in the current dataset. The advantage of high-temperature calorimetry is that it enables the calorimetric study of compounds with high melting points3. However, conducting these experiments is a time-consuming process. For example, a high-temperature calorimetry experiment to determine the enthalpy of formation for just a single compound can take about 18 h. The determination of the enthalpy of formation involves the measurement of the heat of reaction to form the compound, the heat content of the compound itself, and the heat content of a standard reference material for calibration. All measurements are made with multiple samples to quantify the uncertainty of measurements. Due to the large amount of time and resources needed for these measurements, calorimetric measurement results are scarce and non-existent for many systems12. Background & Summary In this work, we present a large dataset of experimentally determined enthalpies of formation from our measurements and literature, including unpublished results obtained in our group at the Illinois Institute of Technology (IIT). Most of the entries are binary intermetallic compounds with transition elements, and rare-earth elements, but ternary and quaternary compounds are being added as they become available. The dataset can also be used to compare experimental measurements of enthalpies of formation of compounds in other critically assessed experimental databases such as the SGTE Substances Database, v3.3 which contains standard enthalpies of formation 3,064 condensed stoichiometric compound phases13. p p The dataset presented is mostly based on the work of O.J. Kleppa’s group at the University of Chicago, and Philip Nash’s group at IIT. The dataset contains 1,276 entries on experimental enthalpy of formation values and structural information such as the space group and Pearson symbol. In the Methods section, the calorimeter setup, calorimetric methods (direct synthesis, solution, solute-solvent drop), and equations used to determine the enthalpies of formation are brieﬂy described. The full dataset is in a JSON ﬁle, described in the Data Records section, accessible from the Figshare-repository (Data Citation 1) and will be updated regularly as more measurements are made. In the Technical Validation section a comparison of measurements made by different research groups is made to demonstrate the reproducibility of the experimental results. The dataset also has Density Functional Theory (DFT) predictions of enthalpies of formation from the Materials Project database14 and the Open Quantum Materials Database (OQMD)15,16 for comparison. The experimental measurements are of great values as benchmarks for ﬁrst principles calculations. SCIENTIFIC DATA \| 4:170162 \| DOI: 10.1038/sdata.2017.162 Data Descriptor: Experimental formation enthalpies for intermetallic phases and other inorganic compounds Received: 11 July 2017 Accepted: 4 September 2017 Published: 24 October 2017 Received: 11 July 2017 Accepted: 4 September 2017 Published: 24 October 2017 George Kim1, S. V. Meschel2, Philip Nash2 & Wei Chen1 George Kim1, S. V. Meschel2, Philip Nash2 & Wei Chen1 The standard enthalpy of formation of a compound is the energy associated with the reaction to form the compound from its component elements. The standard enthalpy of formation is a fundamental thermodynamic property that determines its phase stability, which can be coupled with other thermodynamic data to calculate phase diagrams. Calorimetry provides the only direct method by which the standard enthalpy of formation is experimentally measured. However, the measurement is often a time and energy intensive process. We present a dataset of enthalpies of formation measured by high- temperature calorimetry. The phases measured in this dataset include intermetallic compounds with transition metal and rare-earth elements, metal borides, metal carbides, and metallic silicides. These measurements were collected from over 50 years of calorimetric experiments. The dataset contains 1,276 entries on experimental enthalpy of formation values and structural information. Most of the entries are for binary compounds but ternary and quaternary compounds are being added as they become available. The dataset also contains predictions of enthalpy of formation from ﬁrst-principles calculations for comparison. Design Type(s) data integration objective Measurement Type(s) standard enthalpy of formation Technology Type(s) synthesis method • analytical solution calorimetry • isothermal calorimetry Factor Type(s) protocol Sample Characteristic(s) 1Illinois Institute of Technology, Department of Mechanical, Materials and Aerospace Engineering, Chicago, Illinois 60616, USA. 2Illinois Institute of Technology, Thermal Processing Technology Center, Chicago, Illinois 60616, USA. Correspondence and requests for materials should be addressed to P.N. (email: nash@iit.edu) or to W.C. (email: wei.chen@iit.edu). \| DOI: 10.1038/sdata.2017.162 1 Design Type(s) data integration objective Measurement Type(s) standard enthalpy of formation Technology Type(s) synthesis method • analytical solution calorimetry • isothermal calorimetry Factor Type(s) protocol Sample Characteristic(s) Design Type(s) data integration objective Measurement Type(s) standard enthalpy of formation Technology Type(s) synthesis method • analytical solution calorimetry • isothermal calorimetry Factor Type(s) protocol Sample Characteristic(s) 1Illinois Institute of Technology, Department of Mechanical, Materials and Aerospace Engineering, Chicago, Illinois 60616, USA. 2Illinois Institute of Technology, Thermal Processing Technology Center, Chicago, Illinois 60616, USA. Correspondence and requests for materials should be addressed to P.N. (email: nash@iit.edu) or to W.C. (email: wei.chen@iit.edu). SCIENTIFIC DATA \| 4:170162 \| DOI: 10.1038/sdata.2017.162 www.nature.com/sdata/ Background & Summary Direct synthesis y In the direct synthesis method, the component elements of the compound (in powder form) are mixed in the appropriate molar ratio, compressed into small pellets of about 2 mm in diameter and then dropped from room temperature into the calorimeter3. The high temperature inside the calorimeter enables the components to react completely and the heat of reaction is measured. This reaction is represented in equation (1) with component element A and component element B both in their standard states at 298 K reacting to form compound AαBβ at the reaction temperature T. The s represents the solid state, and α and β represent the mole fractions of the elements. ΔrHT represents the measured heat of reaction at temperature T. Multiple pellets can be dropped into the calorimeter (one at a time and making sure that thermal equilibrium is regained before a consecutive drop). The multiple drops are used to calculate an average and s.d. of the measurement. The pellets which are now composed of the reaction product, compound AαBβ in equation (2), are removed from the calorimeter and the pellets are once again dropped from room temperature into the calorimeter, at the reaction temperature T, to measure the heat content of the compound. The calorimeter temperature for both measurements must be the same as well as the state of the samples at the calorimeter temperature. The heat content of the compound is represented by ΔHT −298(AαBβ) in equation (2). Again, an average and s.d. is calculated from multiple sample measurements. p The reaction that corresponds to the standard enthalpy of formation of the compound AαBβ is obtained by subtracting equation (2) from equation (1), and is given in equation (3). In equation (3) the component elements A and B are both in their standard states at 298 K and they react to form the compound AαBβ in its standard state at 298 K. The standard enthalpy of formation ΔfH298(AαBβ) is determined in equation (4) by subtracting the heat content of the compound, ΔHT −298(AαBβ), from the heat of reaction, ΔrHT(αA+βB). β During the time between the heat of reaction and the heat content experiments, the samples are kept in a vacuum desiccator to prevent reaction with oxygen or moisture. The uncertainty in the standard enthalpy of formation is calculated by adding the s.d. of the heat content measurements, the s.d. of the heat of reaction measurements, and the s.d. Methods h High-temperature calorimetric measurements were performed to collect the enthalpy of formation values in this dataset1. Three different high-temperature calorimetric methods are utilized in this dataset: direct synthesis, solution, and solute-solvent drop calorimetry. The developments in high-temperature calorimetry have enabled the study of compounds with high melting temperatures that require high temperatures to either make the component elements fully react and form the desired solid-state compound or melt and form a liquid phase. The choice between the three methods used in this work depends on experimental considerations. The direct synthesis method measures the enthalpy of formation directly, since the formation of the compound occurs within the calorimeter itself. Whereas in solution calorimetry and solute-solvent drop calorimetry, the compound is synthesized before the calorimetry measurements and the heat of dissolution of the compound is measured3. Solution or solute- solvent drop calorimetry is sometimes used instead of direct synthesis calorimetry because for some compounds, the temperature required for the component elements to react is too high or the reaction rate is too slow for the direct reaction method, resulting in residual unreacted component elements and inaccurate enthalpy of formation results1. py The functioning of a calorimeter relies on the precise measurement of temperature change, good insulation to avoid heat loss to the surroundings, and the presence of protective gas to avoid oxidation of the sample during the experiment17. The experimental setup and methods are described in further detail below, and the precision of the various methods is examined in the Technical Validation section. 2 SCIENTIFIC DATA \| 4:170162 \| DOI: 10.1038/sdata.2017.162 www.nature.com/sdata/ Direct synthesis of the calibration measurements in quadrature. The composition of the reacted compound is examined with energy dispersive spectroscopy (EDS) to check for unreacted or impurity phases. Then the sample is examined by x-ray powder diffraction analysis (XRD) to determine the structure of the phase or phases present5. αA s; 298 K ð Þ þ βB s; 298 K ð Þ ¼ AαBβ s; T ð Þ ΔrHTðαA þ βBÞ ð1Þ AαBβ s; 298 K ð Þ ¼ AαBβ s; T ð Þ HT - 298ðAαBβÞ ð2Þ αA s; 298 K ð Þ þ βB s; 298 K ð Þ ¼ AαBβ s; 298 K ð Þ Δf H298ðAαBβÞ ð3Þ Δf H298 AαBβ ¼ ΔrHTðαA þ βBÞ - HT - 298ðAαBβÞ ð4Þ ð4Þ Experimental setup Th l p p The calorimetric measurements performed by the Kleppa and Nash groups used a Calvet type calorimeter designed and built by O.J. Kleppa. The design of the Kleppa calorimeter is described in ref. 1. The sample crucible is composed of boron nitride. In rare cases when the sample reacts with boron nitride, a beryllium oxide crucible is used. The sample section is further protected from oxidation by stainless steel foils and zirconium ‘gettering’ tubes above the crucible. All experiments are performed under argon gas which is puriﬁed by passing over titanium chips at 1,173 K. The experiments are carried out with the temperature in the calorimeter held at 1,373 K, except for some cases where the calorimeter temperature was lowered to 1,273 K due to experimental considerations such as the vapor pressure of a component element being too high at 1,373 K18. In cases where analysis of reacted samples show unreacted component elements, a commercial Setaram Ligne 96 drop calorimeter is used for measurement at a higher temperature to ensure the reaction goes to completion. The principle and design of both calorimeters is very similar with the thermopile and reference junction sections located vertically on a ceramic tube surrounding the sample reaction crucible. g p The calorimeters have a thermopile embedded in alumina cylinders that surround the sample section and the temperature from the thermopile is recorded over time. The area under the temperature-time curve is proportional to the heat effect. The Kleppa calorimeter calibration is made using pure copper and this is performed once every two weeks. The Setaram calorimeter calibration measurements are performed after each sample drop using a NIST sapphire standard reference material (SRM 720). The latter calibration technique is now also being applied for some of the Kleppa calorimeter measurements. Data Records The dataset of experimentally measured standard enthalpies of formation, crystal structure information and other information is reported in enthalpy_formation.json [Data Citation 1]. The full list of recorded attributes, referred to as keys in the JSON format, is described in Table 1. The dataset contains 1,276 entries and can be retrieved from the Figshare repository [Data Citation 1]. The key ‘notes’ contains text with comments about the experimental results. For example, some compounds in the dataset were revealed by XRD analysis to have multiple crystalline modiﬁcations which are listed. A small amount of second phase can be tolerated as it will not substantially affect the result and this is usually taken as 5% (volume fraction determined by quantitative XRD analysis). If up to 10% of a second phase is present then the entry has a comment in the notes key labeling the entry as an ‘Indicative Result’. y y g y Another important key in Table 1 is the ‘uncertainty’ key. There are three types of measurement uncertainties recorded in the JSON ﬁle due to differing practices used by different sources. Some enthalpy of formation values were published with their measurement uncertainties as standard errors instead of s. d.’s. The s.d. describes how much individual data points differ from the sample mean. The standard error of the mean describes probabilistically how the mean varies given the current sample size. There is also the case where uncertainties are reported simply with an upper and lower limit of the error (i.e., ±0.01 eV/atom) without details on how they were calculated. In this case the uncertainty is simply labeled as ‘experimental_error’. p One of the ‘properties’ obtained from the DFT databases is the ‘Energy above convex hull’ property. If a structure has an energy above convex hull value of 0 the structure is the most thermodynamically stable one at its composition. If the value is positive the structure is predicted to decompose into other more thermodynamically stable compounds. As such it is a useful property to screen compounds based on thermodynamic stability. For example, Ni2CoGa was a compound that was erroneously reported to be thermodynamically stable20, but OQMD’s energy above hull value for the compound indicated that the structure was not stable. OQMD’s prediction was conﬁrmed experimentally in an investigation by Yin, M., Nash, P., Chen, W. and Chen, S21. Data Records Additionally, experimentalists could use this calculated value to discover compounds with polymorph structures by examining structures with small positive values for the energy above the convex hull. Solution calorimetry y The solution calorimetry method relies on rapid solution of both the components and the reacted products in the selected solvent. The solvent is usually a low melting metal such as tin, copper or aluminum3. It is important to check that the solute is completely dissolved. A bubbling tube can be utilized to help the sample dissolve. The heat of dissolution is measured for a mechanical mixture of the component elements. In equation (5), A and B represent the component elements that are dropped from SCIENTIFIC DATA \| 4:170162 \| DOI: 10.1038/sdata.2017.162 www.nature.com/sdata/ 298 K into the solvent that is at temperature T. The heat of dissolution at temperature T of the component elements A and B is ΔsolnHT(αA+βB). The heat of dissolution of the reacted compound is also measured. In equation (6) the compound AαBβ is dropped from 298 K into the solvent which is at the same temperature T as before. The heat of dissolution associated with this reaction is ΔsolnHT(AαBβ). The reaction that corresponds to the standard enthalpy of formation of the compound AαBβ is obtained by subtracting equation (6) from equation (5), and is given in equation (7). In equation (8) the standard enthalpy of formation, ΔfH298(AαBβ), of the compound is determined by subtracting the heat of dissolution from equation (6) from the heat of dissolution from equation (5). αA s; 298K ð Þ þ βB s; 298K ð Þ þ solvent l; T ð Þ ¼ solution l; T ð Þ ΔsolnHT αA þ βB ð Þ ð5Þ AαBβ s; 298K ð Þ þ solvent l; T ð Þ ¼ solution l; T ð Þ ΔsolnHTðAαBβÞ ð6Þ αA s; 298 K ð Þ þ βB s; 298 K ð Þ ¼ AαBβ s; 298 K ð Þ Δf H298ðAαBβÞ ð7Þ Δf H298 AαBβ ¼ ΔsolnHT αA þ βB ð Þ - ΔsolnHT AαBβ ð8Þ ð5Þ Δf H298 AαBβ ¼ ΔsolnHT αA þ βB ð Þ - ΔsolnHT AαBβ ð8Þ ð8Þ The compound AαBβ in equation (6) should be examined by EDS and XRD to conﬁrm there are no additional phases, and to determine its structure. The compound AαBβ in equation (6) should be examined by EDS and XRD to conﬁrm there are no additional phases, and to determine its structure. Solute-solvent drop calorimetry This method was developed in the Kleppa lab at the University of Chicago in 1984 for compounds which have high melting point that it is not possible to achieve complete reaction at 1,473 K by the direct synthesis method19. In this method, an unreacted mixture of the component elements is dropped into the calorimeter with a solid solvent material chosen such that the combination will form a liquid phase in the calorimeter. In a subsequent experiment the compound is dropped into the calorimeter with the same ratio of solvent material. As the liquid phase is formed the heat of dissolution is measured. As with solution calorimetry, the solvent material should not react with the components of the compound9. The formation enthalpy calculation is identical to that of the solution calorimetry method. The reacted compound should be examined by EDS and XRD to conﬁrm there are no additional phases and to determine its structure. Each entry is a JSON object in the dataset and corresponds to a compound. The JSON format is based on a series of key-value pairs. The JSON keys and their descriptions are listed in Table 1. File format Each entry is a JSON object in the dataset and corresponds to a compound. The JSON format is based on a series of key-value pairs. The JSON keys and their descriptions are listed in Table 1. SCIENTIFIC DATA \| 4:170162 \| DOI: 10.1038/sdata.2017.162 www.nature.com/sdata/ Key Value Datatype Description id String Unique ID number for entries in dataset composition JSON object key-value pairs of the component element’s chemical symbol (as a string) and molar fraction (as a ﬂoat). {‘Ag’: 0.66, ‘Sc’: 0.33} formula String Chemical formula of the compound space_group String Space group in Hermann-Mauguin notation without spaces pearson_symbol String Pearson symbol standard_enthalpy_formation Array An array with two nested arrays. The ﬁrst with the value in units of kJ/mole of atoms, and a string ‘kJ/mole of atoms’. The second with the value in units of eV/ atom, and a string ‘eV/atom’ enthalpy_formation Array The enthalpy of formation measured with respect to reaction temperature and not 298 K. An array with two nested arrays. The ﬁrst with the value in units of kJ/mole of atoms, and a string ‘kJ/mole of atoms’. The second with the value in units of eV/ atom, and a string ‘eV/atom’ ref_temp Int The reference temperature in Kelvin. Standard enthalpy of formation has a reference temperature of 298 K uncertainty JSON object with Keys: ‘standard_deviation’, ‘standard_error’, ‘experimental_error’ The value assigned to the key is an array with two nested arrays. The ﬁrst with the uncertainty in units of kJ/mole of atoms, and a string ‘kJ/mole of atoms’. The second with uncertainty in units of eV/atom, and a string ‘eV/atom’ notes String Contains a note, either says Indicative Result or Crystallographic Modiﬁcations citation String The full reference to the article the measurement was published in calorimetry_method String Calorimetric method, either Direct Synthesis, Solute-Solvent Drop or Solution cas_reg_no String Chemical Abstracts Service unique identiﬁer mp_id String Materials Project ID mp_formation_energy Array Enthalpy of formation from the Materials Project database. An array with two nested arrays. The ﬁrst with the value in units of kJ/mole of atoms, and a string ‘kJ/mole of atoms’. The second with the value in units of eV/atom, and a string ‘eV/atom’ mp_e_above_hull Array Energy above convex hull from the Materials Project database. An array with two nested arrays. The ﬁrst with the value in units of kJ/mole of atoms, and a string ‘kJ/mole of atoms’. File format The second with the value in units of eV/atom, and a string ‘eV/atom’ oqmd_id String OQMD ID oqmd_formation_energy Array Enthalpy of formation from the OQMD database. An array with two nested arrays. The ﬁrst with the value in units of kJ/mole of atoms, and a string ‘kJ/mole of atoms’. The second with the value in units of eV/atom, and a string ‘eV/atom’ oqmd_e_above_hull Array Energy above convex hull from the OQMD database. An array with two nested arrays. The ﬁrst with the value in units of kJ/mole of atoms, and a string ‘kJ/mole of atoms’. The second with the value in units of eV/atom, and a string ‘eV/atom’ Table 1. Description of the key-value pairs contained in the JSON ﬁle. The details of the measurement of the enthalpy of formation for each entry in the JSON ﬁle are contained in key-value pairs. The table lists the keys, and a description of the value associated with each key. Table 1. Description of the key-value pairs contained in the JSON ﬁle. The details of the measurement of the enthalpy of formation for each entry in the JSON ﬁle are contained in key-value pairs. The table lists the keys, and a description of the value associated with each key. Comparison of precision of calorimetric methods Figure 1 is a graphical representation of the uncertainty of the experimental results in the dataset, grouped by the calorimetric method used. The uncertainty is quantitatively described by the s.d. of the measured standard enthalpy of formation. The s.d. of the standard enthalpy of formation measurement is calculated by adding the s.d. of the heat of reaction measurements, the s.d. of the heat content measurements, and the s.d. of the calibration measurements in quadrature, using equation (9). σmeasurement ¼ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ ðσheat of reactionÞ2 þ ðσheat contentÞ2 þ ðσcalibrationÞ2 q ð9Þ σmeasurement ¼ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ ðσheat of reactionÞ2 þ ðσheat contentÞ2 þ ðσcalibrationÞ2 q ð9Þ Where σmeasurement is the measurement s.d., σheat of reaction is the s.d. of heat of reaction measurements, σheat content is the s.d. of heat content measurements, and σcalibration is the s.d. of calibration measurements. This method of computing propagated error assumes that the measured quantities are independent and that the measurement errors have a normal distribution. Instrumental errors such as a skewed distribution of measurements or drift in the instrument measurements are both mitigated by the experimental procedure. If the distribution of instrumental errors is skewed, taking the average measurement of the calibration tests will introduce a systematic error. If there is a drift in the instrumental measurements the error of the measurements get worse as the experiment progresses. The effect of a possible skew in the distribution in calibration measurements is mitigated by calculating a modiﬁed mean which is a more robust estimator of central tendency compared to the mean. The possible drift in measurements is mitigated by taking calibration measurements throughout the entire test, one after every sample. Figure 1 demonstrates the reproducibility of the experimental methods used. There are three different calorimetric methods: direct synthesis calorimetry, solution calorimetry, and the solute-solvent drop calorimetry. Figure 1 shows that the three methods are comparable in their measurement precision. 906 measurements by direct synthesis, 94 by solute-solvent drop, and 26 by solution calorimetry are plotted in Fig. 1. The dataset contains experimental results for measurements with the reaction temperature as the reference temperature instead of 298 K. These measurements were omitted in Fig. 1 for consistency. The solid red line represents the mean (μ) of the measurement uncertainty which was 0.0225 eV/atom. The dashed red lines represent one and two s.d.’s away from the mean (1σ, 2σ) respectively. The s.d. was 0.0115 eV/atom. Experimental procedure to limit confounding variables Th i t l d d ib d b Experimental procedure to limit confounding variables The experimental procedures described above ensure reproducible measurements by limiting confounding variables. The reaction of the component elements in the sample crucible occur under a protective atmosphere of Argon gas which is puriﬁed by passing it over titanium chips at 1,173 K. The procedure keeps reaction with oxygen to a minimum. The crucible is made of boron nitride and after every experiment the crucible is checked to make sure that it did not react with the sample. The formation enthalpy is measured through a two-step process. The ﬁrst step measures the heat of reaction when the component elements react to form the compound of interest. The second step measures the heat content of the reacted compound. Between the two steps the samples are kept in a vacuum desiccator to prevent reaction with oxygen or moisture. This ensures that the reacted compound does not form oxides or undergo other side reactions that affect the measurement. Additionally, the weight of the samples is checked after each experiment to check for signiﬁcant mass loss. Each reported enthalpy of formation is based on individual measurements of multiple samples (7 or more). The setup is such that multiple samples of the same compound can be measured in one setting; after each sample is dropped into the crucible through a drop tube in the calorimeter, sufﬁcient time is passed for the calorimeter to reach thermal equilibrium. Additionally, after each sample is dropped in and measured, a reference standard is dropped in for a calibration measurement. The modiﬁed mean of the calibration measurements is calculated by calculating the average after removing the largest and smallest values. This calibration method is used for all Setaram calorimeter measurements and some Kleppa calorimeter measurements. SCIENTIFIC DATA \| 4:170162 \| DOI: 10.1038/sdata.2017.162 www.nature.com/sdata/ Figure 1. Representation of the uncertainty in experimental measurements. Plot of measurement Figure 1. Representation of the uncertainty in experimental measurements. Plot of measurement uncertainties grouped by calorimetric method. The scatterplot x-axis is the measurement uncertainty of the experimental measurements of the standard enthalpy of formation. The y-axis is the experimentally measured standard enthalpy of formation. The solid red line is the mean of the measurement uncertainty (μ = 0.0225 eV/ atom), and the dashed red lines are one and two s.d.’s (σ = 0.0115 eV/atom). Histograms on each axis indicate the number of measurements in each respective bin. Comparison of precision of calorimetric methods Of the 906 direct synthesis measurements, 3.1% were more than 2 s.d.’s away from the mean. Of the 94 solute-solvent drop measurements, 30.8% were more than 2 s.d.’s away from the mean. In the solution calorimetry measurements, there was a single large outlier not plotted in Fig. 1 which was the measurement of Nd2Al using HCl acid as a solvent with a standard enthalpy of formation of −1.073 eV/atom and a s.d. of 0.243 eV/atom. Of the remaining 25 solution calorimetry measurements, 12.0% were more than 2 s.d.’s away from the mean. With a few exceptions, this demonstrates that the three calorimetry methods have comparable precision of measurements. It should be noted however, that the number of solute-solvent drop and solution calorimetry measurements are much smaller than the number of direct synthesis calorimetry measurements, meaning that the description of the spread of the measurements of solute-solvent drop and solution calorimetry may not be as accurate as compared to that of direct synthesis calorimetry. It should also be noted that the vertical patterns visible in Fig. 1 is an artifact of the number of signiﬁcant SCIENTIFIC DATA \| 4:170162 \| DOI: 10.1038/sdata.2017.162 www.nature.com/sdata/ digits used in reporting the s.d.. Most of the literature published reported their uncertainty with a precision of 1 meV/atom. digits used in reporting the s.d.. Most of the literature published reported their uncertainty with a precision of 1 meV/atom. Validation through comparison of experimental measurements by different research groups To demonstrate that the experimental results are reproducible and that measurements made by different research groups are comparable we list in Table 2 calorimetry results of identical compounds measured by different research groups. The number of comparable replication measurements by different research groups in the dataset is small, and Table 2 lists those that are comparable. Of the 76 compounds that have multiple entries, 31 are not comparable because some research groups reported enthalpies of formation with respect to temperatures other than 298 K. There are also 28 compounds that have replication measurements that are comparable, but are reported by members of the same research group, and therefore are not included in Table 2. For calorimetry experiments, it is convention to report enthalpies of formation in units of kJ/mol of atoms and for DFT calculations it is convention to report enthalpies of formation in eV/atom. The dataset has values in both units. Comparison of precision of calorimetric methods For Table 2 the original units of kJ/mol of atoms were converted to eV/atom to follow the rest of the text. 1 kJ/mol of atoms is 0.0103 eV/atom. There are 17 compounds in Table 2, with two measurements for each reported by different sources. The phase, structure, enthalpy of formation (s.d.), and absolute difference is listed. For all the experimental results in Table 2 the absolute difference in experimental measurements has the same order of magnitude or less than the uncertainty of the measurements. This indicates that experimental measurements made by independent research groups are comparable to each other, however, it is noted that the number of comparable replication measurements between different research groups is small and more results published by other research groups could be added. Table 2. Comparison of standard enthalpies of formation between different experimental groups. Measurements of standard enthalpies of formation (and the s.d. of the measurements) of compounds performed by different research groups. There are 17 compounds with two measurements each for a total of 34 measurements being compared. Results were reported in units of kJ/mol of atoms but were converted to eV/ atom to follow the rest of the text. 1 kJ/mol of atoms is 0.0103 eV/atom. The average of the absolute difference in measurements is 0.034 ev/atom of atoms. For all the results in the table the absolute difference has the same order of magnitude or lower than the measurement uncertainty. Comparison of DFT and experimental results In addition to a comparison between replication experiments, a comparison between experimental measurements and ﬁrst-principles calculations based on density functional theory is also made. Figures 2 and 3 compares the experimentally measured standard enthalpy of formation values with the calculated values on OQMD15,16 and Materials Project14 respectively. In Figs 2 and 3 The x-axis of the scatter plot is the difference between the calculated value and the experimentally measured value. The y-axis is the experimentally measured standard enthalpy of formation. The DFT calculated values are with respect to 0 K whereas the standard enthalpy of formation is with respect to 298 K. The solid red line represents the average of the difference in values. The dashed red lines represent one and two s.d.’s away from the mean. The compositions of the compounds and the positions of the plot points were examined for any patterns Phase (structure) Standard enthalpy of formation (s.d.) (eV/atom) Standard enthalpy of formation (s.d.) (eV/atom) Absolute difference (eV/atom) Fe2NiAl (Pm-3m) −0.289 (0.015)19 −0.320 (0.025)20 0.031 FeAl (Pm-3m) −0.275 (0.011)19 −0.244 (0.017)21 0.031 NiAl (Pm-3m) −0.641 (0.012)22 −0.604 (0.011)21 0.036 YMn2 (Fd-3m) −0.029 (0.029)23 −0.025 (0.026)24 0.004 ZrPt (Cmcm) −1.079 (0.019)25 −0.933 (0.104)26 0.146 ZrIr (Pm-3m) −0.888 (0.04)25 −0.840 (0.021)26 0.049 ZrNi (Cmcm) −0.523 (0.016)27 −0.534 (0.021)26 0.010 GdNi (Cmcm) −0.310 (0.011)27 −0.267 (0.019)28 0.042 DyNi (Pnma) −0.365 (0.016)27 −0.346 (0.020)28 0.019 CoTi (Pm-3m) −0.428 (0.009)27 −0.459 (0.005)26 0.031 NiY (Pnma) −0.341 (0.018)12 −0.379 (0.020)29 0.038 NiTi (Pm-3m) −0.343 (0.011)27 −0.352 (0.021)26 0.009 TiPd (Pm-3m) −0.535 (0.026)27 −0.550 (0.031)26 0.016 HfPd (Pm-3m) −0.720 (0.018)30 −0.699 (0.040)31 0.022 LuPd (Pm-3m) −0.944 (0.054)32 −0.985 (0.015)27 0.040 PdZr (Cmcm) −0.635 (0.036)32 −0.685 (0.011)27 0.050 NiB (Cmcm) −0.208 (0.020)33 −0.215 (0.011)34 0.006 Table 2. Comparison of standard enthalpies of formation between different experimental groups. Measurements of standard enthalpies of formation (and the s.d. of the measurements) of compounds performed by different research groups. There are 17 compounds with two measurements each for a total of 34 measurements being compared. Results were reported in units of kJ/mol of atoms but were converted to eV/ atom to follow the rest of the text. 1 kJ/mol of atoms is 0.0103 eV/atom. The average of the absolute difference in measurements is 0.034 ev/atom of atoms. For all the results in the table the absolute difference has the same order of magnitude or lower than the measurement uncertainty. Comparison of DFT and experimental results The most common features shared by these outlier compounds is that they either contain Lanthanide elements or elements from groups 9 and 10 on the periodic table. Figure 3. Comparison between Experimentally Measured Standard Enthalpy of Formation with calculated Enthalpy of Formation values from Materials Project. The x-axis of the scatter plot is the difference between the calculated value and the experimentally measured value. The y-axis is the experimentally measured standard enthalpy of formation. The solid red line represents the average of the difference in values, which is 0.00616 eV/atom. The dashed red lines represent one and two s.d.’s away from the mean. The s.d. is 0.108 eV/ atom. There are 562 points in total, and 25 points that are more than 2 s.d.’s away from the mean. Most of these outliers contain either Lanthanide elements or elements from groups 5, 7, and 10 on the periodic table. re 3. Comparison between Experimentally Measured Standard Enthalpy of Formation with calculated Figure 3. Comparison between Experimentally Measured Standard Enthalpy of Formation with calculated Enthalpy of Formation values from Materials Project. The x-axis of the scatter plot is the difference between the calculated value and the experimentally measured value. The y-axis is the experimentally measured standard enthalpy of formation. The solid red line represents the average of the difference in values, which is 0.00616 eV/atom. The dashed red lines represent one and two s.d.’s away from the mean. The s.d. is 0.108 eV/ atom. There are 562 points in total, and 25 points that are more than 2 s.d.’s away from the mean. Most of these outliers contain either Lanthanide elements or elements from groups 5, 7, and 10 on the periodic table. Figure 3. Comparison between Experimentally Measured Standard Enthalpy of Formation with calculated Enthalpy of Formation values from Materials Project. The x-axis of the scatter plot is the difference between the calculated value and the experimentally measured value. The y-axis is the experimentally measured standard enthalpy of formation. The solid red line represents the average of the difference in values, which is 0.00616 eV/atom. The dashed red lines represent one and two s.d.’s away from the mean. The s.d. is 0.108 eV/ atom. There are 562 points in total, and 25 points that are more than 2 s.d.’s away from the mean. Comparison of DFT and experimental results SCIENTIFIC DATA \| 4:170162 \| DOI: 10.1038/sdata.2017.162 www.nature.com/sdata/ Figure 2. Comparison between Experimentally Measured Standard Enthalpy of Formation with calculated Enthalpy of Formation values from OQMD. The x-axis of the scatter plot is the difference between the calculated value and the experimentally measured value. The y-axis is the experimentally measured standard enthalpy of formation. The solid red line represents the average of the difference in values, which is 0.0183 eV/ atom. The dashed red lines represent one and two s.d.’s away from the mean. The s.d. is 0.123 eV/atom. There are 607 points in total, and 31 points that are more than 2 s.d.’s away from the mean. The most common features shared by these outlier compounds is that they either contain Lanthanide elements or elements from groups 9 and 10 on the periodic table. Figure 2. Comparison between Experimentally Measured Standard Enthalpy of Formation with calculated Figure 2. Comparison between Experimentally Measured Standard Enthalpy of Formation with calculated Enthalpy of Formation values from OQMD. The x-axis of the scatter plot is the difference between the calculated value and the experimentally measured value. The y-axis is the experimentally measured standard enthalpy of formation. The solid red line represents the average of the difference in values, which is 0.0183 eV/ atom. The dashed red lines represent one and two s.d.’s away from the mean. The s.d. is 0.123 eV/atom. There are 607 points in total, and 31 points that are more than 2 s.d.’s away from the mean. The most common features shared by these outlier compounds is that they either contain Lanthanide elements or elements from groups 9 and 10 on the periodic table. Figure 2. Comparison between Experimentally Measured Standard Enthalpy of Formation with calculated Enthalpy of Formation values from OQMD. The x-axis of the scatter plot is the difference between the calculated value and the experimentally measured value. The y-axis is the experimentally measured standard enthalpy of formation. The solid red line represents the average of the difference in values, which is 0.0183 eV/ atom. The dashed red lines represent one and two s.d.’s away from the mean. The s.d. is 0.123 eV/atom. There are 607 points in total, and 31 points that are more than 2 s.d.’s away from the mean. Comparison of DFT and experimental results Most of these outliers contain either Lanthanide elements or elements from groups 5, 7, and 10 on the periodic table. and clusters. Also, compounds in the dataset for which the reference temperature of the reported enthalpy of formation that is not 298 K are omitted from the plots. Figure 2 compares the experimental results with DFT predictions on OQMD. The average difference i l b OQMD d h i l i 0 0183 V/ d h d i 0 123 nd clusters. Also, compounds in the dataset for which the reference temperature of the reported nthalpy of formation that is not 298 K are omitted from the plots. Figure 2 compares the experimental results with DFT predictions on OQMD. The average difference in values between OQMD and the experimental measurements is 0.0183 eV/atom and the s.d. is 0.123 eV/atom. There are 607 compounds compared. Not all compounds in the dataset were found on OQMD. There are 31 outliers that are 2 s.d.’s away from the mean. The most common features shared by the outlier compounds is that they contain lanthanide elements, or elements from groups 9 and 10 in the periodic table (with two exceptions MnSnAu, and FeGe2). In the case of the half-Heusler compound MnSnAu (space group F-43m), which does not contain a lanthanide, group 9, or group 10 element, there SCIENTIFIC DATA \| 4:170162 \| DOI: 10.1038/sdata.2017.162 www.nature.com/sdata/ are three inequivalent atomic arrangements possible22 and it may be the case that the experimentally measured structure and the OQMD structure have inequivalent atomic arrangements. There is only one OQMD entry for MnSnAu with a calculated enthalpy of formation. The speciﬁc atomic arrangements of the half-Heusler compounds were not reported for the experimental measurements. Table 3 lists the top 10 outliers ranked by absolute difference between the experimental and OQMD values. Similar comparisons between calculated values from Materials Project and experimental measure- ments are plotted in Fig. 3. The average of the difference between the experimental measurements and the Materials Project values is 0.0062 eV/atom. The s.d. of the difference is 0.108 eV/atom. There are 562 compounds plotted in total and 25 compounds that are beyond 2 s.d.’s from the mean. The most common features shared within the outlier compounds is that they contain lanthanide series elements, or elements from groups 5, 7 and 10 on the periodic table (with two exceptions Zr5Pb3 and FeGe2). Comparison of DFT and experimental results This is a similar situation with the comparison with the OQMD values where there is some disagreement between experimental measurements and DFT calculated values for intermetallic compounds with lanthanides. Table 4 below lists the 10 compounds with the largest absolute difference between the Materials Project DFT calculated values and the experimentally measured values. In the case of the intermetallic compounds with lanthanide elements the disagreement between the experimental measurements and DFT may be due to the large electronic correlation effects of the 4f- electrons23. We should note that DFT predictions of enthalpies of formation are with respect to a reference temperature of 0 K, and that some of the differences with experimental measurements can be attributed to enthalpic and entropic contributions at ﬁnite temperatures16. The OQMD database and Materials Project database reduced the systematic error introduced by elements with phase transformations between 0 and 298 K. In the submitted experimental dataset, the constituent elements with low temperature phase transformations are Ce, Li, Ti and Sn. The OQMD database calculated the difference between the energies of the ground state and the room-temperature structures. Sn had the largest difference which was 42 meV/atom which has the same order of magnitude as the average experimental uncertainty. The systematic error was reduced by making corrections to the chemical potentials of the elements in the calculation of the enthalpy of formation. The magnitude of the corrections was calculated by solving the chemical potentials using a least squares ﬁtting method using experimental formation enthalpies of compounds containing these constituent elements and the DFT calculated total energies of the compounds16. In comparisons between the experimental results of compounds containing constituent elements with low temperature phase transformations the calculated enthalpies of formation only have an average absolute error of about 0.1 eV/atom in both the Materials Project database and the OQMD database. The average uncertainty of the experimental standard enthalpy of formation measurements are one order of magnitude lower. Of the 607 compounds plotted in Fig. 2, 264 belong to the category of compounds containing lanthanide series elements, 180 belong to the category of compounds containing elements from groups 9 and 10, and 163 belong to the category labelled ‘Others’. Of the 31 outlier compounds, only 1 compound FeGe2 belongs to the ‘Other’ category. Table 3. Ten compounds with the largest absolute difference between the OQMD DFT calculated Table 3. Ten compounds with the largest absolute difference between the OQMD DFT calculated values and the experimentally measured standard enthalpy of formation values. The compound formula, space group structure, experimentally measured standard enthalpy of formation in eV/atom and the absolute difference between OQMD DFT and experimentally measured values are listed. The largest difference is 0.780 eV/atom. Comparison of DFT and experimental results Therefore, containing a lanthanide series element or a group 9 or 10 element is a better predictor for whether a compound might be an outlier. g In Fig. 3, of the 562 compounds plotted in Fig. 3, 215 belong to the category of compounds containing lanthanide series elements, 173 belong to the category of compounds containing elements from groups 5, 7, or 10, and 174 belong to the category labelled ‘Others’. Of the 25 outlier compounds only 2 compounds, Zr5Pb3 and FeGe2 belongs to the ‘Other’ category. Again, containing a lanthanide series element or an element from groups 5, 7, and 10 is a better predictor for whether a compound might be an Compound Spacegroup Standard enthalpy of formation (eV/atom) Absolute difference (eV/atom) HfNiSn F-43m −0.649 0.78 Tb5Pb3 P63/mcm −0.738 0.601 MnSnIr F-43m −0.305 0.562 MnGaIr F-43m −0.424 0.491 Tb5Ge3 P63/mcm −0.847 0.468 TaRh3 Pm-3m −0.145 0.468 PtPb P63/mmc −0.36 0.438 MnSnAu F-43m −0.502 0.423 Tb5Sn3 P63/mcm −0.758 0.409 LuPt Pnma −0.939 0.402 Table 3. Ten compounds with the largest absolute difference between the OQMD DFT calculated values and the experimentally measured standard enthalpy of formation values. The compound formula, space group structure, experimentally measured standard enthalpy of formation in eV/atom and the absolute difference between OQMD DFT and experimentally measured values are listed. The largest difference is 0.780 eV/atom. SCIENTIFIC DATA \| 4:170162 \| DOI: 10.1038/sdata.2017.162 www.nature.com/sdata/ Compound Space group Standard enthalpy of formation (eV/atom) Absolute difference (eV/ atom) HfSnPt F-43m −1.023 0.746 MnSnIr F-43m −0.304 0.599 TaPt3 P121/m1 −0.218 0.503 TaRh3 Pm-3m −0.145 0.469 MnSnAu F-43m −0.501 0.456 Zr5Pb3 P63/mcm −0.524 0.383 PtPb P63/mmc −0.359 0.383 LuB2 P6/mmm −0.308 0.383 ErB2 P6/mmm −0.282 0.34 TmB2 P6/mmm −0.317 0.335 Table 4. Ten compounds with the largest absolute difference between the materials project DFT calculated values and the experimentally measured standard enthalpy of formation values. The compound formula, space group structure, standard enthalpy of formation in eV/atom and the absolute difference between Materials Project DFT and experimentally measured values are listed. The largest difference is 0.746 eV/atom. Table 4. Ten compounds with the largest absolute difference between the materials project DFT calculated values and the experimentally measured standard enthalpy of formation values. The compound formula, space group structure, standard enthalpy of formation in eV/atom and the absolute difference between Materials Project DFT and experimentally measured values are listed. The largest difference is 0.746 eV/atom. outlier. Comparison of DFT and experimental results Other confounding variables that apply more generally and can also contribute to the difference may be human transcription errors, an inaccurate ICSD entry for the crystal structure, or convergence to a magnetic conﬁguration that is not the ground state magnetic conﬁguration. outlier. Other confounding variables that apply more generally and can also contribute to the difference may be human transcription errors, an inaccurate ICSD entry for the crystal structure, or convergence to a magnetic conﬁguration that is not the ground state magnetic conﬁguration. 1. Kleppa, O. J. Evolution and application of high-temperature reaction calorimetry at the University of Chicago from 1952 to 2000. J. Alloys Compd 321, 153–163 (2001). Usage Notes & Ishida, K. Application of the CALPHAD method to material design. Thermochim. Acta 314, 69–77 (1998). 7. Kroupa, A. Modelling of phase diagrams and thermodynamic properties using Calphad method—Development of thermo- dynamic databases Comput Mater Sci 66 3 13 (2013) y p 6. Ohtani, H. & Ishida, K. Application of the CALPHAD method to material design. Thermo 6. Ohtani, H. & Ishida, K. Application of the CALPHAD method to material design. Thermochim. Acta 314, 69–77 (1998). 7. Kroupa, A. Modelling of phase diagrams and thermodynamic properties using Calphad method—Development of thermo- dynamic databases Comput Mater Sci 66 3–13 (2013) 7. Kroupa, A. Modelling of phase diagrams and thermodynamic properties using Calphad method—Development of thermo dynamic databases. Comput. Mater. Sci. 66, 3–13 (2013). 7. Kroupa, A. Modelling of phase diagrams and thermodynam dynamic databases. Comput. Mater. Sci. 66, 3–13 (2013). y p ( ) 8. Du, Y. et al. A thermodynamic description of the Al-Fe-Si system over the whole compositio hybrid approach of CALPHAD and key experiments. Intermetallics 16, 554–570 (2008). y p 8. Du, Y. et al. A thermodynamic description of the Al-Fe-Si system over the whole composition and temperature ranges via h b id h f CALPHAD d k i I lli 16 554 570 (2008) y p 8. Du, Y. et al. A thermodynamic description of the Al-Fe-S 8. Du, Y. et al. A thermodynamic description of the Al-Fe-Si system over the whole composition and temperature ranges via a hybrid approach of CALPHAD and key experiments. Intermetallics 16, 554–570 (2008). y p y p p g hybrid approach of CALPHAD and key experiments. Intermetallics 16, 554–570 (2008). y p T. Progress of CALPHAD. Mater. Trans. JIM 33, 713–722 (1992 . Nishizawa, T. Progress of CALPHAD. Mater. Trans. JIM 33, 7 g 0. Fries, S. G. & Jantzen, T. Compilation of `CALPHAD’ formation enthalpy data: Binary intermetallic compounds in the COST 507 Gibbsian database. Thermochim. Acta 314, 23–33 (1998). Gibbsian database. Thermochim. Acta 314, 23–33 (1998). ( ) 1. Bittner, R. W., Colinet, C., Tedenac, J.-C. & Richter, K. W. Revision of the Ge-Ti phase diagram and structural stability of the new phase Ge4Ti5. J. Alloys Compd 577, 211–216 (2013). 11. Bittner, R. W., Colinet, C., Tedenac, J.-C. & Richter, K. W phase Ge4Ti5. J. Alloys Compd 577, 211–216 (2013). 11. Bittner, R. W., Colinet, C., Tedenac, J.-C. & Richter, K. W. phase Ge4Ti5. J. Usage Notes Usage Notes h d g The dataset presented in this work provides researchers with 1,276 experimentally measured enthalpies of formation of intermetallic compounds with transition elements and rare-earth elements. The dataset also includes metal borides, metal carbides, and metallic silicides. The standard enthalpy of formation of a compound is a fundamental thermodynamic property that correlates with the phase stability and may be used with other thermodynamic data to calculate phase diagrams. In the design of alloys, calculated phase diagrams are useful for guiding areas of research and the availability of experimental thermodynamic data can help optimize calculated phase diagrams. Additionally, this dataset can be used to study trends in the enthalpy of formation, make comparisons with DFT measurements, and make comparisons with other experimental measurements. The precision and reproducibility of the measurements was demonstrated and so we expect the dataset to be a good baseline for comparisons with future DFT calculations and experimental measurements. Most of the entries in the dataset are binary compounds but ternary and quaternary compounds are being added as they become available. 1. Kleppa, O. J. Evolution and application of high-temperature reaction calorimetry at the University of Chicago from 1952 to 2000. J. Alloys Compd 321, 153–163 (2001). y p 2. Miracle, D. B. et al. Exploration and Development of High Entropy Alloys for Structural Applications. Entropy 16, 494–525 (2014). g p y p J y p ( ) 4. Reichmann, T. L., Richter, K. W., Delsante, S., Borzone, G. & Ipser, H. Enthalpies of formation of Cd-Pr intermetallic compounds and thermodynamic assessment of the Cd-Pr system. Calphad 47, 56–62 (2014). g p y p y p ( ) 4. Reichmann, T. L., Richter, K. W., Delsante, S., Borzone, G. & Ipser, H. Enthalpies of formation of Cd-Pr intermetallic compo and thermodynamic assessment of the Cd-Pr system. Calphad 47, 56–62 (2014). 4. Reichmann, T. L., Richter, K. W., Delsante, S., Borzone, G. & Ipser, H. Enthalpies of formatio and thermodynamic assessment of the Cd-Pr system. Calphad 47, 56–62 (2014). , , , , , , , p , p and thermodynamic assessment of the Cd-Pr system. Calphad 47, 56–62 (2014). y y p pencer, P. J. A brief history of CALPHAD. Calphad 32, 1–8 (20 p J y p ( ) 6. Ohtani, H. & Ishida, K. Application of the CALPHAD method to material design. Thermochim. Acta 314, 69–77 (1998). p y p 6. Ohtani, H. Author Contributions h l d h Susan Meschel initiated the work, measured the enthalpies of formation of many compounds and helped organize the topics in this paper. G.K. prepared the dataset, generated ﬁgures and tables, and wrote the report with Susan Meschel. P.N. and W.C. supervised the work and reviewed and revised the manuscript. Acknowledgements h l b d This material is based upon work supported by the National Science Foundation under Grant No. DMR-1607943. This research used resources of the National Energy Research Scientiﬁc Computing Center, a DOE Ofﬁce of Science User Facility supported by the Ofﬁce of Science of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231. G.K. had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. This material is based upon work supported by the National Science Foundation under Grant No. DMR-1607943. This research used resources of the National Energy Research Scientiﬁc Computing Center a DOE Ofﬁce of Science User Facility supported by the Ofﬁce of Science of the U S Department The Creative Commons Public Domain Dedication waiver http://creativecommons.org/publicdomain/ zero/1.0/ applies to the metadata ﬁles made available in this article. Usage Notes Standard molar enthalpy of formation of LaB6 by high-temperature calorimetry. J. Chem. Thermodyn. 16, 993–1002 (1984). , ( ) 20. Webster, P. J. & Ziebeck, K. R. A. Alloys and Compounds of d-elements with Main Group Elements Part 2 (Springer, 1988 20. Webster, P. J. & Ziebeck, K. R. A. Alloys and Compounds of d-elements with Main Group Elements Part 2 (Springer, 1988). 21. Yin, M., Nash, P., Chen, W. & Chen, S. Standard enthalpies of formation of selected Ni2YZ Heusler compounds. J. Alloys Compd 660, 258–265 (2016). 20. Webster, P. J. & Ziebeck, K. R. A. Alloys and Compounds of d elements with Main Group Elements Part 2 (Springer, 1988). 21. Yin, M., Nash, P., Chen, W. & Chen, S. Standard enthalpies of formation of selected Ni2YZ Heusler compounds. J. Alloys Compd 660, 258–265 (2016). , J , y p f p ( p g , 21. Yin, M., Nash, P., Chen, W. & Chen, S. Standard enthalpies of formation of selected Ni2YZ Heusler compounds. J. Alloys Co 660, 258–265 (2016). , ( ) 2. Graf, T., Felser, C. & Parkin, S. S. P. Simple rules for the understanding of Heusler compounds. Prog. Solid State Chem. 39, 1–50 (2011). , ( ) 22. Graf, T., Felser, C. & Parkin, S. S. P. Simple rules for the understanding of Heusler compounds. Prog. Solid State Chem. 3 1–50 (2011). ( ) 23. Topsakal, M. & Wentzcovitch, R. M. Accurate projected augmented wave (PAW) datasets for rare-earth elements (RE = La-Lu). Comput. Mater. Sci. 95, 263–270 (2014). 23. Topsakal, M. & Wentzcovitch, R. M. Accurate projected augmented wave (PAW) datasets for rare-earth elements (RE = La-Lu). Comput. Mater. Sci. 95, 263–270 (2014). Data Citation Data Citation Data Citation 1. Kim, G., Meschel, S. V., Nash, P. & Chen, W. Figshare https://doi.org/10.6084/m9.ﬁgshare.c.3822835 (2017). Usage Notes Alloys Compd 577, 211–216 (2013). p y p 2. Meschel, S. V. & Kleppa, O. J. The standard enthalpies of formation of some intermetallic compounds of transition metals by high temperature direct synthesis calorimetry. J. Alloys Compd 415, 143–149 (2006). 12. Meschel, S. V. & Kleppa, O. J. The standard enthalpies of formation of some interm temperature direct synthesis calorimetry. J. Alloys Compd 415, 143–149 (2006). 12. Meschel, S. V. & Kleppa, O. J. The standard enthalpies temperature direct synthesis calorimetry. J. Alloys Co direct synthesis calorimetry. J. Alloys Compd 415, 143–149 (200 13. Thermo-Calc Software. SSUB Version 3.3 (2017). ( ) 14. Jain, A. et al. Commentary: The Materials Project: A materials genome approach to accelerating materials innovation. AIP. APL Mater 1, 011002 (2013). , ( ) 15. Saal, J. E., Kirklin, S., Aykol, M., Meredig, B. & Wolverton, C. Materials Design and Discovery with High Functional Theory: The Open Quantum Materials Database (OQMD). JOM 65, 1501–1509 (2013). ( ) 15. Saal, J. E., Kirklin, S., Aykol, M., Meredig, B. & Wolverton, C. Materials Design and Discovery with High-Throughput De ( ) 5. Saal, J. E., Kirklin, S., Aykol, M., Meredig, B. & Wolverton, C. Materials Design and Discovery with High-Throughput Density Functional Theory: The Open Quantum Materials Database (OQMD) JOM 65 1501–1509 (2013) 5. Saal, J. E., Kirklin, S., Aykol, M., Meredig, B. & Wolverton, C. Materials Design and Discovery with High Throughput Density Functional Theory: The Open Quantum Materials Database (OQMD). JOM 65, 1501–1509 (2013). Functional Theory: The Open Quantum Materials Database (OQMD). JOM 65, 1501–1509 (2013). 6. Kirklin, S. et al. The Open Quantum Materials Database (OQMD): assessing the accuracy of DFT formation energies. Npj Comput. Mater 1, 15010 (2015). p , ( ) 17. Meschel, S. V. & Nash, P. in Characterization of Materials (ed. Kaufmann, E. N.) 1–11 (J. Wiley, 2012). p 17. Meschel, S. V. & Nash, P. in Characterization of Materials (ed. Kaufmann, E. N.) 1–11 (J. Wiley, 2012). 18. Meschel, S. V. & Kleppa, O. J. Standard enthalpies of formation of some carbides, silicides and germanides of cerium and praseodymium. J. Alloys Compd 220, 88–93 (1995). 10 SCIENTIFIC DATA \| 4:170162 \| DOI: 10.1038/sdata.2017.162 www.nature.com/sdata/ 19. Topor, L. & Kleppa, O. J. Standard molar enthalpy of formation of LaB6 by high-temperature calorimetry. J. Chem. Thermodyn. 16, 993–1002 (1984). 9. Topor, L. & Kleppa, O. J. Additional Information Competing interests: The authors declare no competing ﬁnancial interests. How to cite this article: Kim, G. et al. Experimental formation enthalpies for intermetallic phases and other inorganic compounds. Sci. Data 4:170162 doi: 10.1038/sdata.2017.162 (2017). 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 Interna- tional License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons. org/licenses/by/4.0/ © The Author(s) 2017 SCIENTIFIC DATA \| 4:170162 \| DOI: 10.1038/sdata.2017.162
https://openalex.org/W2888826266	https://digital.csic.es/bitstream/10261/205005/1/199_Observation_BABAR.pdf	English	null	Observation of the Decay <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" display="inline"><mml:msup><mml:mi>D</mml:mi><mml:mn>0</mml:mn></mml:msup><mml:mo stretchy="false">→</mml:mo><mml:msup><mml:mi>K</mml:mi><mml:mo>−</mml:mo></mml:msup><mml:msup><mml:mi>π</mml:mi><mml:mo>+</mml:mo></mml:msup><mml:msup><mml:mi>e</mml:mi><mml:mo>+</mml:mo></mml:msup><mml:msup><mml:mi>e</mml:mi><mml:mo>−</mml:mo></mml:msup></mml:math>	Physical review letters	2,019	cc-by	7,670	Observation of the Decay D0 →K −π + e + e − Kowalewski,64b T. Lueck,64b I. M. Nugent,64b J. M. Roney,64b R. J. Sobie,64a,64b N. Tasneem,64b T. J. Gershon,65 P. F. Harrison,65 T. E. Latham,65 R. Prepost,66 and S. L. Wu66 PHYSICAL REVIEW LETTERS 122, 081802 (2019) PHYSICAL REVIEW LETTERS 122, 081802 (2019) Observation of the Decay D0 →K −π + e + e − Grünberg,48 M. Heß,48 T. Leddig,48 C. Voß A. Pilloni,47a,47b G. Piredda,47a,* C. Bünger,48 S. Dittrich,48 O. Grünberg,48 M. Heß,48 T. Leddig,48 C. Voß,48 R. Waldi,48 T. Adye,49 F. F. Wilson,49 S. Emery,50 G. Vasseur,50 D. Aston,51 C. Cartaro,51 M. R. Convery,51 J. Dorfan,51 W. Dunwoodie,51 M. Ebert,51 R. C. Field,51 B. G. Fulsom,51 M. T. Graham,51 C. Hast,51 W. R. Innes,51,* P. Kim,51 D. W. G. S. Leith,51 S. Luitz,51 D. B. MacFarlane,51 D. R. Muller,51 H. Neal,51 B. N. Ratcliff,51 A. Roodman,51 M. K. Sullivan,51 J. Va’vra,51 W. J. Wisniewski,51 M. V. Purohit,52 J. R. Wilson,52 A. Randle-Conde,53 S. J. Sekula,53 T. Adye,49 F. F. Wilson,49 S. Emery,50 G. Vasseur,50 D. Aston,51 C. Cartaro,51 M. R. Convery,51 J. Dorfan,51 W. Dunwoodie,51 M. Ebert,51 R. C. Field,51 B. G. Fulsom,51 M. T. Graham,51 C. Hast,51 W. R. Innes,51,* P. Kim,51 D. W. G. S. Leith,51 S. Luitz,51 D. B. MacFarlane,51 D. R. Muller,51 H. Neal,51 B. N. Ratcliff,51 A. Roodman,51 W. Dunwoodie, M. Ebert, R. C. Field, B. G. Fulsom, M. T. Graham, C. Hast, W. R. Innes, P. Kim, D. W. G. S. Leith,51 S. Luitz,51 D. B. MacFarlane,51 D. R. Muller,51 H. Neal,51 B. N. Ratcliff,51 A. Roodman,51 M K Sullivan 51 J Va’vra 51 W J Wisniewski 51 M V Purohit 52 J R Wilson 52 A Randle-Conde 53 S J Sekula 53 D. W. G. S. Leith, S. Luitz, D. B. MacFarlane, D. R. Muller, H. Neal, B. N. Ratcliff, A. Roodman, M. K. Sullivan,51 J. Va’vra,51 W. J. Wisniewski,51 M. V. Purohit,52 J. R. Wilson,52 A. Randle-Conde,53 S. J. Sekula,53 H. Ahmed,54 M. Bellis,55 P. R. Burchat,55 E. M. T. Puccio,55 M. S. Alam,56 J. A. Ernst,56 R. Gorodeisky,57 N. Guttman,57 D. R. Peimer,57 A. Soffer,57 S. M. Spanier,58 J. L. Ritchie,59 R. F. Schwitters,59 J. M. Izen,60 X. C. Lou,60 F. Bianchi,61a,61b F. De Mori,61a,61b A. Filippi,61a D. Gamba,61a,61b L. Lanceri,62 L. Vitale,62 F. Martinez-Vidal,63 A. Oyanguren,63 J. Albert,64b A. Beaulieu,64b F. U. Bernlochner,64b G. J. King,64b R. Kowalewski,64b T. Lueck,64b I. M. Nugent,64b J. M. Roney,64b H. Ahmed,54 M. Bellis,55 P. R. Burchat,55 E. M. T. Puccio,55 M. S. Alam,56 J. A. Ernst,56 R. Gorodeisky,57 N. Guttman,57 57 57 58 59 59 60 60 61 61b F. De Mori,61a,61b A. Filippi,61a D. Gamba,61a,61b L. Lanceri,62 L. Vitale,62 F. Martinez-Vidal,63 A. Oyanguren,63 J. Albert,64b A. Beaulieu,64b F. U. Bernlochner,64b G. J. King,64b R. Observation of the Decay D0 →K −π + e + e − Leddig,48 C. Voß,48 R. Waldi,48 T. Adye,49 F. F. Wilson,49 S. Emery,50 G. Vasseur,50 D. Aston,51 C. Cartaro,51 M. R. Convery,51 J. Dorfan,51 W. Dunwoodie,51 M. Ebert,51 R. C. Field,51 B. G. Fulsom,51 M. T. Graham,51 C. Hast,51 W. R. Innes,51,* P. Kim,51 D. W. G. S. Leith,51 S. Luitz,51 D. B. MacFarlane,51 D. R. Muller,51 H. Neal,51 B. N. Ratcliff,51 A. Roodman,51 M. K. Sullivan,51 J. Va’vra,51 W. J. Wisniewski,51 M. V. Purohit,52 J. R. Wilson,52 A. Randle-Conde,53 S. J. Sekula,53 H. Ahmed,54 M. Bellis,55 P. R. Burchat,55 E. M. T. Puccio,55 M. S. Alam,56 J. A. Ernst,56 R. Gorodeisky,57 N. Guttman,57 D. R. Peimer,57 A. Soffer,57 S. M. Spanier,58 J. L. Ritchie,59 R. F. Schwitters,59 J. M. Izen,60 X. C. Lou,60 F. Bianchi,61a,61b F. De Mori,61a,61b A. Filippi,61a D. Gamba,61a,61b L. Lanceri,62 L. Vitale,62 F. Martinez-Vidal,63 A. Oyanguren,63 J. Albert,64b A. Beaulieu,64b F. U. Bernlochner,64b G. J. King,64b R. Kowalewski,64b T. Lueck,64b I. M. Nugent,64b J. M. Roney,64b R. J. Sobie,64a,64b N. Tasneem,64b T. J. Gershon,65 P. F. Harrison,65 T. E. Latham,65 R. Prepost,66 and S. L. Wu66 J. P. Coleman,26 E. Gabathuler,26,* D. E. Hutchcroft,26 D. J. Payne,26 C. Touramanis,26 A. J. Bevan,27 F. Di Lodovico,27 R. Sacco,27 G. Cowan,28 Sw. Banerjee,29 D. N. Brown,29 C. L. Davis,29 A. G. Denig,30 W. Gradl,30 K. Griessinger,30 A. Hafner, K. R. Schubert, R. J. Barlow, G. D. Lafferty, R. Cenci, A. Jawahery, D. A. Roberts, R. Cowan, S. H. Robertson,34a,34b R. M. Seddon,34b B. Dey,35a N. Neri,35a F. Palombo,35a,35b R. Cheaib,36 L. Cremaldi,36 R. Godang,36,¶ D. J. Summers,36 P. Taras,37 G. De Nardo,38 C. Sciacca,38 G. Raven,39 C. P. Jessop,40 J. M. LoSecco,40 K. Honscheid,41 R. Kass,41 A. Gaz,42a M. Margoni,42a,42b M. Posocco,42a G. Simi,42a,42b F. Simonetto,42a,42b R. Stroili,42a,42b S. Akar,43 D. J. Summers, P. Taras, G. De Nardo, C. Sciacca, G. Raven, C. P. Jessop, J. M. LoSecco, K. Honscheid, R. Kass,41 A. Gaz,42a M. Margoni,42a,42b M. Posocco,42a G. Simi,42a,42b F. Simonetto,42a,42b R. Stroili,42a,42b S. Akar,43 43 43 43 43 43 43 43 M. Biasini,44a,44b E. Manoni,44a A. Rossi,44a G. Batignani,45a,45b S. Bettarini,45a,45b M. Carpinelli,45a,45b,** G. Casarosa,45a,45b 45 45 45b 45 45b 45 45 45 45b 45 45b 45 g G. Rizzo,45a,45b J. J. Walsh,45a L. Zani,45a,45b A. J. S. Smith,46 F. Anulli,47a R. Faccini,47a,47b F. Ferrarotto,47a F. Ferroni,47a,47b 47 47b 47 * 48 48 48 48 48 48 48 47a,47b G. Piredda,47a,* C. Bünger,48 S. Dittrich,48 O. Observation of the Decay D0 →K −π + e + e − y J. P. Lees,1 V. Poireau,1 V. Tisserand,1 E. Grauges,2 A. Palano,3 G. Eigen,4 D. N. Brown,5 Yu. G. Kolomensky,5 M. Fritsch,6 H. Koch,6 T. Schroeder,6 C. Hearty,7a,7b T. S. Mattison,7b J. A. McKenna,7b R. Y. So,7b V. E. Blinov,8a,8b,8c A. R. Buzykaev,8a V. P. Druzhinin,8a,8b V. B. Golubev,8a,8b E. A. Kozyrev,8a,8b E. A. Kravchenko,8a,8b A. P. Onuchin,8a,8b,8c S. I. Serednyakov,8a,8b Yu. I. Skovpen,8a,8b E. P. Solodov,8a,8b K. Yu. Todyshev,8a,8b A. J. Lankford,9 J. W. Gary,10 O. Long,10 A. M. Eisner,11 W. S. Lockman,11 W. Panduro Vazquez,11 D. S. Chao,12 C. H. Cheng,12 B. Echenard,12 K. T. Flood,12 D. G. Hitlin,12 J. Kim,12 Y. Li,12 T. S. Miyashita,12 P. Ongmongkolkul,12 F. C. Porter,12 M. Röhrken,12 Z. Huard,13 B. T. Meadows,13 B. G. Pushpawela,13 M. D. Sokoloff,13 L. Sun,13,† J. G. Smith,14 S. R. Wagner,14 D. Bernard,15 M. Verderi,15 D. Bettoni,16a C. Bozzi,16a R. Calabrese,16a,16b G. Cibinetto,16a,16b E. Fioravanti,16a,16b I. Garzia,16a,16b E. Luppi,16a,16b V. Santoro,16a A. Calcaterra,17 R. de Sangro,17 G. Finocchiaro,17 S. Martellotti,17 P. Patteri,17 I. M. Peruzzi,17 M. Piccolo,17 M. Rotondo,17 A. Zallo,17 S. Passaggio,18 C. Patrignani,18,‡ H. M. Lacker,19 B. Bhuyan,20 U. Mallik,21 C. Chen,22 J. Cochran,22 S. Prell,22 A. V. Gritsan,23 N. Arnaud,24 M. Davier,24 F. Le Diberder,24 A. M. Lutz,24 G. Wormser,24 D. J. Lange,25 D. M. Wright,25 J. P. Coleman,26 E. Gabathuler,26,* D. E. Hutchcroft,26 D. J. Payne,26 C. Touramanis,26 A. J. Bevan,27 F. Di Lodovico,27 R. Sacco,27 G. Cowan,28 Sw. Banerjee,29 D. N. Brown,29 C. L. Davis,29 A. G. Denig,30 W. Gradl,30 K. Griessinger,30 A. Hafner,30 K. R. Schubert,30 R. J. Barlow,31,§ G. D. Lafferty,31 R. Cenci,32 A. Jawahery,32 D. A. Roberts,32 R. Cowan,33 S. H. Robertson,34a,34b R. M. Seddon,34b B. Dey,35a N. Neri,35a F. Palombo,35a,35b R. Cheaib,36 L. Cremaldi,36 R. Godang,36,¶ D. J. Summers,36 P. Taras,37 G. De Nardo,38 C. Sciacca,38 G. Raven,39 C. P. Jessop,40 J. M. LoSecco,40 K. Honscheid,41 R. Kass,41 A. Gaz,42a M. Margoni,42a,42b M. Posocco,42a G. Simi,42a,42b F. Simonetto,42a,42b R. Stroili,42a,42b S. Akar,43 E. Ben-Haim,43 M. Bomben,43 G. R. Bonneaud,43 G. Calderini,43 J. Chauveau,43 G. Marchiori,43 J. Ocariz,43 M. Biasini,44a,44b E. Manoni,44a A. Rossi,44a G. Batignani,45a,45b S. Bettarini,45a,45b M. Carpinelli,45a,45b,** G. Casarosa,45a,45b M. Chrzaszcz,45a F. Forti,45a,45b M. A. Giorgi,45a,45b A. Lusiani,45a,45c B. Oberhof,45a,45b E. Paoloni,45a,45b M. Rama,45a G. Rizzo,45a,45b J. J. Walsh,45a L. Zani,45a,45b A. J. S. Smith,46 F. Anulli,47a R. Faccini,47a,47b F. Ferrarotto,47a F. Ferroni,47a,47b A. Pilloni,47a,47b G. Piredda,47a,* C. Bünger,48 S. Dittrich,48 O. Grünberg,48 M. Heß,48 T. PHYSICAL REVIEW LETTERS 122, 081802 (2019) 13University of Cincinnati, Cincinnati, Ohio 45221, USA 14University of Colorado, Boulder, Colorado 80309, USA 15Laboratoire Leprince-Ringuet, Ecole Polytechnique, CNRS/IN2P3, F-91128 Palaiseau, France 16aINFN Sezione di Ferrara, I-44122 Ferrara, Italy 16bDipartimento di Fisica e Scienze della Terra, Universit`a di Ferrara, I-44122 Ferrara, Italy 17INFN Laboratori Nazionali di Frascati, I-00044 Frascati, Italy 18INFN Sezione di Genova, I-16146 Genova, Italy 19Humboldt-Universität zu Berlin, Institut für Physik, D-12489 Berlin, Germany 20Indian Institute of Technology Guwahati, Guwahati, Assam, 781 039, India 21University of Iowa, Iowa City, Iowa 52242, USA 22Iowa State University, Ames, Iowa 50011, USA 23Johns Hopkins University, Baltimore, Maryland 21218, USA 24Laboratoire de l’Acc´el´erateur Lin´eaire, IN2P3/CNRS et Universit´e Paris-Sud 11, Centre Scientifique d’Orsay, F-91898 Orsay Cedex, France 25Lawrence Livermore National Laboratory, Livermore, California 94550, USA 26University of Liverpool, Liverpool L69 7ZE, United Kingdom 27Queen Mary, University of London, London, E1 4NS, United Kingdom 28University of London, Royal Holloway and Bedford New College, Egham, Surrey TW20 0EX, United Kingdom 29University of Louisville, Louisville, Kentucky 40292, USA 30Johannes Gutenberg-Universität Mainz, Institut für Kernphysik, D-55099 Mainz, Germany 31University of Manchester, Manchester M13 9PL, United Kingdom 32University of Maryland, College Park, Maryland 20742, USA 33Massachusetts Institute of Technology, Laboratory for Nuclear Science, Cambridge, Massachusetts 02139, USA 34aInstitute of Particle Physics, Montr´eal, Qu´ebec, Canada H3A 2T8 34bMcGill University, Montr´eal, Qu´ebec, Canada H3A 2T8 35aINFN Sezione di Milano, I-20133 Milano, Italy 35bDipartimento di Fisica, Universit`a di Milano, I-20133 Milano, Italy 36University of Mississippi, University, Mississippi 38677, USA 37Universit´e de Montr´eal, Physique des Particules, Montr´eal, Qu´ebec, Canada H3C 3J7 38INFN Sezione di Napoli and Dipartimento di Scienze Fisiche, Universit`a di Napoli Federico II, I-80126 Napoli, Italy 39NIKHEF, National Institute for Nuclear Physics and High Energy Physics, NL-1009 DB Amsterdam, The Netherlands 40University of Notre Dame, Notre Dame, Indiana 46556, USA 41Ohio State University, Columbus, Ohio 43210, USA 42aINFN Sezione di Padova, I-35131 Padova, Italy 42bDipartimento di Fisica, Universit`a di Padova, I-35131 Padova, Italy 43Laboratoire de Physique Nucl´eaire et de Hautes Energies, IN2P3/CNRS, Universit´e Pierre et Marie Curie-Paris6, Universit´e Denis Diderot-Paris7, F-75252 Paris, France 44aINFN Sezione di Perugia, I-06123 Perugia, Italy 44bDipartimento di Fisica, Universit`a di Perugia, I-06123 Perugia, Italy 45aINFN Sezione di Pisa, I-56127 Pisa, Italy 45bDipartimento di Fisica, Universit`a di Pisa, I-56127 Pisa, Italy 45cScuola Normale Superiore di Pisa, I-56127 Pisa, Italy 46Princeton University, Princeton, New Jersey 08544, USA 47aINFN Sezione di Roma, I-00185 Roma, Italy 47bDipartimento di Fisica, Universit`a di Roma La Sapienza, I-00185 Roma, Italy 48Universität Rostock, D-18051 Rostock, Germany 49Rutherford Appleton Laboratory, Chilton, Didcot, Oxon, OX11 0QX, United Kingdom 50CEA, Irfu, SPP, Centre de Saclay, F-91191 Gif-sur-Yvette, France 51SLAC National Accelerator Laboratory, Stanford, California 94309 USA 52University of South Carolina, Columbia, South Carolina 29208, USA 53Southern Methodist University, Dallas, Texas 75275, USA 54St. (BABAR Collaboration) 1Laboratoire d’Annecy-le-Vieux de Physique des Particules (LAPP), Universit´e de Savoie, CNRS/IN2P3, F-74941 Annecy-Le-Vieux, France 2Universitat de Barcelona, Facultat de Fisica, Departament ECM, E-08028 Barcelona, Spain 3INFN Sezione di Bari and Dipartimento di Fisica, Universit`a di Bari, I-70126 Bari, Italy 4University of Bergen, Institute of Physics, N-5007 Bergen, Norway 5Lawrence Berkeley National Laboratory and University of California, Berkeley, California 94720, USA 6Ruhr Universität Bochum, Institut für Experimentalphysik 1, D-44780 Bochum, Germany 7aInstitute of Particle Physics, Vancouver, British Columbia, Canada V6T 1Z1 7bUniversity of British Columbia, Vancouver, British Columbia, Canada V6T 1Z1 8aBudker Institute of Nuclear Physics SB RAS, Novosibirsk 630090, Russia 8bNovosibirsk State University, Novosibirsk 630090, Russia 8cNovosibirsk State Technical University, Novosibirsk 630092, Russia 9University of California at Irvine, Irvine, California 92697, USA 10University of California at Riverside, Riverside, California 92521, USA 11University of California at Santa Cruz, Institute for Particle Physics, Santa Cruz, California 95064, USA 12California Institute of Technology, Pasadena, California 91125, USA 1Laboratoire d’Annecy-le-Vieux de Physique des Particules (LAPP), Universit´e de Savoie, 0031-9007=19=122(8)=081802(8) 081802-1 Published by the American Physical Society 081802-1 F-91898 Orsay Cedex, France Francis Xavier University, Antigonish, Nova Scotia, Canada B2G 2W5 55 55Stanford University, Stanford, California 94305, USA 56State University of New York, Albany, New York 12222, USA 57Tel Aviv University, School of Physics and Astronomy, Tel Aviv, 69978, Israel 58 ersity of Tennessee, Knoxville, Tennessee 37996, US 59University of Texas at Austin, Austin, Texas 78712, USA 60University of Texas at Dallas, Richardson, Texas 75083, USA 61 ersity of Texas at Dallas, Richardson, Texas 75083, 61 INFN Sezione di Torino, I-10125 Torino, Italy 61bDipartimento di Fisica, Universit`a di Torino, I-10125 Torino, Italy 081802-2 081802-2 F-91898 Orsay Cedex, France 25Lawrence Livermore National Laboratory, Livermore, California 94550, USA 26 26University of Liverpool, Liverpool L69 7ZE, United Kingdom 27 27Queen Mary, University of London, London, E1 4NS, United Kingdom London, Royal Holloway and Bedford New College, Egham, Surrey TW20 0EX, United Kingdom 29 31University of Manchester, Manchester M13 9PL, United Kingdom 32 32University of Maryland, College Park, Maryland 20742, USA nstitute of Technology, Laboratory for Nuclear Science, Cambridge, Massachusetts 02139, USA 34 34aInstitute of Particle Physics, Montr´eal, Qu´ebec, Canada H3A 2T8 34b 34bMcGill University, Montr´eal, Qu´ebec, Canada H3A 2T8 35 35aINFN Sezione di Milano, I-20133 Milano, Italy 35bDipartimento di Fisica, Universit`a di Milano, I-20133 Milano, Italy 36 36University of Mississippi, University, Mississippi 38677, USA Universit´e de Montr´eal, Physique des Particules, Montr´eal, Qu´ebec, Canada H3C 3J7 39NIKHEF, National Institute for Nuclear Physics and High Energy Physics, NL-1009 DB Amsterdam, The Netherlands 39NIKHEF, National Institute for Nuclear Physics and High Energy Physics, NL-1009 DB Amsterdam, The Netherlands 40U i i f N D N D I di 46556 USA of Notre Dame, Notre Dame, Indiana 46556, USA 41Ohio State University, Columbus, Ohio 43210, USA 42 42aINFN Sezione di Padova, I-35131 Padova, Italy 42bDipartimento di Fisica, Universit`a di Padova, I-35131 Padova, Italy Physique Nucl´eaire et de Hautes Energies, IN2P3/CNRS, Universit´e Pierre et Marie Curie-Paris6, Universit´e Denis Diderot-Paris7, F-75252 Paris, France 44 44aINFN Sezione di Perugia, I-06123 Perugia, Italy NFN Sezione di Perugia, I-06123 Perugia, Italy 44bDipartimento di Fisica, Universit`a di Perugia, I-06123 Perugia, Italy 45 45aINFN Sezione di Pisa, I-56127 Pisa, Italy 45bDipartimento di Fisica, Universit`a di Pisa, I-56127 Pisa, Italy 45 45cScuola Normale Superiore di Pisa, I-56127 Pisa, Italy 46 46Princeton University, Princeton, New Jersey 08544, USA 4 7aINFN Sezione di Roma, I-00185 Roma, Italy 47bDipartimento di Fisica, Universit`a di Roma La Sapienza, I-00185 Roma, Italy 48 o di Fisica, Universit`a di Roma La Sapienza, I-00 48 48Universität Rostock, D-18051 Rostock, Germany 49Rutherford Appleton Laboratory, Chilton, Didcot, Oxon, OX11 0QX, United Kingdom 50 50CEA, Irfu, SPP, Centre de Saclay, F-91191 Gif-sur-Yvette, France 51SLAC National Accelerator Laboratory, Stanford, California 94309 USA 52 52University of South Carolina, Columbia, South Carolina 29208, USA 53 uth Carolina, Columbia, South Carolina 29208, U 53Southern Methodist University, Dallas, Texas 75275, USA ethodist University, Dallas, Texas 75275, USA 54St. PHYSICAL REVIEW LETTERS 122, 081802 (2019) Francis Xavier University, Antigonish, Nova Scotia, Canada B2G 2W5 55Stanford University, Stanford, California 94305, USA 56State University of New York, Albany, New York 12222, USA 57Tel Aviv University, School of Physics and Astronomy, Tel Aviv, 69978, Israel 58University of Tennessee, Knoxville, Tennessee 37996, USA 59University of Texas at Austin, Austin, Texas 78712, USA 60University of Texas at Dallas, Richardson, Texas 75083, USA PHYSICAL REVIEW LETTERS 122, 081802 (2019) 13University of Cincinnati, Cincinnati, Ohio 45221, USA niversity of Colorado, Boulder, Colorado 80309, US 14University of Colorado, Boulder, Colorado 80309, USA y f oratoire Leprince-Ringuet, Ecole Polytechnique, CNRS/IN2P3, F-91128 Palaiseau, France 16 q 16aINFN Sezione di Ferrara, I-44122 Ferrara, Italy y partimento di Fisica e Scienze della Terra, Universit`a di Ferrara, I-44122 Ferrara, Italy 17 17INFN Laboratori Nazionali di Frascati, I-00044 Frascati, Italy 18 20Indian Institute of Technology Guwahati, Guwahati, Assam, 781 039, India 21 21University of Iowa, Iowa City, Iowa 52242, USA 22Iowa State University, Ames, Iowa 50011, USA 23Johns Hopkins University, Baltimore, Maryland 21218, USA l’Acc´el´erateur Lin´eaire, IN2P3/CNRS et Universit´e Paris-Sud 11, Centre Scientifique d’Orsay, 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. Funded by SCOAP3. DOI: 10.1103/PhysRevLett.122.081802 Recently, the LHCb Collaboration measured BðD0 →K−πþμþμ−Þ ¼ ð4.17 0.12 0.40Þ × 10−6 in the mass range 0.675 < mðμþμ−Þ < 0.875 GeV=c2, where the decay is dominated by the ρ0 and ω resonances [21]. For modes involving electrons, the CLEO Collaboration set 90% confidence level (C.L.) limits on the branching fractions BðD0 →Xlþl−Þ in the range ð4.5–118Þ × 10−5, where X represents a π0, K0 S, η, ρ0, ω, or ϕ meson and l ¼ e or μ [22]. The E791 Collaboration has reported BðD0 →K−πþeþe−Þ < 38.5 × 10−5 at the 90% C.L. in the full mðK−πþÞ invariant mass range and BðD0 → K−πþeþe−Þ < 4.7 × 10−5 in the mðK−πþÞ mass range within 55 MeV=c2 of the ¯Kð892Þ0 mass [23,24]. Recently, the LHCb Collaboration measured BðD0 →K−πþμþμ−Þ ¼ ð4.17 0.12 0.40Þ × 10−6 in the mass range 0.675 < mðμþμ−Þ < 0.875 GeV=c2, where the decay is dominated by the ρ0 and ω resonances [21]. For modes involving electrons, the CLEO Collaboration set 90% confidence level (C.L.) limits on the branching fractions BðD0 →Xlþl−Þ in the range ð4.5–118Þ × 10−5, where X represents a π0, K0 S, η, ρ0, ω, or ϕ meson and l ¼ e or μ [22]. The E791 Collaboration has reported BðD0 →K−πþeþe−Þ < 38.5 × 10−5 at the 90% C.L. in the full mðK−πþÞ invariant mass range and BðD0 → K−πþeþe−Þ < 4.7 × 10−5 in the mðK−πþÞ mass range within 55 MeV=c2 of the ¯Kð892Þ0 mass [23,24]. The decay D0 →K−πþeþe−[1] is expected to be very rare in the standard model (SM) as it cannot occur at tree level [2]. Short-distance contributions to the D0 → K−πþeþe−branching fraction proceed through loop and box diagrams [3] and are expected to be Oð10−9Þ. However, decays with long-distance contributions, such as D0 →VX, where V is a vector or pseudoscalar meson decaying to two leptons and X is an accompanying particle or particles, could contribute at the level of Oð10−6Þ through photon pole amplitudes or vector meson domi- nance [3–7]. Certain physics models beyond the standard model, such as minimal supersymmetric or R-parity-violating super- symmetric theories, predict branching fractions as high as Oð10−5Þ [3,7–10]. As virtual particles can enter in the one- loop processes, this type of decay can be used to study new physics processes at large mass scales. PHYSICAL REVIEW LETTERS 122, 081802 (2019) PHYSICAL REVIEW LETTERS 122, 081802 (2019) INFN Sezione di Trieste and Dipartimento di Fisica, Universit`a di Trieste, I-34127 Trieste, Italy 66University of Wisconsin, Madison, Wisconsin 53706, USA (Received 30 August 2018; revised manuscript received 11 December 2018; published 27 ed 30 August 2018; revised manuscript received 11 December 2018; published 27 February 2019) We report the observation of the rare charm decay D0 →K−πþeþe−, based on 468 fb−1 of eþe− annihilation data collected at or close to the center-of-mass energy of the ϒð4SÞ resonance with the BABAR detector at the SLAC National Accelerator Laboratory. We find the branching fraction in the invariant mass range 0.675 < mðeþe−Þ < 0.875 GeV=c2 of the electron-positron pair to be BðD0 →K−πþeþe−Þ ¼ ð4.0 0.5 0.2 0.1Þ × 10−6, where the first uncertainty is statistical, the second systematic, and the third due to the uncertainty in the branching fraction of the decay D0 →K−πþπþπ−used as a normalization mode. The significance of the observation corresponds to 9.7 standard deviations including systematic uncertainties. This result is consistent with the recently reported D0 →K−πþμþμ−branching fraction, measured in the same invariant mass range, and with the value expected in the standard model. In a set of regions of mðeþe−Þ, where long-distance effects are potentially small, we determine a 90% con- fidence level upper limit on the branching fraction BðD0 →K−πþeþe−Þ < 3.1 × 10−6. DOI: 10.1103/PhysRevLett.122.081802 Multiple candidates occur in less than 4% of simulated D0 → K−πþπþπ− decays and in less than 2% of D0 → K−πþeþe−decays. If two or more candidates are found in an event, the one with the highest vertex χ2 probability is selected. Monte Carlo (MC) simulation is used to evaluate the level of background contamination and selection efficien- cies. Simulated events are also used to cross-check the selection procedure and for studies of systematic effects. The signal and normalization channels are simulated with the EVTGEN package [27]. We generate the signal channel decay with a phase-space model, while the normalization mode includes two-body and three-body intermediate resonances, as well as nonresonant decays. For background studies, we generate eþe−→q¯q (q ¼ u, d, s, c), dimuon, Bhabha elastic eþe−scattering, B ¯B background, and two- photon events [28,29]. The background samples are pro- duced with an integrated luminosity approximately 6 times that of the data. Final-state radiation is provided by PHOTOS [30]. The detector response is simulated with GEANT4 [31,32]. All simulated events are reconstructed in the same manner as the data. After the application of all selection criteria and correc- tions for small differences between data and MC simulation in tracking and PID performance, the average reconstruction efficiency for the D0 →K−πþπþπ−decay is ˆϵnorm ¼ ð20.1 0.2Þ%, where the uncertainty is due to the limited size of the simulation sample. For the D0 → K−πþeþe−decay, the average reconstruction efficiency ˆϵsig varies between 5.0% and 8.9% depending on the mðeþe−Þ mass range. The remaining background comes predomi- nantly from eþe−→c¯c events. No evidence is found in MC simulation for backgrounds that peak in the mðD0Þ and Δm signal region. Events are required to contain at least five charged tracks. Candidate D0 mesons are formed from four charged tracks reconstructed with the appropriate mass hypothesis for the D0 →K−πþeþe−and D0 →K−πþπþπ−decays. Particle identification (PID) is applied to the charged tracks and the same criteria are applied to the signal and normalization modes [26,33]. The four tracks must form a good-quality vertex with a χ2 probability for the vertex fit greater than 0.005. In the case of D0 →K−πþeþe−, a bremsstrahlung energy recovery algorithm is applied to the electrons, in which the energy of photon showers that are within a small angle (typically 35 mrad) of the initial electron direction are added to the energy of the electron candidate. DOI: 10.1103/PhysRevLett.122.081802 These processes could potentially be detected in regions where the decays of intermediate mesons do not dominate. ð Þ We report here the observation of the decay D0 → K−πþeþe−[1] with data recorded with the BABAR detector at the PEP-II asymmetric-energy eþe−collider operated at the SLAC National Accelerator Laboratory. The data sample corresponds to 424 fb−1 of eþe−collisions col- lected at the center-of-mass energy of the ϒð4SÞ resonance (on peak) and an additional 44 fb−1 of data collected 40 MeV below the ϒð4SÞ resonance (off peak) [25]. The signal branching fraction BðD0 →K−πþeþe−Þ is measured relative to the normalization decay D0 →K−πþπþπ−. The D0 mesons are reconstructed from the decay Dþ →D0πþ produced in eþe−→c¯c events. The use of this decay chain increases the purity of the sample at the expense of a smaller number of reconstructed D0 mesons. Over the last few years there have been a number of measurements of the decays of B mesons to final states involving one or more charged leptons. Some of these measurements suggest a possible deviation from the assumption that all leptons couple equally [11–20]. The possibility therefore exists that a deviation from lepton universality will be seen in D meson decays. The BABAR detector is described in detail in Ref. [26]. Charged particles are reconstructed as tracks with a five- layer silicon vertex detector and a 40-layer drift chamber inside a 1.5 T solenoidal magnet. An electromagnetic calorimeter comprised of 6580 CsI(Tl) crystals is used 081802-3 PHYSICAL REVIEW LETTERS 122, 081802 (2019) masses of the Dþ and D0 candidates is required to satisfy 0.143 < Δm < 0.148 GeV=c2. The regions around the peak positions in mðD0Þ and Δm in data are kept hidden until the analysis steps are finalized. to identify electrons and photons. A ring-imaging Cherenkov detector is used to identify charged hadrons and to provide additional lepton identification information. Muons are identified with an instrumented magnetic-flux return. To reject misreconstructed D0 →K−πþeþe−candidates that originate from D0 hadronic decays with large branch- ing fractions where one or more charged tracks are misidentified by the PID the candidate is reconstructed assuming the kaon or pion mass hypothesis for the leptons. If the resulting candidate mðD0Þ is within 20 MeV=c2 of the known D0 mass [34] and jΔmj < 2 MeV=c2, the event is discarded. After these criteria are applied, the back- ground from these hadronic decays is negligible. DOI: 10.1103/PhysRevLett.122.081802 The electron-positron pair must have an invari- ant mass mðeþe−Þ > 0.1 GeV=c2. The D0 candidate momentum in the PEP-II center-of-mass system p must be greater than 2.4 GeV=c. The requirement for five charged tracks strongly suppresses backgrounds from QED processes. The p criterion removes most sources of combinatorial background and also charm hadrons produced in B decays, which are kinematically limited to less than ∼2.2 GeV=c. The D0 →K−πþeþe−branching fraction is determined relative to that of the normalization decay channel D0 → K−πþπþπ−using BðD0 →K−πþeþe−Þ BðD0 →K−πþπþπ−Þ ¼ ˆϵnorm Nnorm Lnorm Lsig X Nsig i 1 ϵi sig ; ð1Þ ð1Þ where BðD0 →K−πþπþπ−Þ is the branching fraction of the normalization mode [34], and Nnorm and ˆϵnorm are the D0 →K−πþπþπ−fitted yield and the reconstruction effi- ciency calculated from simulated D0 →K−πþπþπ− decays, respectively. The fitted D0 →K−πþeþe−signal yield is represented by Nsig, and ϵi sig is the reconstruction efficiency for each signal candidate i, calculated from MC simulated D0 →K−πþeþe−decays as a function of mðeþe−Þ and mðK−πþÞ. The symbols Lsig and Lnorm represent the integrated luminosities used for the signal D0 →K−πþeþe−decay (468.2 2.0 fb−1) and the nor- malization D0 →K−πþπþπ− decay (39.3 0.2 fb−1), respectively [25]. The signal mode uses both the on-peak and off-peak data samples while the normalization mode uses only a subset of the off-peak data. The candidate Dþ is formed by combining the D0 candidate with a charged pion with a momentum in the laboratory frame greater than 0.1 GeV=c. The pion is required to have a charge opposite to that of the kaon in the D0 decay. A vertex fit is performed with the D0 mass constrained to its known value and the requirement that the D0 meson and the pion originate from the interaction region. The χ2 probability of the fit is required to be greater than 0.005. The D0 meson mass mðD0Þ must be in the range 1.81 < mðD0Þ < 1.91 GeV=c2 and the mass differ- ence, Δm ¼ mðDþÞ −mðD0Þ, between the reconstructed The D0 →K−πþeþe−and D0 →K−πþπþπ−yields are determined from extended unbinned maximum likelihood fits to the Δm and the four-body mass distributions. DOI: 10.1103/PhysRevLett.122.081802 [34], except for the unknown BðD0 →K−πþηÞ, which is estimated to be ð1.8 0.9Þ% from the related decay D0 →K0 Sπ0η. After applying the selection criteria, we expect to find 0.3 0.2 eþe−γ background decays in the 0.675 < mðeþe−Þ < 0.875 GeV=c2 range. FIG. 1. Fits to D0 →K−πþeþe− data distributions for (a) mðK−πþeþe−Þ and (b) Δm mass for candidates with 0.675 < mðeþe−Þ < 0.875 GeV=c2. We test the performance of the maximum likelihood fit by generating ensembles of MC simulation pseudodata samples from both the PDF distributions and the fully simulated MC events. The mean number of signal, nor- malization, and background yields used in the ensembles is taken from the fits to the data sample. The yields are allowed to fluctuate according to a Poisson distribution and all fit parameters are allowed to vary. No significant bias is observed in the normalization mode. The largest fit bias observed in the signal mode is 0.4 0.1. The biases are much smaller than the statistical uncertainties in the yields. The fit biases are subtracted from the fitted yields before calculating the signal branching fractions. The fitted yield for the D0 →K−πþπþπ−normalization data sample is 260870 520. For the D0 →K−πþeþe− signal mode, the fitted yield, after the subtraction of the eþe−γ background, is 68 9 in the range 0.675 < mðeþe−Þ < 0.875 GeV=c2. The significance S ¼ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ −2Δ ln L p of the signal yield in this mass range, including statistical and systematic uncertainties, is 9.7 standard deviations (σ), where Δ ln L is the change in the log- likelihood from the maximum value to the value when the number of D0 →K−πþeþe−signal decays is set to Nsig ¼ 0. g Figure 1 shows the results of the fit to the mðK−πþeþe−Þ and Δm distributions of the D0 →K−πþeþe−signal mode in the mass range 0.675 < mðeþe−Þ < 0.875 GeV=c2. Figure 2 shows the projection of the fit to the D0 → K−πþeþe−signal mode as a function of mðeþe−Þ and mðK−πþÞ, where the background has been subtracted using the sPlot technique [37]. A peaking structure is visible in mðeþe−Þ centered near the ρ0 mass. A broader structure is seen in mðK−πþÞ near the known mass of the ¯Kð892Þ0 meson. Both distributions are similar to the distributions shown in Ref. [21] for the decay D0 →K−πþμþμ−. DOI: 10.1103/PhysRevLett.122.081802 The 081802-4 PHYSICAL REVIEW LETTERS 122, 081802 (2019) 1.82 1.84 1.86 1.88 1.9 ] 2 [GeV/c ) − e + e + π − m(K 5 10 15 20 ) 2 Candidates / (5.00 MeV/c Data Total Signal Background R A B A B (a) 0.143 0.144 0.145 0.146 0.147 0.148 ] 2 [GeV/c m Δ 5 10 15 20 ) 2 Candidates / (0.25 MeV/c Data Total Signal Background R A B A B (b) FIG. 1. Fits to D0 →K−πþeþe− data distributions for (a) mðK−πþeþe−Þ and (b) Δm mass for candidates with 0.675 < mðeþe−Þ < 0.875 GeV=c2. 1.82 1.84 1.86 1.88 1.9 ] 2 [GeV/c ) − e + e + π − m(K 5 10 15 20 ) 2 Candidates / (5.00 MeV/c Data Total Signal Background R A B A B (a) Δm and the four-body mass distributions are not correlated and are treated as independent observables in the fit. For the D0 →K−πþeþe−signal, a Gaussian-like function with different lower and upper widths is used for both Δm and mðK−πþeþe−Þ. This asymmetric function is used in order to describe the imperfect bremsstrahlung energy recovery for the electrons. The background in the D0 →K−πþeþe− channel is modeled with an ARGUS threshold function [35] for Δm and a first-order Chebyshev polynomial for mðK−πþeþe−Þ. For the D0 →K−πþπþπ−normalization mode, the Δm and mðK−πþπ−πþÞ distributions are each represented by two Cruijff functions with shared means [36]. The background is represented by an ARGUS thresh- old function for Δm and a second-order Chebyshev polynomial for mðK−πþπ−πþÞ. All yields and shape parameters are allowed to vary in the fits except for the ARGUS function threshold end point, which is set to the kinematic threshold for the Dþ →D0πþ decay. 0.143 0.144 0.145 0.146 0.147 0.148 ] 2 [GeV/c m Δ 5 10 15 20 ) 2 Candidates / (0.25 MeV/c Data Total Signal Background R A B A B (b) Decays of intermediate mesons to the final state eþe−γ can potentially appear in the mðeþe−Þ spectrum as the photon is not reconstructed. However, the constraint mðD0Þ > 1.81 GeV=c2 is effective in reducing the back- ground from these decays despite their relatively high branching fractions. We investigate the backgrounds by generating simulation samples D0 →K−πþV, with inter- mediate decays ρ0=ω=ϕ →eþe−and η=η0 →eþe−γ. In the simulations, QED radiative corrections are provided by PHOTOS [30]. The branching fractions are taken from Ref. DOI: 10.1103/PhysRevLett.122.081802 To cross-check the normalization procedure, the signal mode D0 →K−πþeþe−in Eq. (1) is replaced with the decay D0 →K−πþ, which has a well-known branching fraction [34]. The D0 →K−πþ decay is selected using the same criteria as used for the D0 →K−πþπþπ−mode, which is used as the normalization mode. The D0 → K−πþ yield is determined using an unbinned maximum likelihood fit to Δm and the two-body invariant mass mðK−πþÞ. Three Crystal Ball functions [38] with shared means are used for the D0 →K−πþ signal Δm and mðK−πþÞ distributions. The backgrounds are represented 081802-5 081802-5 PHYSICAL REVIEW LETTERS 122, 081802 (2019) 0.7 0.75 0.8 0.85 ] 2 [GeV/c ) − e + m(e 0 10 20 ) 2 Entries / (8.00 MeV/c R A B A B (a) 0.6 0.8 1 1.2 ] 2 [GeV/c ) + π − m(K 0 5 10 15 ) 2 Entries / (24.00 MeV/c R A B A B (b) FIG. 2. Projections of the fits to the D0 →K−πþeþe−data distributions onto (a) mðeþe−Þ and (b) mðK−πþÞ for candidates with 0.675 < mðeþe−Þ < 0.875 GeV=c2. The background has been subtracted using the sPlot technique [37]. 0.7 0.75 0.8 0.85 ] 2 [GeV/c ) − e + m(e 0 10 20 ) 2 Entries / (8.00 MeV/c R A B A B (a) the D0 →K−πþeþe−signal parameters for the Δm and four-body distributions fixed to values taken from simu- lation. Alternative fits are also performed with the default peaking and background functions for the signal and normalization modes replaced with alternative functions. The resulting uncertainties are 1.9% and 1.0% for the signal and normalization yields, respectively. In the mass range 0.675 < mðeþe−Þ < 0.875 GeV=c2, we replace the signal phase-space simulation model with a model assuming D0 →¯Kð892Þ0ρ0 with ¯Kð892Þ0 → K−πþ and ρ0 →eþe−and assign half the difference with the default reconstruction efficiency as a systematic uncer- tainty, equivalent to a relative change of 1.8%. We also use this number as an estimate of the relative change in other regions of mðeþe−Þ and mðK−πþÞ where no suitable alternative simulation model exists. DOI: 10.1103/PhysRevLett.122.081802 0.6 0.8 1 1.2 ] 2 [GeV/c ) + π − m(K 0 5 10 15 ) 2 Entries / (24.00 MeV/c R A B A B (b) The systematic uncertainty in the fit bias for the signal yield is taken from the ensemble of fits to the MC pseudodata samples and we attribute a value of half the largest fit bias found, 0.2. To account for imperfect knowledge of the tracking efficiency, we assign an uncer- tainty of 0.8% per track for the leptons and 0.7% for the kaon and pion [39]. For the PID, we estimate an uncertainty of 0.7% per electron, 0.2% per pion, and 1.1% per kaon [26]. A systematic uncertainty of 0.8% is associated with the knowledge of the luminosity ratio, Lnorm=Lsig [25]. g The overall systematic uncertainty in the yields is 5.3% for the signal and 3.6% for the normalization mode. As the PID and tracking systematic uncertainties of the kaons and pions are correlated and cancel, the combined systematic uncertainty in the D0 →K−πþeþe−branching fraction is 3.8%, where the uncertainty in the D0 →K−πþπþπ− branching fraction is excluded [34]. FIG. 2. Projections of the fits to the D0 →K−πþeþe−data distributions onto (a) mðeþe−Þ and (b) mðK−πþÞ for candidates with 0.675 < mðeþe−Þ < 0.875 GeV=c2. The background has been subtracted using the sPlot technique [37]. by an ARGUS function for Δm and a second-order Chebyshev polynomial for mðK−πþÞ. The D0 →K−πþ signal yield is 1881950 1380 with an average reconstruction efficiency of ˆϵsig ¼ ð27.4 0.2Þ%. We determine BðD0 →K−πþÞ ¼ ð3.98 0.08 0.10Þ% using Eq. (1), where the uncertainties are statistical and system- atic, respectively; the current world average is ð3.89 0.04Þ% [34]. Similar compatibility with the BðD0 → K−πþÞ world-average, but with larger uncertainties, is achieved when the normalization mode D0 → K−πþπþπ−in Eq. (1) is replaced with the four-body decay modes D0 →K−Kþπþπ−or D0 →π−πþπþπ−. by an ARGUS function for Δm and a second-order Chebyshev polynomial for mðK−πþÞ. The D0 →K−πþ signal yield is 1881950 1380 with an average reconstruction efficiency of ˆϵsig ¼ ð27.4 0.2Þ%. We determine BðD0 →K−πþÞ ¼ ð3.98 0.08 0.10Þ% using Eq. (1), where the uncertainties are statistical and system- atic, respectively; the current world average is ð3.89 0.04Þ% [34]. DOI: 10.1103/PhysRevLett.122.081802 ‡Present address: Universit`a di Bologna and INFN Sezione di Bologna, I-47921 Rimini, Italy. §Present address: University of Huddersfield, Huddersfield HD1 3DH, United Kingdom. ¶Present address: University of South Alabama, Mobile, Alabama 36688, USA. Also at: Universit`a di Sassari, I-07100 Sassari, Italy. 1] Ch j i i i li d h h We repeat the fit to Δm and mðK−πþeþe−Þ in the continuum mðeþe−Þ region that is predicted to be relatively unaffected by intermediates states, and is defined by excluding the following mðeþe−Þ mass ranges: mðeþe−Þ < 0.2 GeV=c2, 0.675 < mðeþe−Þ < 0.875 GeV=c2, 0.491 < mðeþe−Þ < 0.560 GeV=c2, 0.902<mðeþe−Þ<0.964GeV= c2, and 1.005 < mðeþe−Þ < 1.035 GeV=c2. These corre- spond to ranges dominated by the decays of the π0 and ρ0=ω mesons or potentially affected by the decays of η, η0, and ϕ mesons, respectively. Simulation samples of D0 → K−πþη and D0 →K−πþη0, with η=η0 →eþe−γ, are used to determine the asymmetric mðeþe−Þ mass ranges centered on the known η and η0 masses. These mðeþe−Þ mass ranges exclude 90% of any remaining simulated η and η0 candi- dates that pass the selection criteria. The number of background decays from intermediate states in the con- tinuum region is predicted to be 9.9 0.9, dominated by the decay ρ0=ω →eþe−with mðeþe−Þ less than 0.675 GeV=c2. The fitted yield in the continuum region, after the subtraction of this background, is 19 7, with a statistical significance S ¼ 2.6σ. This corresponds to a branching fraction ð1.6 0.6 0.7Þ × 10−6, where the second uncertainty is systematic and is dominated by our knowledge of the model parametrization. The result is not significant and we determine a 90% C.L. branching fraction upper limit of 3.1 × 10−6. Also at: Universit`a di Sassari, I-07100 Sassari, Italy. [1] Charge conjugation is implied throughout. [2] S. L. Glashow, J. Iliopoulos, and L. Maiani, Phys. Rev. D 2, 1285 (1970). [3] A. Paul, I. I. Bigi, and S. Recksiegel, Phys. Rev. D 83, 114006 (2011). [4] S. Fajfer, S. Prelovšek, and P. Singer, Phys. Rev. D 64, 114009 (2001). [5] S. Fajfer, S. Prelovšek, and P. Singer, Phys. Rev. D 58, 094038 (1998). [6] L. Cappiello, O. Cata, and G. D’Ambrosio, J. High Energy Phys. 04 (2013) 135. [7] A. Paul, A. de la Puente, and I. I. Bigi, Phys. Rev. D 90, 014035 (2014). [8] G. Burdman, E. Golowich, J. A. DOI: 10.1103/PhysRevLett.122.081802 Similar compatibility with the BðD0 → K−πþÞ world-average, but with larger uncertainties, is achieved when the normalization mode D0 → K−πþπþπ−in Eq. (1) is replaced with the four-body decay modes D0 →K−Kþπþπ−or D0 →π−πþπþπ−. The branching fraction BðD0 →K−πþeþe−Þ in the mass range 0.675 < mðeþe−Þ < 0.875 GeV=c2 is determined to be ð4.0 0.5 0.2 0.1Þ × 10−6, where the first uncer- tainty is statistical, the second systematic, and the third comes from the uncertainty in BðD0 →K−πþπþπ−Þ [34]. This result is compatible within the uncertainties with BðD0 →K−πþμþμ−Þ reported in Ref. [21]. ð Þ In the region 0.1 < mðeþe−Þ < 0.2 GeV=c2, the fitted signal yield is 175 14, with the distribution dominated by the decay D0 →K−πþπ0, π0 →eþe−γ, where the photon has not been reconstructed. Figure 3 shows the projection of the signal yield as a function of mðeþe−Þ for the fit to Δm and mðK−πþeþe−Þ in the mass range mðeþe−Þ > 0.2 GeV=c2 above the π0 → eþe−γ decay region, where the background has been subtracted using the sPlot technique. The main sources of systematic uncertainty are associ- ated with the model parametrizations used in the fits and the normalization procedure, signal MC model, fit bias, tracking and PID efficiencies, luminosity, backgrounds from intermediate decays to eþe−γ, and the normalization mode branching fraction. Some of the tracking and PID systematic effects cancel in the branching fraction deter- mination since they affect both the signal and normaliza- tion modes. We determine the signal yield in the region of the ϕ meson by repeating the fit to Δm and mðK−πþeþe−Þ with the mðeþe−Þ distribution restricted to the mass range 1.005 < mðeþe−Þ < 1.035 GeV=c2. This range corre- sponds to 3 times the ϕ mass width, based on simulation and taking into account the detector resolution. The fitted Systematic uncertainties associated with the model parametrization are estimated by repeating the fit with 081802-6 PHYSICAL REVIEW LETTERS 122, 081802 (2019) 0.5 1 ] 2 [GeV/c ) −e + m(e 0 10 20 ) 2 Entries / (10.00 MeV/c R A B A B η ω / 0 ρ ' η φ FIG. 3. Projection of the fits to the D0 →K−πþeþe−data distributions onto mðeþe−Þ for candidates with mðeþe−Þ > 0.2 GeV=c2. The background has been subtracted using the sPlot technique [37]. The shaded bands indicate the mðeþe−Þ regions excluded from the “continuum” region. DOI: 10.1103/PhysRevLett.122.081802 0.5 1 ] 2 [GeV/c ) −e + m(e 0 10 20 ) 2 Entries / (10.00 MeV/c R A B A B η ω / 0 ρ ' η φ ð4.0 0.5 0.2 0.1Þ × 10−6, compatible with the result for BðD0 →K−πþμþμ−Þ [21], and with theoretical pre- dictions for the SM contribution [6] for this mass region. We have placed 90% C.L. branching fraction upper limits on the decay D0 →K−πþeþe−in the mðeþe−Þ mass region of the ϕ meson and in mðeþe−Þ mass regions where long- distance effects are potentially small. ð4.0 0.5 0.2 0.1Þ × 10−6, compatible with the result for BðD0 →K−πþμþμ−Þ [21], and with theoretical pre- dictions for the SM contribution [6] for this mass region. We have placed 90% C.L. branching fraction upper limits on the decay D0 →K−πþeþe−in the mðeþe−Þ mass region of the ϕ meson and in mðeþe−Þ mass regions where long- distance effects are potentially small. We are grateful for the excellent luminosity and machine conditions provided by our PEP-II colleagues, and for the substantial dedicated effort from the computing organiza- tions that support BABAR. The collaborating institutions wish to thank SLAC for its support and kind hospitality. This work is supported by DOE and NSF (USA), NSERC (Canada), CEA and CNRS-IN2P3 (France), BMBF and DFG (Germany), INFN (Italy), FOM (Netherlands), NFR (Norway), MES (Russia), MINECO (Spain), STFC (United Kingdom), BSF (USA-Israel). Individuals have received support from the Marie Curie EIF (European Union) and the A. P. Sloan Foundation (USA). 0.5 1 ] 2 [GeV/c ) −e + m(e 1 ] 2 [GeV/c ) −e + m(e FIG. 3. Projection of the fits to the D0 →K−πþeþe−data distributions onto mðeþe−Þ for candidates with mðeþe−Þ > 0.2 GeV=c2. The background has been subtracted using the sPlot technique [37]. The shaded bands indicate the mðeþe−Þ regions excluded from the “continuum” region. yield is 3.8þ2.7 −1.9, where the uncertainty is statistical only; the statistical significance S is 1.8σ. The branching fraction is determined to be ð2.2þ1.5 −1.1 0.6Þ × 10−7, where the second uncertainty is systematic and is dominated by the uncer- tainty on the model parametrization. We use the frequentist approach of Feldman and Cousins [40] to determine a 90% C.L. branching fraction upper limit of 0.5 × 10−6. *Deceased. †Present address: Wuhan University, Wuhan 430072, China. PHYSICAL REVIEW LETTERS 122, 081802 (2019) PHYSICAL REVIEW LETTERS 122, 081802 (2019) [28] B. F. L. Ward, S. Jadach, and Z. Was, Nucl. Phys. B, Proc. Suppl. 116, 73 (2003). [15] R. Aaij et al. (LHCb Collaboration), Phys. Rev. Lett. 113, 151601 (2014). pp ( ) [29] T. Sjöstrand, Comput. Phys. Commun. 82, 74 (1994). [16] R. Aaij et al. (LHCb Collaboration), Phys. Rev. Lett. 115, 111803 (2015). [30] P. Golonka and Z. Was, Eur. 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https://openalex.org/W2323751316	https://www.scielo.br/j/bdj/a/x56sbXyjVHDLHxy8SZfB8bn/?lang=en&format=pdf	English	null	Study of Anatomical Relationship between Posterior Teeth and Maxillary Sinus Floor in a Subpopulation of the Brazilian Central Region Using Cone-Beam Computed Tomography - Part 2	Brazilian Dental Journal	2,016	cc-by	6,601	Brazilian Dental Journal (2016) 27(1): 9-15 http://dx.doi.org/10.1590/0103-6440201600679 Brazilian Dental Journal (2016) 27(1): 9-15 http://dx.doi.org/10.1590/0103-6440201600679 ISSN 0103-6440 1Department of Stomatologic Sciences, Dental School, UFG - Universidade Federal de Goiás, Goiânia, GO, Brazil 2Department of Oral Sciences, Dental School, UNIC - Universidade de Cuiabá, Cuiabá, MT, Brazil 3Department of Restorative Dentistry, Dental School, USP - Universidade de São Paulo, Ribeirão Preto, SP, Brazil Correspondence: Professor Carlos Estrela, Praça Universitária s/n, Setor Universitário, 74605-220 Goiânia, GO, Brasil. Tel: +55-62-3209-6254. e-mail: estrela3@terra.com.br Study of Anatomical Relationship between Posterior Teeth and Maxillary Sinus Floor in a Subpopulation of the Brazilian Central Region Using Cone- Beam Computed Tomography – Part 2 Carlos Estrela1, Carla A. B. C. M. Nunes1, Orlando Aguirre Guedes2, Ana Helena G. Alencar1, Cynthia R. A. Estrela1, Ricardo Gariba Silva3, Jesus Djalma Pécora3, Manoel Damião Sousa-Neto3 This study evaluated the anatomical relationship between posterior teeth root apices and maxillary sinus floor (MSF) on 202 cone beam computed tomography (CBCT) exams. The distance between the root apices and the MSF, as well as the MSF thickness of the cortical bone closest to root apices and furcation regions were measured. The vertical and horizontal relationships of the MSF with the molar roots were classified into categories adapted from the criteria proposed by Kwak et al. (14). The shortest distances between MSF and the root apices were observed in the mesiobuccal root of the second molar (0.36±1.17 mm) and the palatal root of the first molar (0.45±1.10 mm) and the widest in buccal roots of the first premolars (5.47±4.43 mm). Significant differences were observed between the distance of MSF to the root apices of single-rooted first and second premolars. The cortical thickness ranged from 0.65±0.41 mm over the mesiobuccal root of the second molar to 1.28±0.42 mm over the buccal root of the first premolar. The most observed vertical and horizontal relationships were type II and 2H, respectively. The maxillary molar roots showed greater proximity to the MSF. The thickness of the MSF cortical bone closest to the apices and furcation regions was found to be similar only for premolars. Key Words: anatomy, maxillary sinus floor, maxillary sinusitis, periapical lesion, cone beam computed tomography. 1Department of Stomatologic Sciences, Dental School, UFG - Universidade Federal de Goiás, Goiânia, GO, Brazil 2Department of Oral Sciences, Dental School, UNIC - Universidade de Cuiabá, Cuiabá, MT, Brazil 3Department of Restorative Dentistry, Dental School, USP - Universidade de São Paulo, Ribeirão Preto, SP, Brazil Material and Methods Study Sample For the premolars, the following items were measured: SR: the distance between the apex of single-rooted teeth and the inferior wall of the MSF; BR: the distance between the apex of the buccal root and the inferior wall of the MSF; PR: the distance between the apex of the palatal root and the inferior wall of the MSF; CTSR: the cortical thickness of the inferior wall of the MSF nearest to the apex of single-rooted tooth; CTBR: the cortical thickness of the inferior wall of the MSF closest to the apex of the buccal root; CTPR: the cortical thickness of the inferior wall of the MSF nearest to the apex of the palatal root; CTF: the cortical thickness of the inferior wall of the MSF closest to the furcation area (Figs. 1A and 1B). The present study was performed as a retrospective analysis of CBCT exams selected from the database of a private radiologic center (TCO, Goiânia, GO, Brazil). The inclusion criteria were CBCT exams of the maxilla presenting fully erupted first and second premolars and first and second molars with fully formed apices. Excluded from the study sample were exams presenting image of a device or apparatus of orthodontic retention and presence of external resorption of the root apex, apical periodontitis, bone changes associated with systemic diseases and benign and/or malignant tumors in the posterior area of the maxilla and/or MS. Two hundred and two CBCT exams met the inclusion criteria and were included in this study. Among the selected participants, 128 were females (63.37%) and 74 were males (36.63%), with a mean age of 40.7 years (range: 15-80 years). One thousand and two-hundred maxillary posterior teeth were evaluated (300 first premolars, 300 second premolars, 300 first molars and 300 second molars). Two hundred and sixty-six premolars were single-rooted and 334 were bi-rooted. All molars were tri-rooted teeth. The protocol for the study was approved by the Research Ethical Committee of the Federal University of Goiás (Process number 391.886). Introduction (1-3,8,9). Periapical radiographs were unable to determine the risk of perforation of the maxillary sinus floor (MSF) during periapical surgery (8). The limitation resulting from the two-dimensional images prevents the correct interpretation of the periapical lesions relationship with the MSF (9). Periapical and panoramic radiographs offer little accuracy to the morphometric analysis of the relationship of bone structures with teeth (10). The clinical application of cone beam computed tomography (CBCT) as an aid in the diagnosis and planning has contributed to establish effective therapeutic protocols (11-13). The importance of CBCT scans in the analysis of the morphological characteristics of the MS and its relationship with the roots of the maxillary posterior teeth has been shown (10,14-19). Infection of the root canal system may spread through the periapical tissues and reach important anatomical structures resulting in several complications. The anatomical proximity of the root apices of the maxillary posterior teeth to the maxillary sinus floor (MSF) may favor the development of inflammatory, infectious and/or traumatic alterations in the maxillary sinus (MS) (1-4). In addition, operative procedural errors during root canal therapy (overinstrumentation, overirrigation and overfilling) and aggressive surgical procedures represent potential risk factors for introduction of foreign material into the MS (5). The diagnosis of sinus disease of odontogenic origin is not simple, confounding both patient and the medical and dental professionals (6). The most common causes of odontogenic sinus disease are dental abscesses and periodontal disease that perforated the Schneiderian membrane, and irritation and secondary infection promoted by intra-antral foreign bodies and sinus perforation during tooth extraction (7). The biological constitution of different populations has a variety of genetic characters, which can determine distinct anatomical and topographical relationships. The anatomical knowledge of the structures that compose the middle and lower thirds of the face, especially the MS and its relation with posterior teeth, is of utmost importance not only for the accurate diagnosis of inflammatory alterations that may be established in both the MS and periapical region, but also for the correct establishment of therapeutic, surgical and rehabilitation plans. Thus, the aim of this study was Conventional radiographic exams are commonly used in the study of the anatomical relationship between maxillary posterior teeth and the MS. Introduction However, these exams have limitations that may jeopardize this analysis Braz Dent J 27(1) 2016 to evaluate the anatomical relationship between maxillary posterior teeth root apices and the MSF in a subpopulation of the Brazilian central region by CBCT images. between the inferior wall of the MSF and the root apices of the posterior teeth and the MSF cortical thickness in the region closest to the root apices and in the furcation areas. For the measurements, a specific tool of the I-CAT program was used, and the measurements were performed on the cross-sectional images with slice thickness of 1 mm. C. Estrela et al. The protocol for the study was approved by the Research Ethical Committee of the Federal University of Goiás (Process number 391.886). Material and Methods Study Sample For the molars, the following items were measured: MBR: the distance between the apex of the mesiobuccal root and the inferior wall of the MSF; DBR: the distance between the apex of the distobuccal root and the inferior wall of the MSF; PR: the distance between the apex of the palatal root and the inferior wall of the MSF; CTMBR: the cortical thickness of the inferior wall of the MSF nearest to the apex of the mesiobuccal root; CTDBR: the cortical thickness of the inferior wall of the MSF closest to the apex of the distobuccal root; CTPR: the cortical thickness of the inferior wall of the MSF nearest to the apex of the palatal root; CTF - the cortical thickness of the inferior wall of the MSF closest to the furcation area (Figs. 1C-E). C. Estrela et al. CBCT Image Acquisition and Analysis g q y All CBCT images were acquired using the I-CAT Cone Beam 3D imaging system (Imaging Sciences International, Hatfield, PA, USA) using a 16 cm x 6 cm field of view (FOV). Image volume was reconstructed with isotropic-isometric 0.25 x 0.25 x 0.25 mm voxels. The tube voltage was 120 KVp, tube current was 3.8 mA, and an exposure time of 40 s was used. The images in DICOM format were processed, interpreted and measured by the proprietary software of the CBCT machine (Xoran version 3.1.62; Xoran Technologies, Ann Arbor, MI, USA). The PC workstation used Windows® 7 professional 32-bit with XP Mode operating system (Microsoft Corporation, Redmond, WA, USA) with 2nd Generation Intel® CoreTM i5-2400, 3.1 GHz up to 3.4 GHz with Intel Turbo Boost 2.0, 4 Threads 6 MB Cache (Intel Corporation, USA), card video nVidia GeForce GT610 1 GB, 64-bit (NVIDIA Corporation, USA) and Dell monitor E2211H 21.5 inches – Widescreen resolution of 1920 x 1080 pixels (Dell Corporation, Round Rock, Texas, USA). The vertical relationship between the MSF and the roots of the maxillary molars was evaluated on the CBCT cross-sectional images and classified into five categories according to the criteria described by Kwak et al. (14): Type I: the MSF was located above the level connecting the buccal and palatal root apices; Type II: the MSF was located below the level connecting the buccal and palatal root apices, without an apical protrusion over the MSF; Type III: an apical protrusion of the buccal root apex was observed over the MSF; Type IV: an apical protrusion of the palatal root apex was observed over the MSF; Type V: apical protrusions of the buccal and palatal root apices were observed over the MSF (Figs. 2A-E). The horizontal relationship between the MSF and the roots of the maxillary molars was evaluated on the CBCT cross-sectional images and classified into five categories adapted from the criteria proposed by Kwak et al. (14): Type 1H: the alveolar recess of the MSF was located more towards the buccal side than towards the buccal root; Type 2H: the alveolar recess of the MSF was located between the buccal The anatomical relationship between MSF and maxillary posterior teeth was evaluated by measuring the distances 10 Braz Dent J 27(1) 2016 Figure 1. CBCT Image Acquisition and Analysis A,B: CBCT cross-sections of maxillary premolars with measurements (mm) of the distance between the maxillary sinus floor and the root apices and the maxillary sinus floor cortical thickness in the region closest to the root apices and in the furcation. A: SR and CTSR in single-rooted; B: BR, PR, CTBR, CTPR and CTF bi-rooted. C-E: CBCT cross-sections of maxillary molars with measurements (mm) of the distance between the maxillary sinus floor and the root apices and the maxillary sinus floor cortical thickness in the region closest to the root apices and in the furcation. C: MBR and CTMBR; D: DBR, CTDBR and CTF; E: PR and CTPR. Figure 1. A,B: CBCT cross-sections of maxillary premolars with measurements (mm) of the distance between the maxillary sinus floor and the root apices and the maxillary sinus floor cortical thickness in the region closest to the root apices and in the furcation. A: SR and CTSR in single-rooted; B: BR, PR, CTBR, CTPR and CTF bi-rooted. C-E: CBCT cross-sections of maxillary molars with measurements (mm) of the distance between the maxillary sinus floor and the root apices and the maxillary sinus floor cortical thickness in the region closest to the root apices and in the furcation. C: MBR and CTMBR; D: DBR, CTDBR and CTF; E: PR and CTPR. Figure 1. A,B: CBCT cross-sections of maxillary premolars with measurements (mm) of the distance between the maxillary sinus floor and the root apices and the maxillary sinus floor cortical thickness in the region closest to the root apices and in the furcation. A: SR and CTSR in single-rooted; B: BR, PR, CTBR, CTPR and CTF bi-rooted. C-E: CBCT cross-sections of maxillary molars with measurements (mm) of the distance between the maxillary sinus floor and the root apices and the maxillary sinus floor cortical thickness in the region closest to the root apices and in the furcation. C: MBR and CTMBR; D: DBR, CTDBR and CTF; E: PR and CTPR. 11 Figure 2. CBCT images. Vertical relationship. A: Type I; B: Type II; C: Type III; D: Type IV; E: Type V. Horizontal relationship. F: Type 1H, G: Type 2H, H: Type 3H, I: Type 4H, J: Type 5H. Adapted from Kwak et al. 2004 (14). Figure 2. CBCT images. Vertical relationship. A: Type I; B: Type II; C: Type III; D: Type IV; E: Type V. Horizontal relationship. CBCT Image Acquisition and Analysis F: Type 1H, G: Type 2H, H: Type 3H, I: Type 4H, J: Type 5H. Adapted from Kwak et al. 2004 (14). 11 Braz Dent J 27(1) 2016 and palatal roots; Type 3H: the alveolar recess of the MSF was located more towards the palatal side than towards the palatal root; Type 4H: the alveolar recess of the MSF passes over the roots without establishing relationship with them; Type 5H: the alveolar recess of the MSF is located towards the buccal side and towards the palatal side, and may or may not also extend between the roots (Figs. 2F-J). premolars (p<0.05). The shortest distance was observed for single-rooted second premolar (1.71±2.81 mm) (Table 1). No significant difference was observed between the distance of MSF to the buccal and palatal root apices of bi-rooted first and second premolars (p>0.05). With regards to the molars, the greatest proximity was observed in the mesiobuccal root of the second left (0.36±1.17 mm) and second right (0.44±1.07 mm) molars and the palatal root of the first left molar (0.45±1.10 mm) (Table 2). All measurements and analyzes were performed by two oral and maxillofacial radiologists, with experience in interpreting CBCT exams. The examiners were trained and calibrated using 10% of the sample in a pilot study. In absence of consensus, a third examiner, with the same qualification (oral and maxillofacial radiologist), was called for a final decision. The mean and standard deviation values (mm) of the MSF cortical thickness in the region of the root apices and the furcation area of the maxillary premolars and molars are shown in Tables 3 and 4. The cortical thickness of the MSF inferior wall nearest to the root apices ranged from 0.65±0.41 mm over the mesiobuccal root of the second molar to 1.28±0.42 mm over the buccal root of the first premolar. A statistically significant difference was observed between the cortical thickness of the MSF inferior wall and the root apices of single-rooted first and second premolars. Considering individually each premolar, no statistically significant difference was observed in the mean value of the cortical thickness of the inferior wall of the MSF nearest to the root apex (single, buccal and palatal roots) and the furcation area in all premolars (Table 3). With regards to the molars, significant differences were observed regarding only the cortical bone thickness over the mesiobuccal and distobuccal roots (p>0.05). CBCT Image Acquisition and Analysis Considering each molar individually, statistically significant differences were observed in the mean value of the cortical thickness of the MSF inferior wall nearest to the root apex (mesiobuccal, distobuccal and palatal) and the furcation area in all molars (Table 4). C. Estrela et al. C. Estrela et al. Statistical Analysis The mean and standard deviation of the distances between the root apices and the MSF; and the thickness of the MS cortical bone were obtained. The differences between the distances, as well as between the thicknesses were evaluated by Kruskal-Wallis test. The statistical differences between the types of vertical and horizontal relationships were evaluated by Chi-square test. All statistical analyses were carried out with the Statistical Package for Social Sciences (IBM SPSS 20, IBM Co., New York, NY, USA). Results The mean and standard deviation values (mm) of the distances between the root apices of the maxillary premolars and molars and MSF are shown in Tables 1 and 2. CBCT analysis revealed that the mean value of the distance from the root apices to the MSF ranged from 0.36±1.17 mm for the mesiobuccal root of the second molar to 5.47±4.43 mm for the buccal root of the first premolar. A statistically significant difference was obtained between the distance of root apices to the MSF of single-rooted first and second The frequency distributions of the vertical and horizontal relationships between MSF and roots of maxillary molars are shown in Tables 5 and 6. The most frequent vertical and horizontal relationships were types II and 2H, respectively (p<0.05). Table 2. The mean distances values in mm (SD) between the maxillary molar root apices and MSF Tooth n MBR (X ± SD) DBR (X ± SD) PR (X ± SD) 16 150 0.96 ± 1.79A, b 0.97 ± 1.87A, a 0.79 ± 1.58A, ab 17 150 0.44 ± 1.07A, a 0.74 ± 1.52AB, a 1.00 ± 1.72B, b 26 150 0.75 ± 1.43A, ab 0.66 ± 1.21AB, a 0.45 ± 1.10B, a 27 150 0.36 ± 1.17A, a 0.62 ± 1.53AB, a 0.73 ± 1.63B, ab n = number of teeth; X = mean; SD = standard deviation. Different capital letters indicate significant differences in horizontal lines and different lowercase letters indicate significant differences in the vertical lines; p <0.05 (Kruskal-Wallis); MBR: mesiobuccal root, DBR: distobuccal root, PR: palatal root. Table 1. The mean distances and standard deviation values in mm between the maxillary premolar root apices and MSF Tooth N SR (X ± SD) BR (X ± SD) PR (X ± SD) 14 150 4.25 ± 4.52A, b 5.12 ± 4.14A, b 4.89 ± 4.45A, b 15 150 1.80 ± 2.86A, a 3.19 ± 3.68A, a 2.20 ± 2.90A, a 24 150 4.98 ± 4.97A, b 5.47 ± 4.43A, b 4.39 ± 4.59A, ab 25 150 1.71 ± 2.81A, a 3.31 ± 4.90A, a 2.65 ± 4.36A, ab n = number of teeth; X = mean; SD = standard deviation. Different capital letters indicate significant differences in horizontal lines and different lowercase letters indicate significant differences in the vertical lines; p <0.05 (Kruskal-Wallis); SR: single root, BR: buccal root, PR: palatal root. Table 2. Discussion Jung & Cho (23) analyzed the relationship of the maxillary molars and adjacent structures by CBCT. The authors performed measurements on a sample of 83 patients/332 molars and found that the shortest distance between the root apex and the MS was in the MB root of the second molar. Pagin et al. (22) evaluated qualitatively the close relationship between the MSF and the root apices of the posterior teeth in a Brazilian population by CBCT images. Their sample was composed by 100 MS, 315 teeth, and 601 root apices. Close proximity was observed in 216 roots. Among them, 130 presented root apices in close contact with the MSF with no root protrusion within the MS and no elevation in the sinus floor trajectory. The opposite was observed in 86 roots. The mesiobuccal root of the second molar was frequently found in close proximity to the MSF. With regards to the largest distances, the results of the present study showed that the root apices of first premolars are frequently far away from the MSF, which agrees with the study conducted by Kilic et al. (21). loor e V 67%) 67%) 33%) 33%) 00%) n = number of teeth; X = mean; SD = standard deviation. Different capital letters indicate significant differences in horizontal lines and different lowercase letters indicate significant differences in the vertical lines; p <0.05 (Kruskal-Wallis); CTSR: cortical thickness single root, CTBR: cortical thickness buccal root, CTPR: cortical thickness palatal root, CTF: cortical thickness furcation. n = number of teeth; X = mean; SD = standard deviation. Different capital letters indicate significant differences in horizontal lines and different lowercase letters indicate significant differences in the vertical lines; p <0.05 (Kruskal-Wallis); CTSR: cortical thickness single root, CTBR: cortical thickness buccal root, CTPR: cortical thickness palatal root, CTF: cortical thickness furcation. Table 4. Results The mean distances values in mm (SD) between the maxillary molar root apices and MSF Table 2. The mean distances values in mm (SD) between the maxillary molar root apices and MSF Table 1. The mean distances and standard deviation values in mm between the maxillary premolar root apices and MSF Table 1. The mean distances and standard deviation values in mm between the maxillary premolar root apices and MSF 12 Braz Dent J 27(1) 2016 Braz Dent J 27(1) 2016 Table 3. The mean cortical thickness values in mm (SD) of the maxillary sinus floor in the region of root apices and the furcation area of the maxillary premolars Discussion Molar roots compared to the premolars showed a closer relationship with the MSF. The shortest distance between the root apex and the MSF was observed for the mesiobuccal root of the second left molar. Tooth CTSR (X ± SD) CTBR (X ± SD) CTPR (X ± SD) CTF (X ± SD) 14 1.26 ± 0.37A,b 1.28 ± 0.42A,b 1.15 ± 0.47A,b 1.18 ± 0.33A,b 15 0.92 ± 0.47A,a 1.01 ± 0.52A,a 0.92 ± 0.53A,ab 1.00 ± 0.52A,ab 24 1.17 ± 0.51A,b 1.14 ± 0.34A,ab 1.13 ± 0.47A,b 1.13 ± 0.31A,b 25 0.96 ± 0.49A,a 0.94 ± 0.41A,a 0.77 ± 0.49A,a 0.88 ± 0.35A,a n = number of teeth; X = mean; SD = standard deviation. Different capital letters indicate significant differences in horizontal lines and different lowercase letters indicate significant differences in the vertical lines; p <0.05 (Kruskal-Wallis); CTSR: cortical thickness single root, CTBR: cortical thickness buccal root, CTPR: cortical thickness palatal root, CTF: cortical thickness furcation. The results of the present study are in agreement with those from previous studies that have used computed tomography (CT) (10,14) and CBCT images (20-22). Eberhardt et al. (10) measured the distance between the root apices of posterior teeth and the MS using CT in 38 patients (12 specimens) and obtained results similar to the present study. Jung & Cho (23) analyzed the relationship of the maxillary molars and adjacent structures by CBCT. The authors performed measurements on a sample of 83 patients/332 molars and found that the shortest distance between the root apex and the MS was in the MB root of the second molar. Pagin et al. (22) evaluated qualitatively the close relationship between the MSF and the root apices of the posterior teeth in a Brazilian population by CBCT images. Their sample was composed by 100 MS, 315 teeth, and 601 root apices. Close proximity was observed in 216 roots. Among them, 130 presented root apices in close contact with the MSF with no root protrusion within the The results of the present study are in agreement with those from previous studies that have used computed tomography (CT) (10,14) and CBCT images (20-22). Eberhardt et al. (10) measured the distance between the root apices of posterior teeth and the MS using CT in 38 patients (12 specimens) and obtained results similar to the present study. Discussion The mean cortical thickness values in mm (SD) of the maxillary sinus floor in the region of root apices and the furcation area of the maxillary molars floor in the region of root apices and the furcation area of the maxillary molars Tooth CTMBR (X ± SD) CTDBR (X ± SD) CTPR (X ± SD) CTF (X ± SD) 16 0.88 ± 0.45AB, b 0.85 ± 0.45AB, b 0.78 ± 0.43A, a 0.96 ± 0.22B, a 17 0.71 ± 0.42A, a 0.76 ± 0.42AB, ab 0.81 ± 0.38B, a 0.98 ± 0.27C, a 26 0.85 ± 0.48A, b 0.75 ± 0.39AB, ab 0.72 ±0.43B, a 0.95 ± 0.24AC, a 27 0.65 ± 0.41A, a 0.72 ± 0.45AB, a 0.77 ± 0.46B, a 0.95 ± 0.25C, a n = number of teeth; X = mean; SD = standard deviation. Different capital letters indicate significant differences in horizontal lines and different lowercase letters indicate significant differences in the vertical lines. p <0.05 (Kruskal-Wallis). CTMBR: cortical thickness mesiobuccal root, CTDBR: cortical thickness distobuccal root, CTPR: cortical thickness palatal root, CTF: cortical thickness furcation. Posterior teeth and maxillary sinus n = number of teeth; X = mean; SD = standard deviation. Different capital letters indicate significant differences in horizontal lines and different lowercase letters indicate significant differences in the vertical lines. p <0.05 (Kruskal-Wallis). CTMBR: cortical thickness mesiobuccal root, CTDBR: cortical thickness distobuccal root, CTPR: cortical thickness palatal root, CTF: cortical thickness furcation. n = number of teeth; X = mean; SD = standard deviation. Different capital letters indicate significant differences in horizontal lines and different lowercase letters indicate significant differences in the vertical lines. p <0.05 (Kruskal-Wallis). CTMBR: cortical thickness mesiobuccal root, CTDBR: cortical thickness distobuccal root, CTPR: cortical thickness palatal root, CTF: cortical thickness furcation. Table 5. The vertical relationship between maxillary molar roots and maxillary sinus floor Tooth n Type I Type II Type III Type IV Type V 16 150 39 (26.00%) 67 (44.67%) 12 (8.00%) 25 (16.57%) 7 (4.67%) 17 150 47 (31.33%) 43 (28.67%) 41 (27.33%) 12 (8.00%) 7 (4.67%) 26 150 27 (18.00%) 82 (54.67%) 10 (6.67%) 23 (15.33%) 8 (5.33%) 27 150 38 (25.33%) 52 (34.67%) 37 (24.67%) 15 (10.00%) 8 (5.33%) Total 600 151 (25.16%) 244 (40.67%) 100 (16.67%) 75 (12.50%) 30 (5.00%) n = number of teeth; Chi-square test (p =0.05). Other studies (14,20,21) showed different results from those observed in this study. Kwak et al. (14) analyzed the clinical and morphological features of the MS in a Korean population using CT. The shortest distance between the root apex and the MSF was observed in the distobuccal root of the second molar. Kilic et al. (21) also found a shorter distance between the root apex of distobuccal root of the second molar and the MS, after analyzing 87 right and 89 left MS of Table 6. The horizontal relationship between maxillary molar roots and MSF Tooth n Type 1H Type 2H Type 3H Type 4H Type 5H 16 150 7 (4.67%) 93 (62.00%) 9 (6.00%) 39 (26.00%) 2 (1.33%) 17 150 20 (13.33%) 78 (52.00%) 7 (4.67%) 44 (29.33%) 1 (0.67%) 26 150 7 (4.67%) 106 (70.67%) 8 (5.33%) 29 (19.33%) 0 (0.00%) 27 150 30 (20.00%) 78 (52.00%) 5 (3.33%) 36 (24.00%) 1 (0.67%) Total 600 64 (10.67%) 355 (59.17%) 29 (4.83%) 148 (24.67%) 4 (0.67%) n = number of teeth; Chi-square test (p = 0.00). 13 13 Braz Dent J 27(1) 2016 92 patients using CBCT. Yoshimine et al. Posterior teeth and maxillary sinus (20) analyzed the anatomical characteristics of premolars, molars and MS for planning of dental implant treatment in 30 patients (120 teeth). The shortest distance was found for the palatal root of the first molar. The present results showed that after the mesiobuccal root of the second molar, the root with greater proximity to the MS was the palatal root of the first molar (p>0.05). These results highlight the care that is required in case of periapical surgery involving this area. Maillet et al. (24) noted that odontogenic sinus disease is frequent in patients presenting apical periodontitis in the palatal root of first molar and mesiobuccal root of second molar. the region of the greatest cortical thickness closer to the apex (first premolars), and the region of shortest distance between the apex and the MSF coincided with the lowest cortical thickness closer to the apex (second molars). This could be an indication of a greater chance of spreading infections of dental origin to the MS in the second molars region. Further studies with specific criteria for sample selection are likely to corroborate this hypothesis. All first and second molars (600 teeth) had classified their vertical and horizontal relationship with the MSF. The vertical relationship Type II (MSF located below the level connecting the buccal and palatal root apices without an apical protrusion over the MSF) was the most common. However, contrary to the present results, the vertical relationship most observed in the studies developed by Kwak et al. (14) (Korea) and Kilic et al. (21) (Turkey) were type I (MSF located above the level connecting the buccal and palatal root apices). The difference between these studies may be attributed not only to methodological differences, but also to the characteristics of ethnicity, since the analyzed populations were diverse. A common feature to the I and II types is the absence of protrusion of the roots into the sinus floor. The absence of projection of the roots into the sinus was also observed with high prevalence in other studies (10,15,22). However, Jung and Cho (23) projection of the roots into the MS was the most commonly observed vertical relationship. C. Estrela et al. Posterior teeth and maxillary sinus Some of the studies that analyzed the relationship of the roots of the maxillary posterior teeth with the MS used as reference the presence of protrusion of the roots in MS and assigned negative values for the distance between the apex and MSF, considering the lower portion of the alveolar recess adjacent to the protrusion (21,23). In this study, it was considered that even with the protrusion of the roots into the MS the presence of cortical bone and the mucosa overlying the MSF must be investigated. So, this measure was considered as 0.00 mm when the apex had contact with the floor and also when there was a root protrusion into the MS. For further research, was measured the thickness of the cortical bone of the sinus floor in the region closest to the apex and in the furcation area. Kwak et al. (14) also made measurements of the MSF cortical bone thickness in nearby regions of the root apex and furcation area and pointed out that the thickness of the MSF cortical bone and its relationship to the adjacent teeth is important in determining the prognosis of the orthodontic tooth movement. According to these authors, this information can provide a more appropriate basis for controlling orthodontic tooth movement and forecasting the degree of tooth movement during orthodontic procedures. Yoshmine et al. (20), in a study of the topography of the upper posterior teeth and the MS using CBCT, considered important to know the thickness of the MS cortical bone, in the closest region to the buccal root apex of the posterior teeth. This knowledge helps in the planning of dental implants and obtaining successful aesthetic treatment. In this study, the greatest cortical thickness of the MS floor was found in the region of the first premolar (1.13±0.62 mm) and the smallest in the region of the first molars (0.82±0.28 mm). A similar result was obtained in the present study in which the greatest thickness of the cortical bone of the MS floor was observed in the region of the first premolar apex (1.28±0.42 mm) and the smallest in the region of the second molars apex (0.65±0.41 mm). Although there was no statistically significant difference for these results in this study, it is interesting to note that the region of greatest distance between the apex and the MSF coincided with C. Estrela et al. The type 2H horizontal relationship (alveolar recess of the MSF located between the buccal and palatal roots) was the most frequent. These results are in agreement with the of previous studies (14,23). The Type 1H (alveolar recess of the MSF located towards the buccal side rather than towards the buccal root), showed a higher frequency in the second molars than first molars, agreeing with Jung and Cho (23), and contrasting the results of Kwak et al. (14). The presence of vertical Type II and horizontal Type 2H relationships may contribute to a rapid dissemination of odontogenic infectious processes to the MS and still provide the alveolar extension post extraction, which may jeopardize future rehabilitation by dental implants. Considering the spread of infections originating from maxillary teeth, Obayashi et al. (4) observed that 65.7% of the analyzed cases showed alterations in alveolar cortical bone involving these teeth, the buccal cortical bone being the most affected. Despite these changes being most evident in the anterior teeth, 59% of the analyzed first molars and 42% of second molars showed infection spreading to these cortical plates. Thus, depending on the type of the horizontal relationship, a greater possibility of MS alterations could be present in cases of extensions towards the buccal and palatal sides (Types 1H, 3H, 5H), due to the spread of odontogenic infection. 14 Braz Dent J 27(1) 2016 JD. Characterization of successful root canal treatment. Braz Dent J 2014;25:3-11. JD. Characterization of successful root canal treatment. Braz Dent J 2014;25:3-11. Rational application of CBCT for the evaluation of different aspects involved in an infectious process between the posterior teeth and the MS, and analysis of periapical lesions has favored a better treatment plan, accurate diagnosis compared to conventional radiography and consequently a better therapeutic option (9,13,25). All relationships between teeth and adjacent anatomical structures that may serve to ease spreading an infection should be well studied, especially considering the different features between populations. It was verified (25) that maxillary posterior teeth with periapical radiolucent lesions had the highest frequency of sinus changes. A close spatial relationship between periapical lesion and sinus resulted most frequently in sinus abnormalities. 6. Maloney PL, Doku HC. Maxillary sinusitis of odontogenic origin. J Can Dent Assoc 1968;34:591–603. 7. Brook I. Sinusits of odontogenic origin. Otolaryngol Head Neck Surg 2006; 135:349-355. 8. Oberli K, Bornstein MM, von Arx T. Resumo Avaliou-se a relação anatômica entre dentes posteriores e o soalho do seio maxilar (SSM) por meio da tomografia computadorizada de feixe cônico (TCFC) em 202 exames. A distância entre os ápices radiculares e o SSM, bem como a espessura do osso cortical do SSM próximo dos ápices radiculares e áreas de bifurcação foram medidas. As relações verticais e horizontais do SSM com as raízes dos molares foram classificados em categorias adaptadas a partir dos critérios propostos pelo Kwak et al. (14). A menor distância entre o SSM e os ápices dentários foi observada na raiz mesiovestibular do segundo molar (0,36±1,17 mm) e na raiz palatina do primeiro molar (0,45±1,10 mm), e a maior na raiz vestibular do primeiro pré-molar (5,47±4.43 mm). Diferenças significantes foram observadas entre a distância do SSM e os ápices dentários de primeiros e segundos pré-molares unirradiculares. A espessura da cortical óssea variou de 0,65±0,41 mm na região da raiz mesiovestibular do segundo molar a 1,28±0,42 na raiz vestibular do primeiro pré-molar. As relações vertical e horizontal mais prevalentes foram do tipo II e 2H, respectivamente. As raízes dos molares superiores apresentaram maior proximidade com o SSM. A espessura da cortical óssea do SSM nas regiões mais próximas dos ápices e área de furca foi similar apenas para os pré-molares. 15. Sharan A, Madjar D. 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A new volumetric CT machine for dental imaging based on the cone-beam technique: preliminary results. Eur Radiol 1998;8:1558-1564. In conclusion, the roots of the maxillary molars showed greater proximity with the MS when compared with premolars; the thickness of the cortical bone of the MS floor in the region closest to the apex and furcation area was found to be similar only for premolars. 12. Arai Y, Tammisalo E, Iwai K, Hashimoto K, Shinoda K. Development of a compact computed tomographic apparatus for dental use. Dentomaxillofac Radiol 1999;28:245-248. 13. Estrela C, Bueno MR, Leles CR, Azevedo B, Azevedo JR. Accuracy of cone beam computed tomography and panoramic and periapical radiography for detection of apical periodontitis. J Endod 2008;34: 273-279. 14. Kwak HH, Park HD, Yoon HR, Kang MK, Koh KS, Kim HJ. Topographic anatomy of the inferior wall of the maxillary sinus in Koreans. Int J Oral Maxillofac Surg 2004;33:382-388. Acknowledgements 21. 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Imaging Sci Dent 2012;42:219-224. 2. Maillet M, Bowles WR, McClanahan SL, John MT, Ashmad M. Cone- beam computed tomography evaluation of maxillary sinusitis. J Endod 2011;37:753-757. 2. Maillet M, Bowles WR, McClanahan SL, John MT, Ashmad M. Cone- beam computed tomography evaluation of maxillary sinusitis. J Endod 2011;37:753-757. 24. Maillet M, Bowles WR, McClanahan SL, John MT, Ashmad M. Cone- beam computed tomography evaluation of maxillary sinusitis. J Endod 2011;37:753-757. 3. Nair UP, Nair MK. Maxillary sinusitis of odontogenic origin: cone-beam volumetric computerized tomography-aided diagnosis. Oral Sur Oral Med Oral Pathol Oral Radiol Endod 2010;110:e53-e57. 25. Nunes CABCM, Guedes OA, Alencar AHG, Peters OA, Estrela CRA, Estrela C. Evaluation of periapical lesions and their association with maxillary sinus abnormalities on cone-beam computed tomographic images. J Endod 2016;42:42-46. 4. Obayashi N, Ariji Y, Goto M, Izumi M, Naitoh M, Kurita K, et al.. Spread of odontogenic infection originating in the maxillary teeth: Computerized tomographic assessment. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2004;98:223-231. Received August 2, 2015 Accepted December 10, 2015 Received August 2, 2015 Accepted December 10, 2015 5. Estrela C, Holland R, Estrela CRA, Alencar AHG, Sousa-Neto MD, Pecora 15
https://openalex.org/W4316463283	https://jtuh.org/index.php/jtuh/article/download/1483/1737	Arabic	null	أزمة العلاقات التونسية - الجزائرية 1958-1959	Mağallaẗ ǧāmiʻaẗ tikrīt li-l-ʻulūm al-insāniyyaẗ/Journal of Tikrit university for humanities	2,021	cc-by	14,397	ISSN: 1817-6798 (Print) Journal of Tikrit University for Humanities http://www.jtuh.tu.edu.iq available online at: ISSN: 1817-6798 (Print) Journal of Tikrit University for Humanities http://www.jtuh.tu.edu.iq available online at: © 2021 JTUH, College of Education for Human Sciences, Tikrit University Journal of Tikrit University for Humanities (2021) 28 (9) 290-267 Journal of Tikrit University for Humanities (2021) 28 (9) 290-267 267 Dr. Eyad Ayesh Mohammed University of Anbar- College of Education for Humanities University of Anbar- College of Education for Humanities University for Humanities Tunisian-Algerian relations worsened greatly during the years 1958 and 1959, and the reason for this was due to the attempts of the French authorities to provoke differences between the two countries in order to strike at any unitary project in Maghreb and work to eliminate the great solidarity shown by Tunisia with the Algerian revolution, while at the same time ensuring France's political and economic interests. These conditions were accompanied by the desire of Tunisian President Habib Bourguiba to exploit the situation in order to prove leadership and try to benefit from the developments in the political arena at the time when the Algerian issue was still unresolved. University of Anbar- College of Education for Humanities © 2021 JTUH, College of Education for Human Sciences, Tikrit University DOI: http://dx.doi.org/10.25130/jtuh.28.9.2021.13 تأزمت العالقات التػندية الجدائخية بذكل كبيخ خالل العاميغ1958 و1959 ، وكان الدبب في ذلظ يعػد الى محاوالت الدمصات الفخندية إ ة ثار الخالفات بيغ البمجيغ مغ أ جل ضخب اي مذخوع وحجوي في السغخب العخبي، والعسل عمى القزاء عمى التزامغ الكبيخ الحي تبجيو تػنذ م ع الثػرة الجدائخية , وفي الػقت نفدو ضسان مرالح فخندا الدياسية واالقترادية , ورافق كل ذلظ رغبة الخئيذ التػندي الحبيب بػرقيبة استغالل االوضاع مغ اجل اثبات الدعامة ومحاولة االستفادة مغ تصػرات الداحة الدياسية حيشيا ،في وقت كانت القزية الجدائخية الزالت غيخ محد ػمة , فكانت محاولة الحكػمة التػندية الشطخ الى مرالحيا بسعدل عغ مرمحة الجدائخ وثػرتيا د افعا الى التػجو الى عقج اتفاقات مع فخندا عمى حداب 267 مدتقبل الجدائخ ومرالحيا ،بل والعسل عمى محاولة التعجي عمى حقػق الجدائخ التاريخية وذلظ بالسصالبة بزع بعس أ راضي الرحخاء ال جدائخية و إ ثارة مذاكل حجودية معيا تخجم السرالح التػندية الزيقة وتزخ . بالثػرة الجدائخية ومدتقبل الجدائخ المقدمة لع تدتقخ العالقات التػندية الجدائخية السيسا بعج فذل مؤتسخ ششجة عام1958 والحي عج ًحيشيا مشجداً ميسا ذ ،إ أ قخت االحداب الخئيدية الثالث، حدب االستقالل السغخبي و ال حدب الجستػر ي الججيج التػندي وجبية التحخيخ الػششي الجدائخية خصة مذتخكة لبشاء وحجة مغاربية وتزامغ مع الثػرة , الجدائخية بيج أ ن ذلظ التزامغ مع الثػرة الجدائخية أ قمق الدمصات الفخندية وبالتالي عسمت عمى افذالو, وعشجما جاء شارل ديغػل الى الحكع في فخندا في حديخان1958 عسل عمى شق صف ذلظ التزامغ مغ خالل شخح بعس السذاريع عمى الحكػم تيغ السغخبية والتػندية في عامي1958 و1959 ادت ىحه السذاريع الى تأ زم العالقات الجدائخية التػندية مغ خالل انجالع ازمة ايجمي ، التي كانت عبارة عغ اتفاقية وقعت يا الحكػمية التػندية مع الحكػمة الفخندية , والتي ولجت ردود افعال سمبية عمى الثػرة الجدائخية وكحلظ ازمة الشداعات الحجودية في الرحخاء الجدائخية عشجما شالبت تػنذ بزع بعس االراضي الجدائخية , لحلظ فقج تزسغ البحث محػريغ ، تصخقت في االول الى بعس السؤتسخات التي عقجت لبحث مدألة الػحجة السغخبية ، وتصخقت في الثاني الى اىع االزمات بيغ . الجولتيغ المحور االول : المؤتمرات التي عقدت لبحث مدألة الوحدة المغربية مؤتمر طنجة2 اذار8591 المحور االول : المؤتمرات التي عقدت لبحث مدألة الوحدة المغربية مؤتمر طنجة2 اذار8591 المحور االول : المؤتمرات التي عقدت لبحث مدألة الوحدة المغربية كان حدب االستقالل السغخبي بكيادة عالل الفاسي( 1 ) قج تدعع الجعػة الى الػحجة السغاربية وصخح في مصمع عام1958 " .... االن وقج تحقق االستقالل فسغ واجبشا ان نبحل اقرى مجيػداتشا لتحقيق التعاون الحي كان شعار الحخكات السغاربية ... وان نتجو الى تػحيج السغخب العخبي في دولة واحجة متحجة ألنو لع يعج ىشاك مجال لمعدل ة وال لمػششية الزيقة في ىحا العرخ وقج بيغ التاريخ ان " احدغ عرػرنا ىي التي كانت فييا االقاليع الثالثة مػحجة( 2 ) 268 بالتالي حدب االستقالل السغخبي دعا الى عقج مؤتسخ يزع االحداب الثالثة وذلظ عمى اثخ اجتساع لجشتو التشفيحية في مجيشة ششجة السغخبية في2 اذار1958 لجراسة كل الػسائل الستاحة لتجعيع التزامغ والػحجة بيغ بمجان السغخب العخبي وذلظ بتأسيذ اتحاد حكيقي وفق السصامح الرحيحة يجسع بالتالي حدب االستقالل السغخبي دعا الى عقج مؤتسخ يزع االحداب الثالثة وذلظ عمى اثخ اجتساع لجشتو التشفيحية في مجيشة ششجة السغخبية في2 اذار1958 لجراسة كل الػسائل الستاحة لتجعيع التزامغ والػحجة بيغ بمجان السغخب العخبي وذلظ بتأسيذ اتحاد حكيقي وفق السصامح الرحيحة يجسع 268 التزامغ والػحجة بيغ بمجان السغخب العخبي وذلظ بتأسيذ اتحاد حكيقي وفق السصامح الرحيحة يجسع شعػب السغخب العخبي( 3 ) , وقج القت فكخة عقج السؤتسخ قبػالً لجى الحكػميتيغ التػندية والسغخبية اذ رحب السمظ دمحم الخامذ بالفكخة واضيخ الحب يب بػ رقيبة( 4 ) ارتياحو مشيا اذ دعا الى العسل الى كل ما يحقق وحجة السغخب العخبي( 5 ) اوفج حدب االستقالل السغخبي في17 اذار عام1958 وفجاً الى تػنذ لألعجاد مع قادة الحدب الجستػري لبخنامج عسل السؤتسخ وعقجت عجة اجتساعات في تػنذ بيغ الصخفيغ لمسجة مغ19 الى22 اذ ار عام1958 وتقخر خالليا عقج مؤتسخ ششجة بسذاركة جبية التحخيخ الػششي الجدائخية وحجد مػعج عقج السؤتسخ في نيدان عام1958 وكمف بعس الذخريات باالترال بجبية التحخيخ الػششي( 6 ) , وانقدست جبية التحخيخ الػششي الى قدسيغ بالشدبة لحزػر السؤتسخ لكل قدع رايو الخاص ال قدع االول عارض الحزػر ومذاركة جبية التحخيخ الػششي في السؤتسخ بجعػى انو مؤتسخ قصخي انفرالي ال يعبخ عغ البعج العخبي الحكيقي لمثػرة الجدائخية , اما القدع الثاني فاكج عمى ضخورة حزػر السؤتسخ( 7 ) ,عمى الخغع مغ االعتخاضات والتحفطات لحزػر السؤتسخ قخرت جبية التحخيخ الػششي حزػر السؤتسخ مغ اجل استغاللو لرالح القزية الجدائخية واخحت تحزخ لو بكل ججية( 8 ). المحور االول : المؤتمرات التي عقدت لبحث مدألة الوحدة المغربية مؤتمر طنجة2 اذار8591 التػصية بأنذاء حكػمة جدائخية مؤقتة بعج استذارة تػنذ والسغخب 3 - ان تكػن جبية التحخيخ الػششي ىي السسثل الذخعي الػحيج لمذعب الجدائخي كسا تع االتفاق عمى تػجيو نجاء الى الجول الغخبية مغ اجل الكف عغ مدانجة فخندا في حخبيا ضج الذعب الجدائخي والسيسا دعع الحمف االشمدي( 18 ) , كسا استشكخ السؤتسخ استسخار وجػد القػاعج العدكخية الفخندية في تػنذ والسغخب والسصالبة بجالء القػات الفخندية التي تذارك في الحخب و تشصمق مغ اراضي تػنذ والسغخب , واكج السؤتسخ عمى ىجفو الخئيدي وىػ تػحيج بمجان السغخب العخبي وتحقيق التزامغ بيغ شعػبو, وذلظ مغ خالل اقامة وحجة مذتخكة بيغ البمجان السغخبية الثالث وفق االتحاد الفجرالي( 19 ) , وكانت القزية االساسية لمػفػد السذتخكة في السؤتسخ ىي الق زية الجدائخية وما تحتاجو مغ دعع لتحقيق االستقالل اذ تع التأكيج عمى ان قزية الػحجة السغاربية ال يسكغ ان تتع اال باستقالل الجدائخ( 20 ). اختتع السؤتسخ اعسالو في30 نيدان عام1958 وخخج بعجة بقخارات تاريخية ميسة مثمت ميثاق لمسغخب العخبي واساس لمػحجة السختكبة , وع مى الخغع مغ تمظ القخارات السيسة اال انو بعج اسبػعيغ مغ انتياء السؤتسخ وقع االنقالب الحي انيى الجسيػرية الفخندية الخابعة وقيام الجسيػرية الخامدة في فخندا في13 ايار1958 وعسل ذلظ عمى وضع الثػرة الجدائخية في وضع ججيج في عالقتيا بتػنذ والسغخب مغ جية ومػاجية الحكػمة الفخندية الججيجة مغ جية اخخى( 21 ). كسا تع االتفاق عمى تػجيو نجاء الى الجول الغخبية مغ اجل الكف عغ مدانجة فخندا في حخبيا ضج الذعب الجدائخي والسيسا دعع الحمف االشمدي( 18 ) , كسا استشكخ السؤتسخ استسخار وجػد القػاعج العدكخية الفخندية في تػنذ والسغخب والسصالبة بجالء القػات الفخندية التي تذارك في الحخب و تشصمق مغ اراضي تػنذ والسغخب , واكج السؤتسخ عمى ىجفو الخئيدي وىػ تػحيج بمجان السغخب العخبي وتحقيق التزامغ بيغ شعػبو, وذلظ مغ خالل اقامة وحجة مذتخكة بيغ البمجان السغخبية الثالث وفق االتحاد الفجرالي( 19 ) , وكانت القزية االساسية لمػفػد السذتخكة في السؤتسخ ىي الق زية الجدائخية وما تحتاجو مغ دعع لتحقيق االستقالل اذ تع التأكيج عمى ان قزية الػحجة السغاربية ال يسكغ ان تتع اال باستقالل الجدائخ( 20 ). اختتع السؤتسخ اعسالو في30 نيدان عام1958 وخخج بعجة بقخارات تاريخية ميسة مثمت ميثاق لمسغخب العخبي واساس لمػحجة السختكبة , وع مى الخغع مغ تمظ القخارات السيسة اال انو بعج اسبػعيغ مغ انتياء السؤتسخ وقع االنقالب الحي انيى الجسيػرية الفخندية الخابعة وقيام الجسيػرية الخامدة في فخندا في13 ايار1958 وعسل ذلظ عمى وضع الثػرة الجدائخية في وضع ججيج في عالقتيا بتػنذ والسغخب مغ جية ومػاجية الحكػمة الفخندية الججيجة مغ جية اخخى( 21 ). المحور االول : المؤتمرات التي عقدت لبحث مدألة الوحدة المغربية مؤتمر طنجة2 اذار8591 تع االتفاق عمى عقج السؤتسخ بعج االتراالت والسذاورات بيغ االحداب الثالث وىحا ما عخف بشجوة ششجة التي اسفخت عغ تحجيج مكان وتاريخ عقج السؤتسخ( 9 ) , بعجىا اصجر مسثمػ حدب االس تقالل " الغخبي والحدب الجستػري التػندي بالغاً مذتخكاً جاء فيو إن مسثمي الحدبيغ نطخوا في إبخاز وحجة السغخب العخبي مغ شػر الفكخة الشطخية إلى الصػر الػاقعي التصبيقي وسجمػا وحجة نطخىع في السذاكل القائسة بالذسال اإلفخيقي وعمى رأ سيا ضخورة استقالل ال "جدائخ( 10 ) . بجأت المجشة التحزيخية لمسؤتسخ اعساليا في مجيشة الخباط25 نيدان1958 وبجأت اعسال ًالسؤتسخ رسسيا27 - 30 نيدان عام1958 في مجيشة ششجة السغخبية بخئاسة عالل الفاسي( 11 ) , بمغ ًعجد اعزاء الػفػد السذاركة في السؤتسخ تدعة عذخ عزػا( 12 ) , بعج القاء الخصب مغ قبل مسثمي ا الحداب الثالثة والتي ركدت عمى االىجاف الخئيدية لمسؤتسخ و تسثمت بػحجة الذعػب السغاربية وتقجيع الجعع لمثػرة الجدائخية حتى تتسكغ الجدائخ مغ تحقيق الديادة واالستقالل( 13 ) ,مغ خالل حخب االستقالل الجدائخية والعسل عمى ترفية الشفػذ االستعساري في بمجان السغخب العخبي ( 14 ) , وعمى الخغع مغ التدام وفج جبية التحخيخ الػششي بتشفيح قخارات مؤتسخ ش شجة فان وفجي تػنذ والسغخب لع يت خحا قخار االلتدام وذلظ بحجة انو البج مغ التسثيل الخسسي لحكػمتييسا واضيخا سعييسا لتشفيح قخارات السؤتسخ فأعمغ رئيذ السؤتسخ عالل الفاسي أن جسيع الق خارات ي الت سترجر عغ السؤتسخ ستجج شخيقيا إلى التشفيح عمى يج األح داب أو عمى يج الحكػمات( 15 ) 269 صادق السؤتسخ عمى بعس القخارات اىسيا فيسا يتعمق بحخب التحخيخ الجدائخية واثارىا عمى الػضع في شسال افخيكيا ودعع تػنذ والسغخب إليجاد حل سمسي بيغ فخندا وجبية التحخيخ الػششي وما تعخضت صادق السؤتسخ عمى بعس القخارات اىسيا فيسا يتعمق بحخب التحخيخ الجدائخية واثارىا عمى الػضع في شسال افخيكيا ودعع تػنذ والسغخب إليجاد حل سمسي بيغ فخندا وجبية التحخيخ الػششي وما تعخضت لو تػنذ والسغخب مغ سياسة الترعيج بدبب تزامشيا مع الثػرة الجدائخية( 16 ), وتع اال تفاق عمى ما يأتي لو تػنذ والسغخب مغ سياسة الترعيج بدبب تزامشيا مع الثػرة الجدائخية( 16 ), وتع اال تفاق عمى ما يأتي ( 16 ) ا : - ( 17 ). : - ( 17 ). 1 - ان تقجم االحداب الدياسية التػندية والسغخبية السدانجة لمذعب الجدائخي عمى الرعيج الذعبي . والحكػمي 1 - ان تقجم االحداب الدياسية التػندية والسغخبية السدانجة لمذعب الجدائخي عمى الرعيج الذعبي . والحكػمي 1 - ان تقجم االحداب الدياسية التػندية والسغخبية السدانجة لمذعب الجدائخي عمى الرعيج الذعبي . والحكػمي 2 - . مؤتمر المهدية81 حزيران8591 . نتيجة لمتصػرات االنفة الحكخ تع االتفاق عمى عقج مؤتسخ ججيج وتع االتفاق عمى عقج االجتساع بسجيشة السيجية بتػنذ ما بيغ17 إلى20 حديخان عام1958والتقى مسثمػ الحكػمة التػندية والسغخبية مع مسثل لجشة التشديق والتشفيح( 22 ) عغ الجدائخ وقج افتتح االجتساع لجراسة ججول اعسال السؤتسخ واىسيا تصبيق مقخرات مؤتسخ ششجة ومداعجة ودعع الثػرة الجدائخية واتخاذ مػاقف مذتخكة في االمع الستحجة وادانة سياسة ديغػل( 23 ) وجالء قػات االحتالل مغ بمجان السغخب العخبي وم دألة تذكيل الحكػمة الجدائخية السؤقتة ومدالة اقامة الييئات التي نرت عمييا مقخرا ت مؤتسخ ششجة( 24 ) . مؤتمر المهدية81 حزيران8591 . 270 أسشجت رئاسة الشجوة إلى فخحات عباس( 25 ) وقج تقخر أن تكػف جمداتيا سخية وأن ال تراغ وال تكتب إال بعس السالحطات والقخارات في محاضخ الجمدات وتع خالل الجمدة االولى ب حث مدألة إعانة أسشجت رئاسة الشجوة إلى فخحات عباس( 25 ) وقج تقخر أن تكػف جمداتيا سخية وأن ال تراغ وال تكتب إال بعس السالحطات والقخارات في محاضخ الجمدات وتع خالل الجمدة االولى ب حث مدألة إعانة 270 الجدائخ مغ قبل الحكػميتيغ السغخبية والتػندية ، ثع نػقذت في الجمدة الثانية مدألة جالء القػات االجشبية مغ اراضي السغخب العخبي( 26 ) , وفي اليػم الثاني لمسؤتسخ انتقل الشقاش لمشطخ في مدألة سياسة االدماج التي اثارىا ديغػل في الجدائخ بعج وصػلو لمحكع و التي تعج تخاجعا بالشدبة لمسػ ا قف الدياسية التي تقجمت بيا الحكػمات الفخندية الدابقة وان تمظ الدياسة سػف تؤدي الى زيادة الحخب ضج الذعب الجدائخي وفي اخخ يػم لمسؤتسخ نػقذت مدالة اقامة مؤسدات الػحجة التي اثارتيا مقخرات مؤتسخ ششجة وكحلظ قزية اقامة حكػمة جدائخية م ؤقتة( 27 ) , اضيخ مؤتسخ السيجية محاوالت الحكػميتيغ السغخبية والتػندية لمتشرل والتيخب مغ تشفيح قخارات مؤتسخ ششجة رغع ان السؤتسخ اكج عمى حق الذعب الجدائخي في الديادة واالستقالل فكان مؤتسخ السيجية نياية مأساوية لسقخرات مؤتسخ ششجة فقج ساد االنقدام بيغ االحداب السغخب ية الثالثة حػل مفيػم الػحجة وبج ا الخالف والسذاكل واضحة بيغ االشخاف الثالثة واضيخ مؤتسخ السيجية عجم وفاء تػنذ والسغخب في التداماتيسا في قخارات دعع الثػرة الجدائخية( 28 ) سعت فخندا بعج وصػل الجشخال شارل ديغػل الى افذال قخارات مؤتسخ ششجة مغ خالل التيجيج بتػسيع نصاق الحخب لتذسل االراضي السغخبية والتػندية اذا اس ت سختا في دعع مقخرات ششجة كسا انو في الػقت نفدو عمى احتخام استقالل الحكػميتيغ التػندية والسغخبية( 29 ) , واخح ديغػل يقجم بعس التشازالت كأغخاء لمحكػميتيغ السغخبية والتػندية ففي17 ايار عام1958 وقع اتفاق مع الحكػمة التػندية تزسغ تختيب اندحاب القػات الفخندية مغ كل التخاب التػندي باستثشاء قاعجة بشدرت وفي حديخان عام1958 قخر اجالء السخاكد العدكخية الفخندية السػ جػدة غخب وجشػب السغخب وكان ىجفو دفع حكػمتي السغخب وتػنذ الى التخمي نػعاً ما عغ دعع جبية التحخيخ الػششي الجدائخية( 30 ). 8 - ازمة ايجلي03 حزيران عام8591 . 8 - ازمة ايجلي03 حزيران عام8591 . كانت فخندا قج استغمت ممف قزية الرحخاء الجدائخية لكي تحقق سياستيا في القزاء عمى الثػرة التحخيخية وذلظ مغ خالل تأليب الجول السجاورة لمجدائخ السيسا السغخب و تػنذ ومالي والشيجخ لجعل قزية الرحخاء قزية مذتخكة بيغ تمظ الجول في التػسع والتقديع ،واالستغالل وأن الرحخاء ال تعشي الجدائخ وحجىا فقط( 36 ), لقج كان نفط الرحخاء الجدائخية حافدا كبيخا لبقاء االستعسار الفخندي في الجدائخ في الدشتيغ األولييغ مغ انجالع الثػرة ،الجدائخية ومع مجيء ديغػل أصبح يسثل واقعا يسكغ استغاللو في ترعيج االىتسام الفخندي وتبخيخ سياسة فخندا لمتسدظ بالجدائخ( 37 ), ولتجعيع سياستو قام ديغػل باالترال بعجد مغ رؤساء الجول األوروبية مغ أجل إقشاع الخأي العام بالخسالة الحزارية ل فخندا في الجدائخ وبسدألة اىسية الرحخاء التي ،اكتذفتيا وقج حاول ديغػل يػم 23 تذخيغ االول عام 1958 إقشاع حمفاء فخندا والجول اإلفخيكية بفكخة أن الرحخاء الجدائخية ىي ''صحخاء فخندية''، وىي ''بحخ داخمي تذتخك فيو جسيع الجول السجاورة لمجدائخ( 38 ) . مؤتمر المهدية81 حزيران8591 . 271 واخح ديغػل يعسل عمى تػجيو الخسائل فػجو رسالة الى ممظ السغخب ابجى فييا عدمو عمى اقامة وتشسية عالقات صجاقة بيغ الذعبيغ( 31 ), كسا وجو رسالة الى الخئيذ التػندي الحبيب بػ رقيبة اكج فييا عمى ضخورة تدػية السذاكل العالقة بيغ تػنذ وفخندا( 32 ) ,عسمت فخندا بعج ذلظ عمى زرع الزغيشة بيغ بمجان السغخب العخبي والثػرة الجدائخية مغ خالل تقجيع عخوض اىسيا استغالل الشفط والغاز الصبيعي في الرحخاء الجدائخية( 33 ), كسا ا ثار ديغػل قزايا الحجود بيغ الجدائخ وجيخانيا في السغخب العخبي مغ خالل جعل الرحخاء ال تخزع لديادة دولة معيشة بل تذتخك فييا جسيع الجول السجاورة كسا انيا اتفقت مع تػنذ في حديخان1958 عمى تسخيخ الشفط الجدائخي عبخ االراضي التػندية عمى ان تدتفيج تػنذ( 34 ), ولج كل ذ لظ سػء تفاىع بيغ جبية التحخيخ الػششي الجدائخية والحكػمة التػندية و بحلظ فقج قادة ازمة الحجود بيغ الجدائخ والسغخب في صيف عام1958 الى حجوث اشتباكات مدمحة في السشاشق الحجودية( 35 ) . وسػف نتصخق الى اىع االزمات الخاصة بسػضػع البحث وكسا يأتي المحور الثاني : االز. مات التي اثرت على العالقات بين البلدين واخح ديغػل يعسل عمى تػجيو الخسائل فػجو رسالة الى ممظ السغخب ابجى فييا عدمو عمى اقامة وتشسية عالقات صجاقة بيغ الذعبيغ( 31 ), كسا وجو رسالة الى الخئيذ التػندي الحبيب بػ رقيبة اكج فييا عمى ضخورة تدػية السذاكل العالقة بيغ تػنذ وفخندا( 32 ) ,عسمت فخندا بعج ذلظ عمى زرع الزغيشة بيغ بمجان السغخب العخبي والثػرة الجدائخية مغ خالل تقجيع عخوض اىسيا استغالل الشفط والغاز الصبيعي في الرحخاء الجدائخية( 33 ), كسا ا ثار ديغػل قزايا الحجود بيغ الجدائخ وجيخانيا في السغخب العخبي مغ خالل جعل الرحخاء ال تخزع لديادة دولة معيشة بل تذتخك فييا جسيع الجول السجاورة كسا انيا اتفقت مع تػنذ في حديخان1958 عمى تسخيخ الشفط الجدائخي عبخ االراضي التػندية عمى ان تدتفيج تػنذ( 34 ), ولج كل ذ لظ سػء تفاىع بيغ جبية التحخيخ الػششي الجدائخية والحكػمة التػندية و بحلظ فقج قادة ازمة الحجود بيغ الجدائخ والسغخب في صيف عام1958 الى حجوث اشتباكات مدمحة في السشاشق الحجودية( 35 ) . وسػف نتصخق الى اىع االزمات الخاصة بسػضػع البحث وكسا يأتي المحور الثاني : االز. مات التي اثرت على العالقات بين البلدين المحور الثاني : االز. مات التي اثرت على العالقات بين البلدين 8 - ازمة ايجلي03 حزيران عام8591 . وقج استيجفت فخندا مغ خالل استخاتيجيتيا االقترادية واستغالليا لشفط الجدائخ في تحقيق أىجافيا االستخاتيجية ضج السذاريع الػحجوية السغاربية وذلظ مغ خالل ضخب التزامغ السغاربي لمثػرة التحخيخية عغ شخيق استسالة تػنذ والسغخب حػل األشساع االقترادية واالستفادة مغ نفط الرحخاء الجدائخية , وحاولت فخندا ابخام اتفاق مع ليبيا لتسخيخ انبػب ايجمي عمى االراضي الميبية وعخضت السذخوع عمييا يف مصمع عام1958 واقتخحت الحكػمة الميبية عمى جبية التحخيخ الػششي ان تتقاسع معيا االرباح لكغ الجبية رفزت فشدلت ليبيا عشج مػقف الجبية فخضت السذخوع جسمة وتفريال بالخغع مغ الفػائج السالية واالقترادية التي يسكغ أن تعػد بيا ىحه الرفقة عمى ،ليبيا اذ كأ ج السمظ ادريذ الدشػسي( 39 ) رفزو ل تمظ االتفاقية وألح عمى ضخورة التسدظ بقخارات مؤتسخ ششجة وتقجيع الجعع لمثػرة الجدائخية مزيفا" بأن ليبي ا لغ تخضى بأي قخار يزخ بالثػرة ال جدائخية مغ قخيب أو بعيج" ( 40 ), كسا رفزت السغخب تكخيخ الشفط ولع تػقع االتفاق في القشيصخة ال ألنو نفط جدائخي ولكغ األيجي التي تخيج استثساره أيج فخندية( 41 ) ,فالرحخاء الجدائخية كانت تذكل في نطخ االستعسار الفخندي خصخ ًا يجب احتػاءه عغ شخيق فرميا عغ الذسال حتى تفقج الجدائخ حجودىا السذتخكة مع مالي وذلظ مغ اجل قصع التزامغ ،اإلفخيقي وتكدخ العسػد الفقخي لمتزامغ بيغ شسال إفخيكيا وجشػبيا وتػجو بحلظ ضخبة قاسسة لمتزامغ اإلفخيقي ولفكخة الحياد في القارة اإلفخيكية بأكسميا ( 42 ) ,وبخرػص تسخيخ نفط الرحخاء الجدائخية إلى ،فخندا اقتخح وزيخ الرحخاء الفخندي نقمو إلى تػنذ ًقائال " لقج فكخنا في تػصيمو إلى تػنذ أو إلى الجدائخ وعشجما صخت وزيخا لمرحخاء اقتخحت عمى الحكػمة تػصيمو إلى تػنذ ألسباب مادية فمقج كان ذلظ أقرخ ندبيا وكحلظ ألن ذلظ كان في إمكانو مداعجة تػنذ وربصيا بفخندا اقتراديا وبسجخد أن صار ذلظ مسكشا تقشيا وأن ىشاك مرمحة سياسية تزاف إلى ذلظ صار البج مغ إقامتو'' ( 43 ) ,وفي رسالة وردت مغ الدفارة الفخندية بتػنذ في10 ايار عام1957 الى قائج الجيػش العدكخية 272 272 272 و القائج األعمى لمفخق العدكخية في تػنذ تزسشت تكميف ميشجسيغ فخندييغ لجراسة الصخق السحتسمة لتسخيخ خصػط أنابيب إيجمي إلى قابذ. 8 - ازمة ايجلي03 حزيران عام8591 . وتبيغ ىحه الخسالة أن فخندا بجأت أشغاليا لتسخيخ أنبػب إيجمي عبخ األراضي الجشػبية التػندية قبل إمزاء اتفاقيا مع تػنذ يػم 30 حديخان عام1958 أعمشت خاللو الحكػمة الفخندية في باريذ عغ تػقيع اتفاقية مذتخكة بتسخيخ أنبػب غاز إيجمي عبخ التخاب التػندي إلى ميشاء قابذ ( 44 ) لقج ضيخت ليػنة بػرقيبة في التعامل مع فخندا الستغالل ثخوات الجدائخ فيػ لع يكغ يسانع الدياسة الفخندية االستغاللي ة ، بل كان يبحث ليا عغ الحمػل لكي يزسغ ليا بقاء تمظ الثخوات تحت سيصختيا وىحا ما صخح وب سابقاً في خصاب ألقاه في15 اب عام1957 قال " فيو... وىػ نفذ ما قمشاه لفخندا فيسا يخز الرحخاء عشجما ادعت أنيا بعج أن اكتذفت في بصػنيا الحىب األسػد أو األورانيػم يمدميا أن ترخ عمى البقاء بالجدائخ أكثخ مغ أي وقت مزى وىحا ما جعميا تسعغ في القزاء عمى الثػرة الجدائخية لتصسئغ عمى بقاء تمظ الثخوات بيجىا بيشسا نحغ نخى عكذ ما تخاه فخندا باعتبار أن أىسية الثخوات التي أنتجتيا الرحخاء مغ شأنيا أن تفخض عمى فخندا تغييخ سياستيا لتتسكغ مغ استثسارىا في ىجوء واشسئشان السيسا وان لجييا ما يزسغ ليا شػل انتفاعيا بالشريب الخاجع ليا مشيا بدبب أن أىالي تمكع الجية غيخ قادريغ اآلن عمى استغالل تمظ الثخوات بالصخق السججية'' ( 45 ) لقج كان ا خص ب بػرقيبة ممتػي ًا نػعاً ما فيػ يجعػا فخندا أن تسشح االستقالل ،لمجدائخ لكي تتسكغ مغ استثسار خيخات ،البالد فإذا كان استقالل البالد بجون استقالل ثخوات البالد فكيف يسكغ أن ندسي ىحا استقالل. 8 - ازمة ايجلي03 حزيران عام8591 . استسخت فخندا بالعسل عمى تفكيظ وحجة التزامغ السغاربي , وعسمت فخندا بعج فذل مؤتسخ السيجية عمى تػجيو تػنذ والسغخب نحػ التشرل مغ مقخرات مؤتسخ ششجة( 46 ) ,فاتجيت فخندا الى اقامة (مذاريع اقترادية مع تػنذ والسغخب ألبعادىا عغ دعع الثػرة الجدائخية فعخض عمى تػنذ الجخػل في السشصقة السذتخكة الستغالل االراضي الرحخاوية (o.c.r.s ( 47 ) , وكان اليجف مغ ذلظ دفع تػنذ الى تػقيع اتفاقية ايجمي في30 حديخان1958 التي اخخجت تػن ذ مغ التداماتيا في مقخرات ششجة بعج ان ( سسحت لمذخكة الفخندية ستخابداSTRAPSA ) ( 48 ) بتسخيخ انبػب لشقل الشفط مغ ايجمي الػاقعة جشػب شخق الجدائخ ليرل عبخ االراضي التػندية الى ميشاء قابذ الػاقع شسال شخق تػنذ( 49 ) , رأت الحكػمة التػندية بان ليا سيادة عمى السشاشق الغش ية بالشفط الن االنبػب يرل الى السيشاء التػندي ثع الى البحخ الستػسط وىحا يحقق ليا ارباح كبيخة والسيسا انو يسخ باألراضي التػندية وحاول بػرقيبة في اكثخ مغ مخة ان يرػر الشداع بخرػص الحقػق الديادية عمى ابار الشفط في ايجمي عمى انو قزية تػندية وحاول ايجاد حل لمقز ية الشفصية في اشار تػندي فخندي فقط واخخاج الجدائخ مغ محػر ىحا الشداع( 50 ) ، وبعج االعالن عغ ىحه االتفاقية تأزم السػقف بيغ جبية التحخيخ الػششي الجدائخية والحكػمة التػندية اال ان تػنذ خالفت تعيجاتيا في مؤتسخ ششجة وساعجت في خمق خصخ عمى الكفاح الػششي الجدائخي( 51 ). 273 273 حاولت لجشة التشديق والتشفيح قبل تػقيع االتفاقية بأسبػع ان تقشع الحكػمة التػندية بعجم التػقيع عمييا وارسمت محكخة الى الحبيب بػ رقيبة في23 حديخان عام1958 اعخبت فييا عغ قمقيا مغ دفع تػنذ الى عقج تمظ االتفاقية مبيشة االخصار الستختبة عمى االتفاقية( 52 ) , واىسيا خخوج تػنذ مغ التدامات اتفاق مؤتسخ ششجة كسا انيا عجت ضخب ة لمذعب الجدائخي حيث ان نفط ايجمي سػف يدتخجم في الحخب مع الجدائخ كسا ان االتفاقية تعشي االعتخاف بحق فخندا في الترخف بالرحخاء الجدائخية وثخواتيا وان السذخوع يعشي زيادة رؤوس االمػال االجشبية وىحا بجوره يخجم الدياسية االستعسارية الفخندية وحخبيا في الجدائخ( 53 ). لع تشجح لجشة التشديق والتشفيح في مشع الحكػمة التػندية مغ تػقيع االتفاقية رغع كل جيػدىا ولع تسشع مقخرات مؤتسخ ششجة الحكػمة التػندية مغ السزي في ذلظ ونتيجة الزغػط واالغخاءات الفخندية عمى الحكػم ة التػندية تع التػقيع عمى االتفاقية في30 حديخان عام1958 وبحلظ تسكشت فخندا مغ تحقيق نرخ سياسي بزخب التزامغ السغاربي وخمق خالف بيغ تػنذ والجدائخ وفي الػقت نفدو ًاستغالل نفط ايجمي بتكاليف ضئيمة ججا( 54 ) . 8 - ازمة ايجلي03 حزيران عام8591 . تأزمت العالقات بيغ الحكػمة التػندية وجبية التحخيخ الػ ششي الجدائخية بعج عقج االتفاقية واصجرت لجشة التشديق والتشفيح في11 تسػز عام1958 بيان ادانت فيو االتفاقية الفخندية التػندية( 55 ) , وىجدت بانيا سػف تفجخ انابيب الشفط السارة عبخ االراضي الجدائخية وانيا ستعارض اي استغالل لمشفط الجدائخي ما دامت الحخب قائسة وانيا سػف تشقل مكاتبيا الى شخابمذ( 56 ) , حاولت الرحافة التػندية تبخيخ االتفاقية بجواعي اقترادية كػنيا ستحقق دخالً مالياً وفخص عسل لمتػندييغ وسػف تعسل عمى تحديغ االوضاع االقترادية في كل شسال افخيكيا( 57 ) , ردت جبية التحخيخ الػششي عمى ذلظ بسقال افتتاحي نذختو جخيجة السجا ىج الجدائخية عشػانو ( الخبد السدسػم ) انتقجت فيو السػقف التػندي واالتفاقية جاء فيو " ان الجماء التي دفعيا شعبشا في السغخب العخبي بدخاء لع يبحليا في سبيل الخبد اليػمي " السمصخ بالجم والسحلة والجخائع االستعسارية وانسا بحليا مغ اجل ىجف اجل واعطع( 58 ), دفع مقا ل صحيفة السجاىج الحكػمة التػندية الى حجد العجد28 مغ صحيفة السجاىج في السصبعة في22 تسػز عام1958 واضصخت بعس الرحف في تػنذ لمتػقف بعج ان اخزعت بخامجيا لمسخاقبة مغ قبل الحكػمة التػندية( 59 ) , كسا وجو فخحات عباس العزػ ف ي جبية التحخيخ الػششي كمسة لػم عاتب فييا الحكػمة التػندية والذعب التػندي قائالً " ان انبػب ايجمي وان كان سيجر عميكع السديج مغ السال اال ان مخروه ىحا سيكػن " عمى جثث اخػانكع الجدائخييغ السخابصيغ في الجبال دفاعاً وجياداً مغ اجل تحخيخ وششيع( 60 ) ,كل ذلظ ادى الى حجوث ازمة حكيكية بيغ الجدائخ وتػنذ دفعت الحكػمة التػندية الى اخخاج الجخحى والسخضى الجدائخييغ مغ مدتذفياتيا وبقػا امام ابػاب السدتذفيات بال رعاية مسا دفع بالكيادة الجدائخية الى نقميع الى بعس مخاكد العالج في الجبال السجاورة رغع الشقز الكبيخ في االشباء والسسخضيغ واالدوية نتيجة 274 274 نقز الػاردات( 61 ) , وحجدت الدمع واإلعانات السػجية لميالل األحسخ الجدائخي خالل شيخ تسػز 1958 , فزالً عغ مصالبة جبية التحخيخ الػششي بالتخمي عغ أي سمصة تسثميا ذات شابع قشرمي واالدعاء بان الجدائ خ, ييغ اصبحػا ال يحتخمػن الديادة التػندية كسا تع حجد كسيات كبيخة مغ االسمحة (حػالي5070 بشجقية و2037 بشجقية رشاشة وبعس السجافع والحخيخة ,كسا ضايقت جير التحخيخ وقامت بحسالت اعتقال ضج بعس الثػار( 62 ) , لع تدتفد كل تمظ الترخفات الجدائخييغ عمى الخغع مغ زيادة نذاط الرحافة الفخندية حػل مجح اتفاقية ايجمي والحكػمة التػندية والحبيب بػ رقيبة( 63 ) . 8 - ازمة ايجلي03 حزيران عام8591 . دامت االزمة قخابة ثالثة شيػر وكانت قج عسمت عمى خشق الثػرة الجدائخية مغ خالل صعػبة التشقل عبخ الجبال التػندية( 64 ) , فقج مثمت اتفاقية ايجمي دعساً سياسياً ومادياً لفخندا التي عسمت عمى عدل الثػرة الجدائخية عغ جيخانيا وىحا االمخ ضيق نذاط الثػرة الجدائخية في الس شاشق الحجودية( 65 ) , كسا ان تمظ االتفاقية عسمت عمى تأخيخ استقالل الجدائخ وذلظ ألنيا فتحت السجال لالستثسارات االجشبية االوربية في الرحخاء الجدائخية( 66 ). يتزح مسا تقجم انو بالخغع مغ تححيخ جبية التحخيخ الػششي الجدائخية مغ خصػرة السذخوع قبل تػقيع االتفاقية اال ا ن بػرقيبة انحاز الى مرمحتو الخاصة واغخاءات السذخوع وسارع الى تػقيع اتفاقية . ايجمي مسا زاد مغ تػتخ االمػر وبالتالي اثخت سمباً عمى العالقات التػندية الجدائخية : كان التفاقية ايجمي انعكاسات خصيخة عمى الثػرة الجدائخية اىسيا- ( 67 ) : كان التفاقية ايجمي انعكاسات خصيخة عمى الثػرة الجدائخية اىسيا- ( 67 ) 1 - ان ىحا االتفاق تجعيسا سياسيا وماديا غيخ مباشخ مغ شخف الحكػمة التػندية لالستعسار الفخندي ومبخر لقسع الذعب الجدائخي . 275 1 - ان ىحا االتفاق تجعيسا سياسيا وماديا غيخ مباشخ مغ شخف الحكػمة التػندية لالستعسار الفخندي ومبخر لقسع الذعب الجدائخي . 2 - استغالل االتفاقية مغ شخف فخندا لسحاولة تطميل الخاي العام العالسي عمى ان الثػرة الجدائخية مخفػضة حتى مغ شخف جيخانيا الحيغ يتعاممػن مع فخندا بذكل عادي . 3 - ىحا االتفاق تجعيع لسذخوع فخندا لفرل الرحخاء عغ الجدائخ . 4 - خمق مبخر ججيج لتػاجج ومزاعفة اعجاد الجير الفخندي عمى الحجود الجدائخية التػندية مغ اجل تذجيج الحخاسة عمى انبػب ايجمي وىحا ما يعخقل نذاط جير التحخيخ الػششي الجدائخي . 5 - التأثيخ الدمبي عمى معشػيات اعزاء جير التحخيخ الجدائخي السيسا الستػاججيغ في الحجود الجدائخية التػندية اذ عجت جبية التحخيخ الػششي ان التأثيخ الدمبي لالتفاق سيكػن عمى حداب كل بمجان السغخب .العخبي أرادت جبية التحخيخ الػششي احتػاء ذلظ الخالف لمحفاظ عمى العالقات بيغ البمجيغ ومرالحيا في ،تػنذ ونجحت قيادة جبية التحخيخ الػششي الجدائخية في حل خالفاتيا ومذاكميا مع الحكػمة التػندية واستصاعت أن تتفادى أي نػع مغ الرخاع أو الرجام الحي يسكغ أن يؤثخ سمبا عمى مدار القزية الجدائخية السيسا وأنيا كانت في أمذ الحاجة إلى مدانجتيا لكفاح الذعب الجدائخي ضج االس تعسار 2 - استغالل االتفاقية مغ شخف فخندا لسحاولة تطميل الخاي العام العالسي عمى ان الثػرة الجدائخية مخفػضة حتى مغ شخف جيخانيا الحيغ يتعاممػن مع فخندا بذكل عادي . 3 - ىحا االتفاق تجعيع لسذخوع فخندا لفرل الرحخاء عغ الجدائخ . 4 - خمق مبخر ججيج لتػاجج ومزاعفة اعجاد الجير الفخندي عمى الحجود الجدائخية التػندية مغ اجل تذجيج الحخاسة عمى انبػب ايجمي وىحا ما يعخقل نذاط جير التحخيخ الػششي الجدائخي . : كان التفاقية ايجمي انعكاسات خصيخة عمى الثػرة الجدائخية اىسيا- ( 67 ) 5 - التأثيخ الدمبي عمى معشػيات اعزاء جير التحخيخ الجدائخي السيسا الستػاججيغ في الحجود الجدائخية التػندية اذ عجت جبية التحخيخ الػششي ان التأثيخ الدمبي لالتفاق سيكػن عمى حداب كل بمجان السغخب .العخبي أرادت جبية التحخيخ الػششي احتػاء ذلظ الخالف لمحفاظ عمى العالقات بيغ البمجيغ ومرالحيا في ،تػنذ ونجحت قيادة جبية التحخيخ الػششي الجدائخية في حل خالفاتيا ومذاكميا مع الحكػمة التػندية واستصاعت أن تتفادى أي نػع مغ الرخاع أو الرجام الحي يسكغ أن يؤثخ سمبا عمى مدار القزية الجدائخية السيسا وأنيا كانت في أمذ الحاجة إلى مدانجتيا لكفاح الذعب الجدائخي ضج االس تعسار 275 الفخندي باإلضافة إلى تجشب تعخيس الثػرة الجدائخية إلى اإلجياض في أىع قاعجة ليا وىي القاعجة الذخقية ، لحلظ سعت جبية التحخيخ الػششي إلى عقج العجيج مغ االجتساعات بيجف الشطخ في السدائل , العالقة وإعادة العالقات بيغ البمجيغ إلى شبيعتيا ومغ بيغ ىحه السداعي قيام جبية التحخيخ الػششي باالجتساع مع الحكػمة التػندية إلعادة الشطخ في السدائل العالقة والبحث عغ حل لمخالف الحي يسكغ ان يدبب في قصيعة في العالقات بيغ البمجيغ, االمخ الحي ميج الجتساع لػفج لجشة التشديق والتشسية بالحكػمة التػندية بجاية اب عام1958 لسعالجة م ػضػع الخالف الخئيدي وىػ أنبػب إيجمي واتفق الصخفان عمى عػدة عالقات التعاون والتفاىع بيشيسا، وتع االتفاق عمى حل وسط ال يغزب الصخفيغ الجدائخي والتػندي وذلظ بأن تتعيج الحكػمة التػندية بعجم تذغيل األنبػب إلى أن تدتقل الجدائخ وأن يكػن استغاللو لرالح الذعبيغ الذك يقيغ( 68 ) . انتيت االتراالت بيغ جبية التحخيخ الػششي والحكػمة التػندية حػل حادثة ،إيجمي بالتػافق حػل البحث عغ حمػل تخضي الصخفيغ قبل اإلعالن عغ الحكػمة السؤقتة لمثػرة ،الجدائخية حتى وإن لع تػضح تمظ االجتساعات آليات حل ،الخالف إال أن قخار تػنذ بتجسيج اتفاق إيجمي إلى ما بعج استقالل الجدائخ قج أعاد السياه إلى مجارييا وأكدب الثػرة الجدائخية الجعع الدياسي التػندي الزخوري( 69 ), أعخبت الحكػمة التػندية عمى مػاصمة واستسخارية دعسيا لمقزية الجدائخية والثػرة ،التحخيخية كسا قام بػرقيبة الحقا بتبخيخ مػقفو في خصاب ألقاه في شباط1959 حػل تمظ األزمة قائال '' وقج كشا نعتقج أن اكتذاف الثخوات والخيخات في جػف الرحخاء سيعجل بشياية الحخب في الجدائخ وألجل ذلظ قبمشا مخور أنابيب البتخول عبخ بالدنا ألدراك تمظ الغاية بالحات " ( 70 ). : كان التفاقية ايجمي انعكاسات خصيخة عمى الثػرة الجدائخية اىسيا- ( 67 ) كسا أصجرت جبية التحخيخ الػششي بيان اكجت فيو أن الصخفان قاما بػضع إجخاءات تيجف الى حل الخالف القائع بيغ الصخفيغ حيث قامت الحكػمة التػندية بحل الخالف مغ خالل تأكيجىا بأن تػنذ لغ تدسح لديخ الديت في أنابيبيا حتى تشال الجدائخ استقالليا ( 71 ) ,وبعج أول اجتساع لألمانة الجائسة السغخبية السشعقج بتػنذ مغ 30 اب الى1 ايمػل1959 تع االتفاق بيغ الصخفيغ الجدائخي والتػندي عمى تجسيج قزية أنبػب إيجمي الى ما بعج استقالل الجدائخ( 72 ). وتػصل الصخفان الجدائخي والتػندي فيسا بعج إلى اتفاق حػل مدألة الحجود يشز عمى أن قزية الرحخاء ال يسكغ إيجاد حل ليا إال بعج استقالل الجدائخ, ويبجو أن الزغط الفخندي الستدايج عمى تػنذ وإصخار الجدائخ عمى أن الرحخاء قزية جدائخية ىػ الحي جعل بػرقيبة يغيخ مػقفو تج اه تمظ السدألة فخأى أنو مغ واجبو مصالبة فخندا بسا كان يعتقج أنو حق لتػنذ، وأن يجتاز مغ اآلن حخبو معيا عػض أن يجخل مدتكبال في صخاع مع الجدائخ متعيجاً باالنتطار إلى ما بعج االستقالل لخسع الحجود نيائيا مع الجدائخ وفق ما تع االتفاق عميو( 73 ) . 276 ومغ ىشا يتزح لشا أن قيادة جبية التحخيخ الػششي قج ادركت مشح وقت مبك خ الفخ الحي وضعو ديغػل ،لمتفخقة بيغ أقصار السغخب العخبي وأبانت عغ مقجرة كبيخة في تدييخ عالقاتيا مع بمجان السغخب العخبي كسا حخصت عمى إضيار شعػب السغخب العخبي ككتمة واحجة ومػحجة وىػ ما جشبيا الػقػع في شباك السؤامخة الفخندية التي كادت أن تعصي ثسارىا لػال جخأة قيادة الجبية وسياستيا القائسة عمى ضخورة السحافطة عمى وحجة الرف السغاربي وتجاوز جسيع خالفاتيا مع تػنذ وذلظ بإقشاع الحكػمة التػندية بتأجيل مدألة الحدع في ىحه القزايا إلى ما بعج استقالل الج.دائخ 2 - ازمة الخالفات الحدودية بين تونس والجزائر1959 . استعسمت فخندا مذاكل الرحخاء الجدائخية كػرقة تأثيخ سياسي لمجول السجاورة لمجدائخ والسيسا تػنذ والسغخب مغ اجل دفع الجول السجاورة لمجدائخ الى التخمي عغ دعع الثػرة الجدائخية( 74 ) , اذ رفزت فخندا التفاوض مع الثػار الجدائخييغ فيسا يخز مدتقبل الرحخاء وعشجما فذمت في الزغط عمى جبية التحخيخ الػششي الجدائخية حاولت جعل الرحخاء الجدائخية تخزع الى قدسة الجول االفخيكية وان تكػن فخندا ىي الحكع في ذلظ( 75 ) . فقج ذكخ بػ رقيبة في خصاب ألقاه يػم11 نيدان عام1957 قائالً '' ومغ السعمػم أن الحجود التػندية لع تكغ مزبػشة عشجما دخمت الجيػش الفخندية لتػنذ. وإذا ألقيشا أالن نطخة إلى خخيصة إفخيكيا الذسالية فساذا نجج؟ نجج كال مغ ليبيا والجدائخ والسغخب األقرى ليا حجود مت رمة بالرحخاء الكبخى ما عجا تػنذ فقج قصع بيشيا وبيغ السشافح لمفزاء الفديح فكأنسا قج عمقت في اليػاء وكأنسا جارتيا قج حالت بيشيا وبيغ التشفذ وما مغ سبب ليحه الػضعية الذاذة إال اتفاق ضباط السكاتب العدكخية في الجشػب التػندي وفي الجشػب الجدائخي عمى تزخيع السجال الخاجع لمدمصة الفخندية التي يخون أن بقاءىا أثبت وأدوم في الجدائخ مسا ىػ في تػنذ أو في السغخب وليحا كان ىسيع ييجف دائسا لتشقيل التخاب الجدائخي ''يسيشا وشساال عمى حداب كل مغ التخاب التػندي والتخاب السغخبي( 76 ) .ردت الدمصات الفخندية عمى تمظ السصالب بقػليا " ان رسع الحجود بيغ تػنذ والجدائخ قج تع عمى الذكل الحي يحفع حقػق تػنذ ولع " ًيشقز مشيا شيئا( 77 ) جخت محادثات بيغ الحكػمة التػندية والحكػمة الفخندية بتا ريخ24 كانػن الثاني عام1959 تع خالليا تػقيع اتفاقية بخوتػكػل األمالك الجولية التػندية، أثيخت خالليا مشاقذات حػل رسع الحجود الرحخاوية بيغ تػنذ والجدائخ, وفي نفذ اليػم أرسمت الخارجية التػندية رسالة إلى الدمصات الفخندية تؤكج فييا مصالبة الحكػمة التػندية إعادة الشطخ حػل رسع الحجود التػندية ، وأن ضبط الحجود جشػبا ىػ رسع وقتي قامت بو الدمصات الفخندية مع الحكػمة التػن دية أثشاء تػقيع اتفاقية الحكع الحاتي عام 1955 حػل مخاقبة الحجود التػندية الجدائخية فقط( 78 ) , واوضح بػرقيبة بانو مدتعج لعخض القزية امام محكسة العجل الجولية لكشو يفزل ان يقع حل ىحا الخالف بالصخق الػدية مع الحكػمة الفخندية( 79 ) . 2 - ازمة الخالفات الحدودية بين تونس والجزائر1959 . 277 كان الحبيب بػ رقيبة يصسح في زعامة السغخب العخبي ولسا كانت مداحة تػنذ مقارنة بسداحة جيخانيا الجدائخ وليبيا ىي مداحة صغيخة كسا ان تػنذ كانت تفتقخ الى السػارد الصبيعية( 80 ) ,لحلظ عسل بػ رقيبة عمى السصالبة باألحكية في الرحخاء الجدائخية فصالب بسداحات صحخاوية لزسيا الى حجود تػنذ( 81 ) , وفي5 شباط عام1959 القى بػرقيبة خصاب امام السجمذ الػششي التػندي اكج عمى ضخورة اعادة رسع الحجود مع الجدائخ عمى اعتبار ان الرحخاء الكبخى تذتخك بيا كل الجول السجاورة وان حجود تػنذ قج عيشت شخقاً وغخباً لكشيا تخكت دون تحجيج مغ جية الجشػب( 82 ) , وصخح قائ الً " .... رأيشا مغ الزخوري التػصل الى تدػية نيائية لسذكمة حجودنا الجشػبية ... وانو البج لتػنذ مغ مشفح عمى " الرحخاء الكبخى لسشصقة خمفية تذتخك فييا أفخيكيا الذسالية( 83 ) , وعخف مذكل صحخاء تػنذ بسذكل ''حجود الرحخاء'' قائال: '' وندتصيع أن ندسي ىحا السذكل بسذك ل حجود الرحخاء وىػ مذكل قجيع لكغ جخيا عمى معتقجنا وشبيعتشا وحدب خصتشا الستبعة في عجم تكجيذ السذاكل ندعى لصخق السػاضيع وفس السذاكل واحجا بعج آخخ وكل حل يريب أحجىا يؤثخ عمى الحي يمييا وكسا قمت في خصاب سابق فإن تػنذ ال تصالب بحجودىا القجيسة التي كانت عمى العي ج الفاشسي. 2 - ازمة الخالفات الحدودية بين تونس والجزائر1959 . والحفري مثال والتي ترل إلى القاىخة شخقا وإلى فاس غخبا وإنسا تصالب بالحجود السػروثة التي اختصتيا الدمصات الفخندية عشجما كانت صاحبة الديادة بتػنذ ومغ حدغ الحع أنو وقع الذخوع في ضبط الحجود بيغ تػنذ والخارج بػاسصة الدمصات الفخندية نفديا سػاء ما يترل مشيا بميبيا أو ما يترل مشيا بالجدائخ ورغع أن مغ حقشا أن نصعغ فيسا ابخمو الفخنديػن عغ الجانبيغ باسع تػنذ وباسع الجدائخ غيخ مخاعيغ فيو السرمحة الفخندية ''فحدب لكغ رضيشا بسا تع إبخامو وأقيع كحجود فاصمة( 84 ) وقج أكج بػرقيبة في نفذ الخصاب أن الجولة التػندية لغ تتػقف عغ مصالبتيا بحجودىا الرحخاوية التي تعبخ حق شبيعي ال يجب الدكػت عشو وقال بيحا الرجد: '' ىحا ما أردت أن أعخفكع بو لتعمسػا مػقف تػنذ وأن الجولة ال يسكشيا أن تقف مكتػفة األيجي أو أن تغس الصخف والػاجب عمييا أن تجافع عغ كيانيا وتأخح قدصيا مغ الرحخاء ال ألج ل العثػر عمى البتخول وإنسا ألنو حق شبيعي ال يسكغ الدكػت عشو ويتأكج ضبصو بالتي ىي أحدغ بكيفية تحفع حقػق الجسيع وبػدنا أن يقع فس ىحه السذاكل في نصاق السرالح السذتخكة مغ غيخ ان يؤول إلى معخكة مغ أجل الحجود أو مذكمة الحجود فإنشا نػد حميا باتفاق مع فخندا وفي ''دائخة التعقل واالتدان( 85 ) وحاول بػرقيبة ان يزفي عمى مصمبو شخعية تاريخية فاستذيج بأحكام السعاىجة التػندية العثسانية السعقػدة في19 اذار1910 التي لع تكغ فييا الحجود التػندية محجدة في قدسيا الجشػبي الغخبي , كسا استشجت تػنذ الى وثيقة عبارة عغ اتفاق كان قج تع ابخامو بيغ الدمصات الفخندية في تػنذ والجدائخ عام1901 )ضبصت عمى اثخىا الحجود التػندية الجدائخية حتى مشصقة اسسيا ( بئخ رومان( 86 ) , اذ قال في ىحا الرجد: '' ...فتذكمت لجشة ...خالل عام1901 تتخكب مغ نػاب فخندييغ عغ الجدائخ ونػاب فخندييغ عغ تػنذ وضعػا خخيصة مبشية عمى رغبة كل شق في تػسيع الحج إلى الشاحية األخخى وتذيخ إلى مػقع الحج حدب وجية الشطخ الجدائخية والى الحج الػسط الستفق عميو وىػ السدصخ بالمػن األحسخ وضبصت الخخيصة السػاقع واألسساء إلى أن يرل الحج جشػبا إلى بئخ الخومان الحي مازال إلى اآلن مػجػدا باسسو وأ مامي اآلن األمخ السمكي الحي ضبط االتفاقية وأعصاىا القانػن التػندي الرادر مغ األميخ دمحم اليادي وىػ يقتزي تعييغ الحجود الفاصمة بيغ أيالتي الجدائخ وتػنذ والتي تبجأ أوال بيغ جبل ''الغخة وفخن عائذة'' بعج شبخقة وثانيا بيغ ''ذراع الدػائل و فج الذتاء'' وثالثا بيغ ''عقمة العفخيت وخط بخ الرػف'' والسيع أن الحج الحي وقفت عشجه المجشة يشتيي عشج شخقي الرحغ يػجج بئخ الخومان وىي نقصة عمى خط بخ الرػف السػازي وىحه السدألة انتيت مشح عام1901 وال يدتصيع أحج تجاىميا أو السجادلة ''فييا( 87 ) . 2 - ازمة الخالفات الحدودية بين تونس والجزائر1959 . وردا عمى خصاب بػرقيبة وجيت الحكػمة الفخندية رسالة في شباط عام1959 عبخت فيو عغ ومػقفيا اتجاه مصالب الحكػمة التػندية، ورد فييا بأن تػنذ وافقت خالل تػقيعيا عمى اتفاقية3 حديخان عام1955 عمى الخسع الحالي لمحجود السبيغ حدب الخخائط السخفقة باالتفاقية( 88 ) , وفي اجتساع لجشة الذؤون الخارجية لمبخلسان الفخندي يػم13 شباط1959 تجخل وزيخ الخارجية الفخندي كػف دي مخفيل Couve de Murville واعتبخ أن مدألة الحجود قج سػيت نيائياً مشح عام1950 و اكج ان الدمصات التػندية قامت بسسارسة ضغػط عمى الدكان الجدائخييغ القاششيغ ف ي السشاشق الحجودية الذخقية، فقج تعخض سكان وادي سػف الخحل البالغ عجدىع80 ألف ندسة إلى مزايقات مغ الدمصات التػندية أثشاء تػاججىع بأماكغ رعػية مشح مئات الدشيغ بجػار الحجود الميبية وفي نفذ الػقت عسجت إلى اعتقال وحبذ البعس مغ البجو واالستيالء عمى قصعان مػا ,شييع بشية إبعادىع عغ األماكغ السحكػرة أعاله وأمام ىحه الزغػشات قامػا بسخاسمة رئيذ الجسيػرية الفخندية شارل ديغػل ويصالبػه بالتجخل لحفع حقػقيع التاريخية، والحيمػلة دون تقجم تػنذ نحػ مشاشقيع الغخبية، وأرفقػا مخاسمتيع لو بقائسة بأسساء الشقاط الحجودية بيغ البم جيغ( 89 ) كان بػ رقيبة يصسح مغ وراء السصالبة بسداحة ال تتجاوز20 كع فتح ثغخة يػسعيا فيسا بعج ويصالب بإلغاء الحجود الرحخاوية وجعل السشصقة الخمفية بسا في ذلظ حقل ايجمي بحخاً داخمياً لتػنذ وىحا دعع لمسخصط الفخندي وانكار لمصابع الجدائخي لمرحخاء, وحاول دعع مػقف و ايزاً بحجة ان الفخندييغ اعتجوا عمى شيء مغ االراضي التػندية( 90 ). 2 - ازمة الخالفات الحدودية بين تونس والجزائر1959 . اعتبخت الجدائخ مػقف بػ رقيبة مشاقس لتػصيات التزامغ السغاربي في مؤتسخ ششجة وعجت ذلظ اعتخافاً تػندياً بييسشة فخندا عمى الجدائخ في الػقت الحي وضعت فيو الجدائخ بعج احجاث "ساقية "سيجي يػسف( 91 ) اسمح تيا وجيذيا تحت ترخف تػنذ لحساية سيادة وتخاب تػنذ( 92 ) , كسا استيجغ الجدائخيػن ذلظ السػقف و عجوه ال يقل فجاحة عغ ازمة ايجمي مؤكجيغ ان " ىحه السػاقف التي ابجاىا بػ رقيبة في الػقت الحي كان يدقط فيو االف الجدائخييغ يػمياً ألنياء سيادة فخندا عمى الجدائخ قج شعخ بيا "الجدائخيػن وكأنيا شعشات خشجخ في الطيخ( 93 ) 279 279 قجم بػرقيبة حالن ليحه السدالة التي تيع دول الجػار االول التسجد الصبيعي ألراضي ىحه البمجان حتى الرحخاء والثاني اعتبار الرحخاء كبحخ داخمي تتع استغالل ثخواتو مغ شخف الجسيع وصخح بالقػل " ما ال يسكغ القبػل بو ىػ ان يغمق باب الرحخاء في وجػىشا بحجة ان كل ما يػجج خمف الباب ىػ " لفخندا( 94 ) , وارسمت الحكػمة التػندية حذجاً مغ مػضفييا الى داخل التخاب الجدائخي لػضع العمع التػندي فارسل جير التحخيخ الػششي الجدائخي مجاىجيغ لسشعيع لكغ الحي ترجى ليع ىػ الجير الفخندي وذكخت بعس السرادر ان كخيع بالقاسع( 95 ) قج خصط الغتيال بػرقيبة لكغ تخاجعو عغ مػقفو وعػدتو لسدانجة الثػرة الجدائخية ادى الى تخاجع بالقاسع عغ مخصصو( 96 ) . فعمى الخغع مغ مصالبة بػرقيبة بأجداء ميسة مغ التخاب الجدائخي كان يبجي في الػقت نفدو حخصاً شجيجاً عمى استقالل الجدائخ ويطيخ استعجاده لمتزحية مغ اجل ذلظ ففي17 شباط1959 عقج "بػ رقيبة نجوة صحفية في تػنذ صخح مغ خالليا قائالً " نحغ نقبل بتدميع "بشدرت( 97 ) لفخندا بذخط ان " تشيي حخبيا مع الجدائخ وتدتجيب لسصالب الذعب الجدائخي( 98 ) , كسا صخح بػرقيبة لرحيفة البالد العخاقية في13 ايمػل1959 بالقػل " لقج كشت مدتعج لمتشازل عغ بشدرت مق ابل استقالل الجدائخ لكغ فخندا لع تعط مػافقتيا لحلظ سأسانج كفاح الجدائخ ضج االستعسار الفخن دي"( 99 ). يبجو ان بػرقيبة اجل مصالبو الحجودية وعسل عمى ان يكػن مبادر وشخف في مدالة انياء حخب الجدائخ مع فخندا ومغ ثع يربح شخيكاً اساسياً في السفاوضات السؤدية الى استقالل الجدائخ وفي ىحا الػقت يسكشو استخجام قزية تدميع بشدرت لمعسل عمى مقايزتيا بأجداء في الرحخاء الجدا ئخية تحتػي عمى ابار نفصية , مغ اجل ذلظ بقيت االمػر عمى حاليا حتى زار بػرقيبة فخندا واثار مدالة الحجود . 2 - ازمة الخالفات الحدودية بين تونس والجزائر1959 . الرحخاوية التػندية مغ ججيج الخاتمة تػصمت مغ خالل البحث الى بعس الشتائج أ ىسيا , إ ن رغب ة فخندا في احباط وقتل أ ي مذخوع وحجوي يخبط بمجان السغخب العخبي ومحاولة تزييق الخشاق عمى الثػرة الجدائخية وقصع الجعع السادي والسعشػي كان وراء اثارة االزمات بيغ بمجان السغخب العخبي , وفي الػقت نفدو كانت فخندا ذكية في استغالل رغبة وشسػح الخئيذ بػ رقيبة في الدعامة , كسا لسدت مغ خالل البحث حخص عجد مغ الدعامات الجدائخية عمى الػحجة وعجم شق الرف فترخفت بحكسة وبيجوء مع االزمات التي اثارتيا فخندا وتػنذ كسا لسذ البحث عجم انجخار السػاقف الذعبية التػندية والجدائخية وراء رغبات مثيخي االزمات , كسا ان قزية الثػرة الجدائخية وشسػح الذعب الجدائخي بالحخية واالستقالل استصاع ان يغصي عمى جسي ع االزمات السفتعمة واجبخ جسيع االشخاف الى تأجيل كل االزمات الى ما بعج استقالل الجدائخ . 280 الهوامش الهوامش ( 1 ) : عالل الفاسي ولج في مجيشة فاس عام1910 ، مغ اسخة اشتيخت بالعمع والتقػى ،اكسل دراستو بجامعة القخوييغ 1931 ، واسيع بتأسيذ كتمة العسل الػششي عام1934 ، اسذ الحدب الػششي1937 ، نفي مغ قبل الدمصات الفخندية الى الغابػن في افخيكيا ،بعجىا التحق بالحخكة الػششية السغاربية في القا ىخة ،اسيع في تأسيذ حدب االستقالل واصبح رئيداً لو عام1944 ، كان لو نذاط بارز في اتراالتو بجامعة الجول العخبية ودور بارز في مكتب السغخب العخبي دافع عغ حقػق بالده في ىيئة االمع الستحجة عام1952 ونذط في الجعاية لقزية استقالل السغخب ، واستسخ في نذاشو الدياسي بعج االستقالل وتػلى وزارة الذؤون االسالمية ،وتػفي في13 ايار1974 . لمسديج يشطخ: نرخ الجيغ سعيجوني , معجع مذاىيخ السغاربة , ج1 , 1995 , ص2018 ؛ مػسػعة اعالم العخب،ج1،ص365 - 366 . ( 2 ) دمحم عمي ،داىر د سات را في الحخكات الػششية واالتجاىات الػحجوية في السغخب ،العخبي مشذػ رات اتحاد الكتاب ،العخبي ،دمذق 2004 , ص169 ( 3 ) , معسخ العايب , مؤتسخ ششجة السغاربي دراسة تحميمية تقييسية , دار الحكسة لمشذخ , الجدائخ2010 , ص125 ( 4 ) : الحبيب بػ رقيبة ولج في13 آب عام1903، في بمجة السشدتخ جشػبي تػنذ، تمقى تعميسو االبتجائي في السجرسة الرادقية ثع في (الميديو كارنػ) وىي مجرسة انذئت وفق الشطام التخبػي الفخندي. حرل عمى الميدانذ في القانػن ودبمػم في العمػم الدياسية مغ فخندا. عاد الى تػنذ عام1927 ، وعسل محامياً ثع عسل بالدياسة، وكتب في صحف (العمع التػندي)و(صػت تػنذ)و(العسل التػندي)، اصبح اميشاً عاماً لمحدب الحخ الجستػري الججيج عام 1934 ، وسجغ في العام نفدو وافخج عشو عام1936 ، ثع سجغ ثانية في عام1938 ، وافخج عشو عام1942 ، ثع اعتقمتو الدمصات الفخندية عام1952 ، وافخجت عشو فيسا بعج عام1955 ً، كان مؤيجا لدياسة السفاوضات مع فخندا بصخيقة السخاحل او سياسة الحمػل السشقػصة، اصبح في عام1957 ، اول رئيذ لمجسيػرية التػندية واعيج انتخابو عجة مخات تػفي في6 نيدان2000 . نقالً عغ : ىيثع عبج الخزخ معارج , مػقف االمع الستحجة مغ قزايا استقالل بمجان السغخب العخبي1948 - 1962 , , اشخوحة دكتػراه غيخ مشذػرة , جامعة بغجاد كمية التخبية ابغ رشج2009 , ص 189 . الهوامش ( 1 ) : عالل الفاسي ولج في مجيشة فاس عام1910 ، مغ اسخة اشتيخت بالعمع والتقػى ،اكسل دراستو بجامعة القخوييغ 1931 ، واسيع بتأسيذ كتمة العسل الػششي عام1934 ، اسذ الحدب الػششي1937 ، نفي مغ قبل الدمصات الفخندية الى الغابػن في افخيكيا ،بعجىا التحق بالحخكة الػششية السغاربية في القا ىخة ،اسيع في تأسيذ حدب االستقالل واصبح رئيداً لو عام1944 ، كان لو نذاط بارز في اتراالتو بجامعة الجول العخبية ودور بارز في مكتب السغخب العخبي دافع عغ حقػق بالده في ىيئة االمع الستحجة عام1952 ونذط في الجعاية لقزية استقالل السغخب ، واستسخ في نذاشو الدياسي بعج االستقالل وتػلى وزارة الذؤون االسالمية ،وتػفي في13 ايار1974 . لمسديج يشطخ: نرخ الجيغ سعيجوني , معجع مذاىيخ السغاربة , ج1 , 1995 , ص2018 ؛ مػسػعة اعالم العخب،ج1،ص365 - 366 . ( 2 ) دمحم عمي ،داىر د سات را في الحخكات الػششية واالتجاىات الػحجوية في السغخب ،العخبي مشذػ رات اتحاد الكتاب ،العخبي ،دمذق 2004 , ص169 ( 3 ) , معسخ العايب , مؤتسخ ششجة السغاربي دراسة تحميمية تقييسية , دار الحكسة لمشذخ , الجدائخ2010 , ص125 ( 4 ) : الحبيب بػ رقيبة ولج في13 آب عام1903، في بمجة السشدتخ جشػبي تػنذ، تمقى تعميسو االبتجائي في السجرسة الرادقية ثع في (الميديو كارنػ) وىي مجرسة انذئت وفق الشطام التخبػي الفخندي. حرل عمى الميدانذ في القانػن ودبمػم في العمػم الدياسية مغ فخندا. عاد الى تػنذ عام1927 ، وعسل محامياً ثع عسل بالدياسة، وكتب في صحف (العمع التػندي)و(صػت تػنذ)و(العسل التػندي)، اصبح اميشاً عاماً لمحدب الحخ الجستػري الججيج عام 1934 ، وسجغ في العام نفدو وافخج عشو عام1936 ، ثع سجغ ثانية في عام1938 ، وافخج عشو عام1942 ، ثع اعتقمتو الدمصات الفخندية عام1952 ، وافخجت عشو فيسا بعج عام1955 ً، كان مؤيجا لدياسة السفاوضات مع فخندا بصخيقة السخاحل او سياسة الحمػل السشقػصة، اصبح في عام1957 ، اول رئيذ لمجسيػرية التػندية واعيج انتخابو عجة مخات تػفي في6 نيدان2000 . نقالً عغ : ىيثع عبج الخزخ معارج , مػقف االمع الستحجة مغ قزايا استقالل بمجان السغخب العخبي1948 - 1962 , , اشخوحة دكتػراه غيخ مشذػرة , جامعة بغجاد كمية التخبية ابغ رشج2009 , ص 189 . ( 5 ) زىخة ،دلباني وساشة تػنذ والسغخب لحل القزية الجدائخية سمسيا 1956 - 1962 , مجمة اول نػفسبخ , العجد 183 , , السؤسدة الػششية لمشذخ , الجدائخ2017 , ص29 . الهوامش ( 13 ) , الدبتي غيالني , عالقة جبية التحخيخ الػششي الجدائخية بالسسمكة السغخبية اثشاء الثػرة التحخيخية الجدائخية , اشخوحة دكتػراه غيخ مشذػرة , قدع التاريخ , جامعة الحاج لخزخ2011 , ص186 . ( 14 ) عبج القادر العخيبي , تػنذ وعالقاتيا مع بمجان السغخب العخبي1947 - 1980 , , اشخوحة دكتػراه غيخ مشذػرة , قدع التاريخ , الجامعة التػندية1999 , ص253 . ( 13 ) , الدبتي غيالني , عالقة جبية التحخيخ الػششي الجدائخية بالسسمكة السغخبية اثشاء الثػرة التحخيخية الجدائخية , اشخوحة دكتػراه غيخ مشذػرة , قدع التاريخ , جامعة الحاج لخزخ2011 , ص186 . ( 14 ) عبج القادر العخيبي , تػنذ وعالقاتيا مع بمجان السغخب العخبي1947 - 1980 , , اشخوحة دكتػراه غيخ مشذػرة , قدع التاريخ , الجامعة التػندية1999 , ص253 . ( 15 ) ,دمحم بالقاسع , وحجة السغخب العخبي فكخة وواقعاً , البرائخ الججيجة لمشذخ , الجدائخ2013 , ص334 ؛ معسخ العايب , السرجر الدابق , ص145 . ( 16 ) عبج هللا حسادي , التػجو السغاربي في ذاكخة الحخكة الػششية الجدائخية , مجمة الحاكخة الػششية لمسشجوبية الدامية , لجير التحخيخ2002 , ص313 . ( 17 ) شارل انخي فافخود , الثػرة الجدائخية , تخجسة كابػية عبج الخحس , غ , مشذػرات دحمب2010 , ص378 . ( 18 ) عامخ رخيمة , السرجر الدابق , ص161 . ( 19 ) اسساعيل دبر , الدياسة العخبية والسػاقف الجولية تجاه الثػرة الجدائخية1954 - 1962 , , دار ىػمة لمشذخ , الجدائخ2003 , ص238 . ( 20 ) جخيجة السجاىج , الجدء االول , مغ ششجة الى السيجية , العجد26 , , 2 تسػز1958 , ص13 . ( 21 ) احسج سعيػد , العسل الجبمػماسي لجبية التحخيخ الػششي1954 - 1962 , , دار الذخوق لمشذخ , د. م2008 , ص151 . ( 22 ) لجشة التشديق والتشفيح : انبثقت ىحه المجشة عغ السجمذ الػششي لمثػرة الجدائخية الحي شكل في مؤتسخ الرػمام 20 اب1956 تكػنت في البجاية مغ خسدة اعزاء العخبي بغ مييجي , كخيع بمقاسع , عبان رمزان , بغ يػسف بغ خجه , سعج دحمب وىي السدؤولة عغ ادارة جسيع شؤون الثػرة واجيدتيا العدكخية والدياسية امام السجمذ الػششي لمثػرة كان مقخىا في البادية في الجدائخ ثع اضصخت الى مغاد رتيا الى الخارج . يشطخ : عثامشية فاشسة , بػرقيبة والثػرة الجدائخية1954 - 1962 , رسالة ماجدتيخ , كمية العمػم االندانية واالجتساعية , جامعة8 ماي1945 , قالسة2018 , ص80 . الهوامش ( 6 ) , عامخ رخيمة الثػرة الجدائخية والسغخب العخبي , السرادر, العجد1 , السخكد الػششي لمجراسات والبحث في الحخكة الػششية وثػرة اول نػفسبخ1954 , 1999 , ص160 . ( 7 ) مخيع صغيخ , مػاقف الجول العخبية مغ القزية الجدائخية1954 - 1962 , , دار الحكسة لمشذخ , الجدائخ 2010 , 163 ( 8 ) معسخ العايب , السرجر الدابق , ص133 . ( 9 )ال ا ال مة ا339 ( 5 ) زىخة ،دلباني وساشة تػنذ والسغخب لحل القزية الجدائخية سمسيا 1956 - 1962 , مجمة اول نػفسبخ , العجد 183 , , السؤسدة الػششية لمشذخ , الجدائخ2017 , ص29 . ( 6 ) , عامخ رخيمة الثػرة الجدائخية والسغخب العخبي , السرادر, العجد1 , السخكد الػششي لمجراسات والبحث في الحخكة ( 5 ) زىخة ،دلباني وساشة تػنذ والسغخب لحل القزية الجدائخية سمسيا 1956 - 1962 , مجمة اول نػفسبخ , العجد 183 , , السؤسدة الػششية لمشذخ , الجدائخ2017 , ص29 . ( 5 ) ز خ ب ي و ػ ذ خب و ل ي ي جد خي 1956 1962 , ج ول ػ بخ , 183 , , السؤسدة الػششية لمشذخ , الجدائخ2017 , ص29 . ( 6 ) , عامخ رخيمة الثػرة الجدائخية والسغخب العخبي , السرادر, العجد1 , السخكد الػششي لمجراسات والبحث في الحخكة الػششية وثػرة اول نػفسبخ1954 , 1999 , ص160 . ( 6 ) , عامخ رخيمة الثػرة الجدائخية والسغخب العخبي , السرادر, العجد1 , السخكد الػششي لمجراسات والبحث في الحخكة الػششية وثػرة اول نػفسبخ1954 , 1999 , ص160 . ( 7 ) مخيع صغيخ , مػاقف الجول العخبية مغ القزية الجدائخية1954 - 1962 , , دار الحكسة لمشذخ , الجدائخ 2010 , 163 ( 8 ) معسخ العايب , السرجر الدابق , ص133 . ( 9 ) عامخ رخيمة , السرجر الدابق ,ص339 . 281 ( 10 ) جخيجة السجاىج , الجدء االول , شخيق الػحجة السغخبية , العجد21 , 1 نيدان1958 , ص2 . ( 11 ) دمحم بجاوي , الثػرة الجدائخية والقانػن1960 - 1961 , ط2 , , دار الخائج لمكتاب , الجدائخ2005 , ص171 . ( 10 ) جخيجة السجاىج , الجدء االول , شخيق الػحجة السغخبية , العجد21 , 1 نيدان1958 , ص2 . ( 11 ) دمحم بجاوي , الثػرة الجدائخية والقانػن1960 - 1961 , ط2 , , دار الخائج لمكتاب , الجدائخ2005 , ص171 . ( 12 ) معسخ العايب , السرجر الدابق , ص137 . الهوامش ( 23 ) شارل ديغػل : ولج في مجيشة ليل الفخندية عام1890 التحق بالجير الفخندي بعج حرػلو عمى شيادة البكالػريا تخخج مغ السجرسة العدكخية عام1911 شارك في الحخب العالسية االولى ووقع اسيخا لجى االلسان , شارك في الحخب العالسية الثانية ثع عيغ مداعج لػزيخ الجفاع في حكػمة بػل ريشػ عام1940 رفس اليجنة وقاد السقاومة الفخندية مغ لشجن , عسل في الدياسة واسذ ال مجشة الػششية لفخندا الحخة عام1941 , تخاس حكػمة فخندا عام1944 وشكل حدب التجسع الذعبي الفخندي عام1947 وعاد الى سجة الحكع عام1958 بعج انقالب عام1958 واصبح رئيدا ً لمجسيػرية الفخندية الخامدة , تعخض لسحاولة اغتيال عام1961 ًمغ قبل مشطسة الجير الدخي بقي رئدا 282 282 لمجسيػرية الخامدة لغاية عام1969 اعتدل بعجىا الحياة الدياسية تػفي عام1970 . لمسديج يشطخ : عبج اوىاب الكيالي واخخون , السػسػعة الدياسية , ط1 , , السؤسدة العخبية لمجراسات والشذخ , بيخوت1974 ,ص272 - 273 . لمجسيػرية الخامدة لغاية عام1969 اعتدل بعجىا الحياة الدياسية تػفي عام1970 . لمسديج يشطخ : عبج اوىاب الكيالي واخخون , السػسػعة الدياسية , ط1 , , السؤسدة العخبية لمجراسات والشذخ , بيخوت1974 ,ص272 - 273 . ( 24 ) عبج هللا مقالتي , العالقات الجدائخية السغاربي ة واالفخيكية ,ج2 , , دار الدبيل لمشذخ والتػزيع , الجدائخ2009 , ص236 . ( 24 ) عبج هللا مقالتي , العالقات الجدائخية السغاربي ة واالفخيكية ,ج2 , , دار الدبيل لمشذخ والتػزيع , الجدائخ2009 , ص236 . ( 25 ) : فخحات عباس ولج في بمجة الذحشة في الجدائخ عام1899 درس الريجلة وانطع عام1933 الى حدب الذعب الجدائخي ،اسذ حدب االتحاد الجيسقخاشي عام1943 ، اعتقل بعجىا وافخج عشو ثع انطع الى جبية التحخيخ الجدائخي عام1956 ، انتخب رئيداً لمحكػمة السؤقتة في19 ايمػل1958 والتي اتخحت القاىخة مقخاً ليا ، ثع انتقمت الى تػنذ في عام1959 ، بقي في رئاسة الحكػمة السؤقتة حتى آب1961 انتخب عشج استقالل الجدائخ رئيداَ لمسجمذ الػششي التذخيعي تػفي عام1985 . لم سديج يشطخ :احسج عصية هللا ،القامػس الدياسي،ط3، دار الشيزة ا ، لعخبية ، القاىخة1968 ، ص1607 ،؛ سامي صالح الرياد، غيالن سسيخ شو فخحات عباس ودوره في الدياسة الجدائخية 1895 - 1985 ، مجمة جامعة تكخيت لمعمػم االندانية، ج19 ، العجد1 ، عام2012 . ( 26 ) رضا ميسػني , دور الػششييغ السغاربة في حخكة تحخيخ تػنذ والجدائخ مغ نياية الحخب العالسية الثانية الى غاية االستقالل , رسالة ماجدتيخ غيخ مشذػرة , كمية العمػم االندانية واالجتساعية والعمػم االندانية , جامعة الحاج , لخزخ , الجدائخ2012 , ص108 . الهوامش , ر الكمسة لمشذخ, بيخوت1981 , ص ( 37 ) , خ, بيخو ػب , ر ح ول و ب خبي بيغ خب ي ي , دمحم 1981 , ص 75 . ( 38 ) دمحم العخبي الدبيخي , ديغػل والرحخاء , كتابات سمدمة وممتكيات فرل الرحخاء والد , ياسة االستعسارية1998 , ص199 ،؛ لالشالع عمى التصػر االقترادي خالل حكبة االستقالل في تػنذ يشطخ: مؤيج محسػد حسج السذيجاني غيالن سسيخ شو، التجخبة االقترادية واالشتخاكية والخصط التش سػية في تػنذ1962 - 1970 ، مجمة جامعة تكخيت لمعمػم االندانية، ج28 ،، العجد الخابع2021 . ( 39 ) ولج دمحم ادريذ بغ السيجي الدشػسي في12 اذار1890 في واحة الجغبػب في ليبيا تعمع مغ صغخه القخاءة والكتابة وحفع القخان وتمقى دروس في الفقو والمغة وعشجما احتمت ايصاليا ليبي ا بقي في ليبيا وفي عام1913 ذىب لمحج وعاد الى ليبيا عام1915 اصبح اميخاً عمى بخقة عام1920 ثع سافخ لديارة ايصاليا ورجع عام1922 قبل بيعة اىل شخابمذ بعج عػدتو وىاجخ الى مرخ وفي عام1940 تحالف مع بخيصانيا وجشج السياجخيغ الميبييغ في مرخ في ما يدسى بجير التح خيخ الميبي الحي اشتخك في الحخب العالسية الثانية ضج ايصاليا ونجحت بخيصانيا بسداعجة جير التحخيخ الميبي مغ االنترار عمى ايصاليا واخخاجيا مغ ليبيا وفي24 كانػن االول1951 اصبح ممكاً عمى ليبيا واستسخ حكسو حتى عام1969 اذ قامت مجسػعة مغ الزباط العدكخييغ بأسقاط حكسو وتشحيتو عغ مشربو لجأ بعجىا الى مرخ وبقي فييا حتى وفاتو عام1983 . لمسديج يشطخ : صادق فاضل زغيخ الدىيخي , محسػد السشترخ ودوره الدياسي في ليبيا1903 - 1970 , , رسالة ماجدتيخ غيخ مشذػرة , جامعة بغجاد , كمية التخبية ابغ رشج2010 ص38 ( 40 ) , دمحم السيمي السغخب العخبي بيغ حدابات الجول ومصامح الذعػب , السرجر الدابق , ص75 ؛ جخيجة السجاىج , الخبد السدسػم , السرجر الدابق , ص1 . ( 40 ) , دمحم السيمي السغخب العخبي بيغ حدابات الجول ومصامح الذعػب , السرجر الدابق , ص75 ؛ جخيجة السجاىج , الخبد السدسػم , السرجر الدابق , ص1 . ( 41 ) جخيجة السجاىج , بتخول السغخب العخبي مذاكمو اليػم وغجاً , العجد27 , تسػز1958 , ص2 . ( 42 ) جخيجة السجاىج , االشساع االستعسارية ف ي الرحخاء , الجدء الخابع , العجد98 , 19 حديخان1961 , ص4 . ( 43 ) رشيج اوعيدى , كخاسات ىاتسػت الفيانز , حخب الجدائخ حدب فاعمييا الفخندييغ , تخجسة احسج وعسخ السعخاجي , دار القرة لمشذخ , الجدائخ , ص153 . الهوامش ( 27 ) حبيب حدغ المػلب , التػندييغ والثػرة الجدائخية1954 - 1962 , اشخوحة دكتػراه غيخ مشذػرة , كمية العمػم , االجتساعية واالندانية , جامعة الجدائخ2007 , ص136 . ( 28 ) عبج هللا مقالتي , العالقات الجدائخية السغاربية واالفخيكية , السرجر الدابق , ص243 - 246 . ( 29 ) زىخة دلباني ً, وساشة تػنذ والسغخب في حل القزية الجدائخية سمسيا1956 - 1962 , , مجمة اول نػفسبخ العجد183 , ,السؤسدة الػششية لمشذخ , الجدائخ2017 , ص30 - 31 ( 30 ) عسخ بػ ضخبة , تصػر الشذاط الخارجي لمثػرة الجدائخية1954 - 1962 , اشخوحة دكتػراه غبخ مشذػرة , قدع التاريخ , جامعة , جياللي ليابذ , سيجي بمعباس2010 ,ص177 - 178 ( 31 ) جخيجة السجاىج , الجدء االول , مؤتسخ تػنذ كبف بجأ وكيف انتيى , العجد26 , 2 , تسػز1958 , ص1 . ( 32 ) معسخ العايب , السرجر الدابق , ص195 . ( 33 ) , بذيخ سعجوني , الثػرة الجدائخية في الخصاب العخبي , الجدء االول دار مجني ل مشذخ والتػزيع , د.م , د. ت ص48 . ( 28 ) عبج هللا مقالتي , العالقات الجدائخية السغاربية واالفخيكية , السرجر الدابق , ص243 - 246 . ( 33 ) , بذيخ سعجوني , الثػرة الجدائخية في الخصاب العخبي , الجدء االول دار مجني ل مشذخ والتػزيع , د.م , د. ت ص48 . ( 34 ) دمحم لحدغ ازغيجي , مؤتسخ الرػمام وتصػر ثػرة التحخيخ الػششي الجدائخية1956 – 1962 , دار ىػمة لمشر , والتػزيع , الجدائخ2009 , ص256 . ( 35 ) احسج سعيػد , السرجر الدابق , ص110 . ( 36 ) دمحم قشصاري , استخاتيجية الدياس ية الفخندية في محاولة فرل الرحخاء الجدائخية , كتاب سمدمة ممتكيات فرل , الرحخاء في الدياسة االستعسارية1954 , ص165 . ( 34 ) دمحم لحدغ ازغيجي , مؤتسخ الرػمام وتصػر ثػرة التحخيخ الػششي الجدائخية1956 – 1962 , دار ىػمة لمشر , والتػزيع , الجدائخ2009 , ص256 . ( 35 ) احسج سعيػد , السرجر الدابق , ص110 . ( 36 ) دمحم قشصاري , استخاتيجية الدياس ية الفخندية في محاولة فرل الرحخاء الجدائخية , كتاب سمدمة ممتكيات فرل , الرحخاء في الدياسة االستعسارية1954 , ص165 . 283 283 ( 37 ) , دمحم السيمي , السغخب العخبي بيغ حدابات الجول ومصامح الذعػب , دار الكمسة لمشذخ, بيخوت1981 , ص 75 . الهوامش ( 53 ) عبج هللا مقالتي , العالقات الجدائخية السغاربية ابان الثػرة التحخيخية , السرجر الدابق , ص403 . ( 54 ) دمحم السيمي , السغخب العخبي بيغ حدابات الجول ومصامح الذعػب , السرجر الدابق , ص76 ؛ جخيجة السجاىج , الجدء االول , الخبد السدسػم . السرجر الدابق , ص1 . ( 55 ) , دمحم حخبي , جبية التحخيخ الػششي االسصػرة والػاقع , تخجسة كسيل قيرخ داغخ , دار الكمسة لمشذخ , بيخوت 1983 , 178 . ( 52 ) جخيجة الس جاىج , الجدء االول , الخبد السدسػم , العجد27 , 22 تسػز1958 , ص1 . ( 53 )ال ابق ج ال ي ية ال القات ال دائ ية ال غا بية ابان الث ة الت عبج هللا قالت03 ( 52 ) جخيجة الس جاىج , الجدء االول , الخبد السدسػم , العجد27 , 22 تسػز1958 , ص1 . ( 53 ) عبج هللا مقالتي , العالقات الجدائخية السغاربية ابان الثػرة التحخيخية , السرجر الدابق , ص403 . ( 55 ) , دمحم حخبي , جبية التحخيخ الػششي االسصػرة والػاقع , تخجسة كسيل قيرخ داغخ , دار الكمسة لمشذخ , بيخوت 1983 , 178 . ( 55 ) , دمحم حخبي , جبية التحخيخ الػششي االسصػرة والػاقع , تخجسة كسيل قيرخ داغخ , دار الكمسة لمشذخ , بيخوت 1983 , 178 . ( 55 ) , دمحم حخبي , جبية التحخيخ الػششي االسصػرة والػاقع , تخجسة كسيل قيرخ داغخ , دار الكمسة لمشذخ , بيخوت 1983 , 178 . ( 56 ) عبج هللا مقالتي , العالقات الجدائخية , السرجر الدابق , ص405 . ( 57 ) دمحم السيمي , السغخب العخبي , السرجر الدابق , ص76 . ( 58 ) جخيجة السجاىج , الجدء االول , الخبد السدسػم , السرجر الدابق , ص1 . (59) Redah Malek, L’Algérie à Ḗvain histoire des négociations secretes 1956-1962, ed Dahlab, 1994, p141. (59) Redah Malek, L’Algérie à Ḗvain histoire des négociations secretes 1956-1962, ed Dahlab, 1994, p141. (59) ( 60 ) , مجني بغ العخبي بجاوي , محكخات مجني بجاوي مجاىج وشاىج , دار ىػمو لمشذخ والتػزيع , الجدائخ2012 , ص215 . ( 60 ) , مجني بغ العخبي بجاوي , محكخات مجني بجاوي مجاىج وشاىج , دار ىػمو لمشذخ والتػزيع , الجدائخ2012 , ص215 . ( 61 ) . السرجر نفدو ( 62 ) دمحم حخبي , جبية التحخيخ الػششي , ص178 - 180 . الهوامش ( 41 ) جخيجة السجاىج , بتخول السغخب العخبي مذاكمو اليػم وغجاً , العجد27 , تسػز1958 , ص2 . ( 42 ) جخيجة السجاىج , االشساع االستعسارية ف ي الرحخاء , الجدء الخابع , العجد98 , 19 حديخان1961 , ص4 . ( 43 ) رشيج اوعيدى , كخاسات ىاتسػت الفيانز , حخب الجدائخ حدب فاعمييا الفخندييغ , تخجسة احسج وعسخ السعخاجي , دار القرة لمشذخ , الجدائخ , ص153 . ( 44 ) اسساعيل دبر , الدياسة العخبية والسػاقف الجولية تجاه الثػرة الجدائخية1954 - 1962 ,, دار ىػمة لمشذخ , الجدائخ2003 , ص111 . ( 44 ) اسساعيل دبر , الدياسة العخبية والسػاقف الجولية تجاه الثػرة الجدائخية1954 - 1962 ,, دار ىػمة لمشذخ , الجدائخ2003 , ص111 . ( 45 ) حػرية دمان , االستخاتيجية الفخندية في مػاجية الجعع السغاربي لمثػرة التحخيخية الجدائخية1954 - 1962 , اشخوحة دكتػراه غيخ مشذػرة , كمية , العمػم االندانية واالجتساعية , جامعة جيالني بػنعامة خسذ مميانة2017 , ص449 . ( 46 ) عبج هللا مقالتي , العالقات الجدائخية السغاربية ابان الثػرة التحخرية الجدائخية1954 - 1962 ’ اشخوحة دكتػراه غيخ مشذػرة , قدع التاريخ واالثار , جامعة مشتػري , , قدشصيشة2008 , ص385 . ( 47 ) أنذأت ىحه السشطسة بعج إصجار البخلسان الفخندي قانػن فرل ،الرحخاء وذلظ بتاريخ 10 كانػن الثاني 1957 ويخى واضعػىحا القانػن الحي جاء في 13 مادة ان اليجف الستػخي مغ إصجار ىحه الييئة ىػ العسل عمى 284 التصػيخ االقترادي والخقي االجتساعي لسشاشق الجسيػرية الفخندية وىي الجدائخ مػريتانيا والدػدان والتذاد وفيسا بعج تػنذ والسغخب :, يشطخ معسخ العايب , السرجر الدابق , ص192 . ( 48 ) ( شخكة ستخايداSTRABSA ) ىي جدء مغ شخكة كخيذ التي تسمظ الحكػمة الفخندية76 . مغ اسيسيا% يشطخ : جخيجة السجاىج , الخبد السدسػم , السرجر الدابق , ص1 . ( 49 ) , الحديغ بغ عيدى , البػرقيبية واليػية صخاع مذاريع , مكتبة تػنذ , تػنذ2015 , ص222 ( 50 ) , فيخنخ روف , بػ رقيبة والدياسية الخارجية لتػنذ السدتقمة , تخجسة الرحبي الثابت , السصبعة العرخية , تػنذ , د.ت , د ط ص248 . ( 51 ) , محفػظ قجاش , وتحخرت الجدائخ , تخيػ بشػر , دار االمة لمشذخ والتػزيع , الجدائخ2011 , . ؛ دمحم شصيبي العالقات الجدائخية التػندية ابان الثػرة التحخرية1954 - 1962 , , رسالة ماجدتيخ , قدع التاريخ , جامعة مشتػري , قدشصيشة2009 , ص111 . ( 52 ) جخيجة الس جاىج , الجدء االول , الخبد السدسػم , العجد27 , 22 تسػز1958 , ص1 . الهوامش ( 80 ) فاروق جياب , الحبيب بػرقيبة وسياستو تجاه الثػرة التحخيخية الجدائخية , اشخوحة دكتػراه غيخ مشذػرة, كمية , العمػم االندانية واالجتساعية ,جامعة بي بكخ بمقايج , تمسدان2017 , ص223 . ( 81 ) عبج هللا مقالتي , العالقات الجدائخية الس غاربية , السرجر الدابق , ص407 . ( 82 ) صالح العقاد , السغخب العخبي دراسة في تاريخو واوضاعو السعاصخة الجدائخ– تػنذ– , السغخب االقرى , مكتبة االنجمػ السرخية , القاىخة1985 , ص488 . ( 83 ) فيخنخ روف , بػرقيبة والدياسية الخارجية , ص240 . ( 84 ) الحبيب بػ رقيبة , , خصب ,الجدء الثامغ , نذخيات كتابة الجولة لألعالم1977 , ص143 - 144 . ( 85 ) السرجر نفدو , ص148 . ( 86 ) الصاىخ بمخػخة , الحبيب بػ رقيبة سيخة زعيع شاىج عمى العرخ , ط1 , , الجار الثقافية لمشذخ , القاىخة1999 , ص54 . ( 67 ) عثسانية فاشسة , بػرقيبة والثػرة الجدائخية1954 - 1962 , رسالة ماجدتيخ غيخ مشذػرة , كمية العمػم االندانية واالجتساعية , جامعة8 ماي1945 , قالسة2018 , ص80 - 81 . ( 68 ) عبج هللا , مقالتي , العالقات الجدائخية السغخبية , ص408 – 409 . ( 69 ) حػرية دمان , االستخاتيجية الفخندية , ص455 . ( 70 ) الحبيب بػرقيبة , خصب , الجدء الثامغ , السرجر الدابق , ص149 . ( 71 ) ,جػان جمدي , ثػرة الجدائخ , تخجسة عبج الخحسغ صجقي ابػ شالب , الجار السرخية لمتأليف والتخجسة , مرخ د.ت , ص215 . 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( 82 ) صالح العقاد , السغخب العخبي دراسة في تاريخو واوضاعو السعاصخة الجدائخ– تػنذ– , السغخب االقرى , مكتبة االنجمػ السرخية , القاىخة1985 , ص488 . ( 83 ) فيخنخ روف , بػرقيبة والدياسية الخارجية , ص240 . ( 84 ) الحبيب بػ رقيبة , , خصب ,الجدء الثامغ , نذخيات كتابة الجولة لألعالم1977 , ص143 - 144 . ( 85 ) السرجر نفدو , ص148 . ( 83 ) فيخنخ روف , بػرقيبة والدياسية الخارجية , ص240 . ( 84 ) الحبيب بػ رقيبة , , خصب ,الجدء الثامغ , نذخيات كتابة الجولة لألعالم1977 , ص143 - 144 . ( 85 ) السرجر نفدو , ص148 . ( 86 ) الصاىخ بمخػخة , الحبيب بػ رقيبة سيخة زعيع شاىج عمى العرخ , ط1 , , الجار الثقافية لمشذخ , القاىخة1999 , ص54 . الهوامش ( 63 ) دمحم تػفيق اسكشجر , الحخكة الجولية لجبية التحخيخ الػششي1954 - 1962 , مشذػرات الدائحي , , الجدائخ 2016 , ص99 . ( 64 ) مجني بغ العخبي بجاوي , السرجر الدابق , ص216 ( 65 ) اسساعيل دبر , الدياسة العخبية والسػاقف الجولية , ص111 - 112 . ( 66 ) دمحم السيمي , السرجر الدابق , ص79 . 62 ) دمحم حخبي , جبية التحخيخ الػششي , ص178 - 180 . 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( 91 ) : ساقية سيجي يػسف قخية تػندية تقع عمى الحجو د التػندية الجدائخية وليا اىسية كػنيا مخكد لسخور االسمحة ولمتسػيغ واالستخاحة شيجت القخية اشتباكات بيغ جير التحخيخ الػششي الجدائخي وقػات الجير الفخندي راح ضحيتيا عجد مغ الذيجاء وذكخت الدمصات الفخندية ان عجد قتالىا في ىحه االشتباكات ستة عذخ قتيال وخسذ اسخى اعتبخت الحكػمة الفخندية الحكػمة التػندية متػاشئة مع السقاوميغ الجدائخييغ وان االسخى الفخندييغ محتجديغ في تػنذ وردت عمى ذلظ بيجػم جػي عشيف في8 شباط1958 استسخ لداعة استخجمت فيو ما يقارب خسذ وعذخون شائخة حخبية في قرف القخية وقج قجر عجد ضحايا اليجػم بسا يقار ب ثسان وتدعػن شييجا بيشيع اشفال ونداء وعذخات مغ الجخحى وتجميخ مجرسة القخية وسػقيا ومشازليا . لمسديج يشطخ : امال ججي , خػلة بػزيان , العجوان الفخندي عمى , ساقية سيجي يػسف واثخه عمى السػقف التػندي اتجاه الثػرة , رسالة ماجدتيخ غيخ مشذػرة , جامعة العخبي التبدي , كمية العمػم االندانية واالجتساعية2016 ص45 – 48 . ىخ ذ وا ي خ با يا ن خ ي و خ ي غ بخ الحكػمة الفخندية الحكػمة التػندية متػاشئة مع السقاوميغ الجدائخييغ وان االسخى الفخندييغ محتجديغ في تػنذ وردت عمى ذلظ بيجػم جػي عشيف في8 شباط1958 استسخ لداعة استخجمت فيو ما يقارب خسذ وعذخون شائخة حخبية في قرف القخية وقج قجر عجد ضحايا اليجػم بسا يقار ب ثسان وتدعػن شييجا بيشيع اشفال ونداء وعذخات مغ الجخحى وتجميخ مجرسة القخية وسػقيا ومشازليا . لمسديج يشطخ : امال ججي , خػلة بػزيان , العجوان الفخندي عمى , ساقية سيجي يػسف واثخه عمى السػقف التػندي اتجاه الثػرة , رسالة ماجدتيخ غيخ مشذػرة , جامعة العخبي التبدي , كمية العمػم االندانية واالجتساعية2016 ص45 – 48 . 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https://openalex.org/W3043266001	http://www.scielo.br/j/rbeaa/a/mt7ZQY5cLsRjBrdCMncyFxs/?format=pdf&lang=en	English	null	Growth, yield and gas exchanges of ‘D’Angola’ plantain under different plant densities	Revista Brasileira de Engenharia Agrícola e Ambiental	2,020	cc-by	5,871	Growth, yield and gas exchanges of ‘D’Angola’ plantain under different plant densities Vagner A. Rodrigues Filho1, Sérgio L. R. Donato2, Alessandro M. Arantes2, Mauricio A. Coelho Filho3 & Marcelo B. Lima3 1 Sítio Barreiras Fruticultura Ltda./Setor Técnico, Missão Velha, CE, Brasil. E-mail: vagner@sitiobarreiras.com.br (Corresponding author) - ORCID 0000-0001-7702-7445 1 Sítio Barreiras Fruticultura Ltda./Setor Técnico, Missão Velha, CE, Brasil. E-mail: vagner@sitiobarreiras.com.br (Corresponding author) - ORCID: 0000 0001 7702 7445 2 Instituto Federal de Educação, Ciência e Tecnologia Baiano/Setor de Agricultura, Guanambi, BA, Brasil. E-mail: sergio.donato@ifbaiano.edu.br - ORCID: 0000-0002-7719-4662; alessandro.arantes@ifbaiano.edu.br - ORCID: 0000-0002-7520-9891 b 3 Embrapa Mandioca e Fruticultura, Cruz das Almas, BA, Brasil. E-mail: mauricio-antonio.coelho@embrapa.br - ORCID: 0000-0002-4667-5535; marcelo.lima@embrapa.br – ORCID: 0000-0002-9073-7246 ABSTRACT: Information about production, crop systems and economic viability for technical grown of plantain are scarce in Brazil. Few technologies developed specifically for plantain are available; thus, there are many adaptations of information on banana crops extrapolated to plantain. The objective of this study was to evaluate the growth, nutritional status, gas exchanges, water use efficiency and yield of ‘D’Angola’ plantain under different plant densities, in the first production cycle. The treatments consisted of six plant densities (1,111; 2,500; 2,777; 3,125; 3,571; and 4,166 plants ha-1), which were evaluated in a randomized block design with four repetitions. Vegetative growth, leaf nutrient concentrations at the flowering stage, gas exchanges (monthly) at two reading times, fruit yield and water use efficiency at harvest were evaluated. The nutritional status is not dependent on plant density. The vegetative growth varied, regardless of the plant density, whereas the leaf area index increased as the plant density was increased. The leaf temperature increased as the plant density was increased. The water use efficiency for fruit yield, as a function of plant density, fitted to a quadratic model, with the maximum value at 3,301 plants ha-1. The use of 3,333 plants ha-1 is recommended for plantain. Key words: Musa spp., dense planting, leaf area index, photosynthesis, mineral nutrition Editor responsible: Hans Raj Gheyi Ref. 224126 – Received 17 May, 2019 • Accepted 05 May, 2020 • Published 30 May, 2020 Revista Brasileira de Engenharia Agrícola e Ambiental Campina Grande, PB, UAEA/UFCG – http://www.agriambi.com.br ISSN 1807-1929 v.24, n.7, p.490-496, 2020 Introduction considering the reference evapotranspiration (ETo), calculated by the Penman-Monteith method modified by the FAO, using the crop coefficient of 1.4 after the flowering stage, according to Coelho et al. (2012), and a total water depth of 2,546 mm per cycle, complementing the rainfall depth (602 mm) that occurred during the experiment.h Information about production, crop systems and economic viability of plantain (Musa spp.) crops are scarce in Brazil. Few technologies developed specifically for plantain are available; thus, there are many adaptations of information on banana crops extrapolated to plantain. Although both the crops can be treated similarly, plantain have different growth habit, size, cycle and genetic. Treating plantain, technically, as banana can lead to inadequate experimental and productive results. The vegetative growth of the plants was evaluated monthly over the production cycle up to the flowering stage, totaling 10 evaluations, considering: plant height (PH), corresponding to the pseudostem length measured from the base to the apex; pseudostem perimeter at the ground level (PPGL); number of functional leaves in the plant (NLP), corresponding to leaves with at least 50% preserved area; total leaf area (TLA), considering the width and length of the leaf 3, according to the expression of Zucoloto et al. (2008) [TLA = 0.5187 (L×W×N) + 9603.5; R² = 0.89 (p ≤ 0.05)], where L and W are, respectively, the length and width of the leaf 3, and N is the number of leaves; and the leaf area index (LAI), considering the ratio between total leaf area and area per plant. Plantain production in Brazil needs technological solutions, such as determinations of spacings for high-density planting, which allows the expression of the productive potential of cultivars in use by producers, definition of irrigation and fertilization management practices, and mitigation of damages caused by pests and diseases. According to Rosales et al. (2008), the use of only one production cycle with high plant densities, up to 5,000 plants ha-1, has been recommended as a technical management for plantain crops in some great producing countries; and this short crop cycle is due to the vulnerability of these plants to damages in roots and rhizomes caused by nematodes and borers, which naturally limit the crop longevity. h The relative growth rates for PH (PHRGR), PPGL (PPGLRGR), and TLA (TLARGR) were determined using Eq. 1. Crescimento, produtividade e trocas gasosas de plátano ‘D’Angola’ sob diferentes densidades de plantio RESUMO: No Brasil, são escassas informações acerca dos dados de produção, sobre sistemas de cultivo e viabilidade econômica para exploração tecnificada de plátanos. Há pouca disponibilidade de tecnologias desenvolvidas especificamente para os plátanos, assim, há apenas adaptações de todo o conhecimento gerado para a cultura da bananeira extrapolada aos plátanos. Objetivou-se com o presente estudo avaliar o crescimento, o estado nutricional, as trocas gasosas, a eficiência de uso da água e a produtividade de Plátano ‘D’Angola’ submetido a diferentes densidades de plantio, no primeiro ciclo de produção. Os tratamentos, seis densidades de plantio: 1.111; 2.500; 2.777; 3.125; 3.571; 4.166 plantas ha-1, foram dispostos em um delineamento em blocos casualizados, com quatro repetições. Avaliaram-se: o crescimento vegetativo, os teores de nutrientes nas folhas no florescimento, as trocas gasosas (mensalmente) em dois horários de leitura, os caracteres de rendimento e a eficiência de uso da água na colheita. O estado nutricional independe da densidade de plantio. As características de crescimento vegetativo variaram independentemente das densidades de plantio, enquanto o índice de área foliar aumenta com a densidade de plantio. A temperatura foliar aumenta com o acréscimo da densidade de plantio. A eficiência de uso da água e a produtividade ajustam-se ao modelo quadrático em função da densidade de plantio, sendo o valor máximo com 3.301 plantas ha-1. Recomenda-se a utilização prática de 3.333 plantas ha-1. Palavras-chave: Musa spp., plantio adensado, índice de área foliar, fotossíntese, nutrição mineral Editor responsible: Hans Raj Gheyi Ref. 224126 – Received 17 May, 2019 • Accepted 05 May, 2020 • Published 30 May, 2020 Growth, yield and gas exchanges of ‘D’Angola’ plantain under different plant densities Growth, yield and gas exchanges of ‘D’Angola’ plantain under different plant densities 491 where: where: RGR - relative growth rate for PH, PPGL (cm cm-1 d-1), or TLA (m2 m-2 d-1); RGR - relative growth rate for PH, PPGL (cm cm-1 d-1), or TLA (m2 m-2 d-1); P1 - the PH, PPGL (cm), or TLA (m2) in the previous evaluation; P2 - the PH, PPGL (cm), or TLA (m2) of the current evaluation; and, Introduction ( ) ln P2 ln P1 RGR t − = ∆ (1) (1) Therefore, increases in plant density may increase fruit yield and water use efficiency for the plantain cultivar D'Angola, without losses in fruit quality. Thus, the objective of this study was to evaluate the growth, nutritional status, gas exchanges, water use efficiency and yield of ‘D’Angola’ plantain under different densities, in the first production cycle. Material and Methods macronutrient concentrations found were above the sufficiency ranges for plantain (Borges et al., 2016), which are (g kg-1): 23.3- 30.8 for N, 1.5-1.9 for P, 21.5-26.6 for K, 0.8-1.9 for S, 4.6-8.3 for Ca, and 3.0-4.2 for Mg. g g Gas exchanges were measured monthly in the plants, from the 90th to the 360th day after transplanting (replications), at two reading times (8:00 h and 14:00 h). The leaf 3 or 4 was considered for evaluations, according to its exposition to the Sun, using one plant per experimental unity and five strokes in the reading device after stabilization. A infrared gas analyzer (IRGA; LCPro+®; ADC BioScientific Ltda) was used to evaluate photosynthesis rate (A; µmol CO2 m-2 s-1); transpiration (E; mmol H2O m-2 s-1); stomatal conductance (gs; mol m-2 s-1); solar radiation on the leaf (Qleaf; µmol photons m-2 s-1); temperature on the leaf (Tleaf; °C); substomatal CO2 concentration (Ci; μmol CO2 mol-1); instantaneous water use efficiency (A/E); carboxylation efficiency (A/Ci); and photosynthesis quantum or photochemical efficiency (A/Qleaf). The micronutrient concentrations found (Table 2) were within the sufficiency ranges for plantain (Borges et al., 2016), except for Cu and Fe, which were above this range. These ranges are (g kg-1): 19.8-37.4 for B, 3.8-5.2 for Cu, 53.2-92.1 for Fe, 44.0-428.4 for Mn, and 13.7-19.6 for Zn. The plant density had no effect on plant height (PH) of the ‘D’Angola’ plantain evaluated, which increased linearly as a function of days after transplanting (DAT) (Figure 1A). The PH varied from 20.8 cm (60 DAT) to 357.97 cm (341 DAT). The estimated model showed an increase in PH of 1.08 cm for each DAT. Faria et al. (2010) evaluated PH of ‘D’Angola’ plantain plants at the flowering stage, in the same site, using a plant density of 1,111 plants ha-1, and found maximum PH of 337 cm, which was confirmed by the results found in the present study for the same density. These PH were higher than those found by Cavalcante et al. (2014) (279 cm) for the same plant density and phenological stage. The water use efficiency was also determined, considering the ratio between bunch and hand weights and total water depth applied to the crop over the production cycle (Coelho et al., 2013). Material and Methods ∆t - the time between evaluations (days). The experiment was conducted from October 2014 to January 2016 in an area of the Instituto Federal Baiano, in Guanambi, BA, Brazil (14º 17’ 27” S, 42º 46’ 53” W, and altitude of 537 m). The area had a Red Yellow Oxisol, mean texture, hypoxerophytic caatinga vegetation, and plain to slightly wavy relief. The Table 1 presents the chemical attributes of the soil. The mean annual rainfall depth of the region was 680 mm, and the mean annual temperature was 26 ºC. The weights of bunches and hands, number of fruits and hands per bunch, fruit weight, and diameter and external length of the fruit were determined after harvest (Santos et al., 2019). The central fruits of the external rows of the second hand were used for the measurements. Fruit yield was determined using Eq. 2 (Robinson & Galán Saúco, 2012). Pd FY 12 C   =     (2) The treatments consisted of six planting densities: 1,111 (2.0 × 4.5 m), 2,500 (2.0 × 2.0 m), 2,777 (2.0 × 1.8 m), 3,125 (2.0 × 1.6 m), 3,571 (2.0 × 1.4 m), and 4,166 plants ha-1 (2.0 × 1.2 m), arranged in a randomized block design with four repetitions, totaling 24 experimental plots. Each plot consisted of 20 plants (four rows of five plants), and the six plants of the two central rows were used for the evaluations. (2) FY - the fruit yield in Mg ha-1 cycle-1; Micro-propagated seedlings from ‘D’Angola’ plantain plants were used for the planting. Cultural practices were done as recommended for the crop by Rosales et al. (2008). The irrigations were done based on the crop evapotranspiration, Leaves were sampled at the flowering stage of the first production cycle, according to Rodrigues et al. (2010). Simple leaf samples were collected from each plant in the evaluation SOM - soil organic matter; CEC - cation exchange capacity; BS - base saturation Table 1. Chemical attributes of the soil of the experimental area before the planting of ‘D’Angola’ plantain R. Bras. Eng. Agríc. Ambiental, v.24, n.7, p.490-496, 2020. Vagner A. Rodrigues Filho et al. 492 area of each replication to form a composite sample. The samples were used to determine the N, P, K, Ca, Mg, S (g kg-1), B, Cu, Fe, Mn, and Zn (mg kg-1) concentrations. Material and Methods The statistical analyses of the data of the characteristics evaluated was done considering the following procedures: a) a randomized block design with a 6×10 split-plot arrangement was adopted for the results of vegetative characteristics, corresponding to six planting densities (plots) and 10 evaluation times (subplots); the data were subjected to analysis of variance, the interactions were evaluated according to their significance, and, then, the data were fitted to regression models or response surface; b) a randomized block design with six treatments (planting densities) was adopted for the results of fruit yield and leaf nutrient contents; the data were subjected to analysis of variance and, when significant, fitted to regression models; c) a randomized block design was adopted, consisting of a 6 × 2 factorial arrangement, six planting densities, and two reading times for the physiological characteristics evaluated at the flowering stage; the results were subjected to analysis of variance and the interactions were evaluated according to their significance; the means were compared by the F test and fitted to regression models. In all cases, the fitting to regression models considered the adequacy to the biological phenomenon studied, the R2 value, and the significance of regression coefficients by the t test. The plant density had no effect on the pseudostem perimeter at the ground level (PPGL) of the ‘D’Angola’ plantain plants; the means varied, with linear increases as a function of DAT (Figure 1B). The mean PPGL varied from 10.38 cm (60 DAT) to 84.84 cm (341 DAT). The estimated model showed an increase of 0.2696 cm in PPGL for each DAT. Arantes et al. (2010) found positive correlations between PPGL, bunch weight, and hand weight for plantain. Material and Methods Moreover, cultivars with higher PPGL usually have better root system development, and are less susceptible to tipping (Silva et al., 2002) and pseudostem breakage, which are current problems in plantain crops; thus, this is a desirable characteristic when selecting a plant density.hf The plant density had no effect on the number of leaves per plant (NLP) (Figure 1C) of the ‘D’Angola’ plantain plants; the means of the treatments as a function of DAT fitted to a quadratic model, with maximum NLP of 14.90 at 341 DAT.hl The NLP at the beginning of flowering stage is related to number of hands per bunch, whereas the NLP at full flowering is related to weights of hands and bunches (Robinson & Galán Saúco, 2012); at least six functional leaves are required for plantain at flowering stage (Barrera et al., 2009), whereas Prata- Anã banana at flowering require 10 to 12 leaves (Rodrigues et al., 2009) and Grande Naine banana require less than 12 leaves (Rodríguez González et al., 2012). Faria et al. (2010) evaluated plantain with spacing of 3.00 × 3.00 m (1.111 plants ha-1) and R. Bras. Eng. Agríc. Ambiental, v.24, n.7, p.490-496, 2020. Growth, yield and gas exchanges of ‘D’Angola’ plantain under different plant densities 493 Growth, yield and gas exchanges of ‘D’Angola’ plantain under different plant densities 493 - Significant at p ≤ 0.01 by t test Figure 1. Growth of plantain plants of the cultivar D'Angola under different plant densities as a function of days after transplanting (DAT) ficant at p ≤ 0.01 by t test gi p y Figure 1. Growth of plantain plants of the cultivar D'Angola under different plant densities as a function of days after transplanting (DAT) ntain plants of the cultivar D'Angola under different plant densities as a function of days after transplanting Figure 1. Growth of plantain plants of the cultivar D'Angola under different plant densities as a function of days after transplanting (DAT) found that ‘D’Angola’ plantain plants stand out with a mean of 14 leaves per plant at flowering, as confirmed by the data of the present study.hf was expected, since the RGR shows increases in PH, PPGL, and TLA expressed in units per DAT. This lower gain in these characteristics as a function of DAT is due to increases in shading caused by increases in leaf area index, which reduces the net assimilation rate (net photosynthetic rate minus the respiration and the photorespiration), which is essential to increase these plant attributes (Taiz et al., 2017).h The plant density had no effect on the total leaf area (TLA) of ‘D’Angola’ plantain plants; the means of the treatments increased linearly as a function of DAT (Figure 1D). The mean TLA varied from 1.18 m2 (60 DAT) to 14.49 m2 (341 DAT). The estimated model showed an increase in TLA of 0.0486 m2 for each DAT. The leaf area index (LAI) of the ‘D’Angola’ plantain plants increased linearly as the plant density and DAT were increased, showing a response surface (Figure 1E). The response surface estimated LAIs at 341 DAT of 2.79; 3.76; 3.95; 4.19; 4.50; and 4.92 for populations of 1,111; 2,500; 2,777; 3,125; and 4,166 plants ha-1, respectively. Regarding the relative growth rate (RGR), PHRGR and PPGLRGR data fitted to an inverse quadratic model as a function of DAT, while TLARGR fitted to a cubic model (Figures 1A, B and D). The higher PHRGR and PPGLRGR were found at 90 DAT, and the lowest at 300 DAT; there was a slight increase in the RGR after 300 DAT up to 341 DAT, which is a similar result to that of TLARGR. Results and Discussion The leaf nutrient concentrations of ‘D’Angola’ plantain in the different planting densities were similar (Table 2). The Table 2. Leaf macro and micronutrient concentrations in ‘D’Angola’ plantain grown under different plant densities Growth, yield and gas exchanges of ‘D’Angola’ plantain under different plant densities Growth, yield and gas exchanges of ‘D’Angola’ plantain under different plant densities Growth, yield and gas exchanges of ‘D’Angola’ plantain under different plant densities Photosynthetically active radiation absorption is higher in denser crops due to their higher LAI (Turner et al., 2007), which increases chlorophyll concentrations. However, the radiation on the plant base decreases, consequently decreasing the development of suckers (Israeli, 1995). Despite the linear increases in PH, PPGL, and TLA, in absolute values, the RGR presented different results, which R. Bras. Eng. Agríc. Ambiental, v.24, n.7, p.490-496, 2020. 494 Vagner A. Rodrigues Filho et al. Vagner A. Rodrigues Filho et al. for 3,301 plants ha-1; and maximum number of hands of 4.61 for 2,614 plants ha-1. According to Turner et al. (2007), the LAI of plantain varies from 2 to 5, and a plantain crop with LAI near 4.5, as found for the populations equal to or higher than 3.125 plants ha-1, intercepts approximately 90% of the radiation; and increasing the LAI to values above 4.5 has little benefit to the crop, since most radiation is already intercepted. The hand weight yield for plant density of 2,000 plants ha-1 was 12.06 Mg ha-1, which was a similar result to that found by Coelho et al. (2013), who reported 11.9 Mg ha-1 for the same plant density. The higher water use efficiencies found were 7.6 kg ha-1 cycle-1 mm-1 with plant density of 3,392 plants ha-1 for bunch weight yield (Figure 2D), 6.07 kg ha-1 cycle-1 mm-1 with 3,301 plants ha-1 for hand weight yield (Figure 2E).h The higher the LAI, the lower the cost with weed control, because the shading will be fast, decreasing weed emergence and maintaining a more favorable microclimate under the canopy of the crop.h The other variables evaluated at harvest - hand weight, number of hands, number of fruits, fruit external length, fruit diameter, fruit mean weight, number of days for harvest, number of days for flowering, and flowering to harvest period - as a function of plant densities did not fit to any model. The means found were 4.86 kg for hand weight, 4.23 for number of hands, 23.87 for number of fruits, 23.69 cm for fruit external The bunch (Figure 2A) and hand (Figure 2B) weights, water use efficiency for bunches (Figure 2C) and hands (Figure 2D), and number of hands (Figure 2E) of ‘D’Angola’ plantain plants as a function of plant density fitted to quadratic models.h i The estimates of the models showed a maximum bunch weight yield of 19.35 Mg ha-1 cycle-1 for plant density of 3,392 plants ha-1; maximum hand weight yield of 15.47 Mg ha-1 cycle-1 R. Bras. Eng. Agríc. Ambiental, v.24, n.7, p.490-496, 2020. p g y g y - Significant at p ≤ 0.01 by t test Figure 2. Fruit yield and water use efficiency of plantain plants D’Angola cultivar as a function of plant densities - Significant at p ≤ 0.01 by t test R. Bras. Eng. Agríc. Vagner A. Rodrigues Filho et al. Ambiental, v.24, n.7, p.490-496, 2020. Significant at p ≤ 0.01 by t test Figure 2. Fruit yield and water use efficiency of plantain plants D’Angola cultivar as a function of plant densities R. Bras. Eng. Agríc. Ambiental, v.24, n.7, p.490-496, 2020. gi p y Figure 2. Fruit yield and water use efficiency of plantain plants D’Angola cultivar as a function of plant densities R. Bras. Eng. Agríc. Ambiental, v.24, n.7, p.490-496, 2020. R. Bras. Eng. Agríc. Ambiental, v.24, n.7, p.490-496, 2020. Growth, yield and gas exchanges of ‘D’Angola’ plantain under different plant densities 495 air layer on the leaf, probably increasing leaf temperature in plants grown under high plant density. However, the thermal comfort in the interior of the canopy probably increases as the plant density is increased, because the leaf temperature is measured on the third leaf at the top of the canopy. length, 40.44 mm for fruit diameter, 256.64 g for fruit mean weight, 379.19 for number of days for harvest, 305.41 for number of days for flowering, and 73.78 for number of days from flowering to harvest. These results were lower than those found by Faria et al. (2010) in the same experimental site for a population of 1,111 plants ha-1. Photosynthesis rates, transpiration, stomatal conductance, solar radiation incidence on the leaf, substomatal CO2 concentration, carboxylation efficiency, instantaneous water use efficiency, and quantum efficiency of photosynthesis varied between reading times (Table 3). The photosynthetically active radiation incidence on the leaf (Qleaf) and internal CO2 concentration (Ci) were similar in both reading times. The Qleaf found were within the range that provides the maximum photosynthesis rates - 1,500 to 2,000 µmol photons m-2 s-1 (Turner et al. 2007).h Hand weight and number of hands are the main interests to the producer, since they are the unities used for marketing (Silva et al., 2006). However, hand weight yield represents the net fruit yield of the unit for marketing and should be used to define the better plant density, provided that the fruit diameter and length in the density chosen assure a suitable fruit for marketing. h The highest net fruit yield was found for the plant density of 3,301 plants ha-1, i.e., an planting spacing of 1.51×2.0 m, which resulted in a LAI of 4.32 at 341 DAT, which is a similar value to that recommended (4.5) by Turner et al. Vagner A. Rodrigues Filho et al. The optimum temperature for CO2 carboxylation in plants with C3 photosynthetic mechanism, such as plantain, is approximately 22 °C, whereas for growth and development it is approximately 27 °C (Robinson & Galán Saúco, 2012). The leaf temperature in ‘D’Angola’ plantain varied from 34 to 36 °C at 8:00 h, and from 41 to 43 °C at 14:00 h, as a function of plant densities (Figure 3), which were above of the optimum temperature for photosynthesis. According to Donato et al. (2015), increasing plant density is a technic that improves thermal comfort for banana plants; however, the leaf temperature of ‘D’Angola’ plantain increased as the plant density was increased. The increase in plant density decreases the exchanges of sensible heat within the plantain crop, because it decreases the air movement and removes the bordering - Significant at p ≤ 0.01 by t test - Significant at p ≤0.01 by t test The optimum temperature for CO2 carboxylation in plants with C3 photosynthetic mechanism, such as plantain, is approximately 22 °C, whereas for growth and development it is approximately 27 °C (Robinson & Galán Saúco, 2012). The leaf temperature in ‘D’Angola’ plantain varied from 34 to 36 °C at 8:00 h, and from 41 to 43 °C at 14:00 h, as a function of plant densities (Figure 3), which were above of the optimum temperature for photosynthesis. Carboxylase and oxygenase activities of rubisco are balanced by its kinetics, temperature, and CO2 and O2 substrate concentration. Under normal CO2 concentrations, increases in temperature change the kinetic constants of the rubisco and increase the oxygenation rate preferentially to carboxylation, consequently increasing photorespiration and decreasing net photosynthesis (Taiz et al., 2017), as also found by Arantes et al. (2016; 2018). - Significant at p ≤ 0.01 by t test Figure 3. Leaf temperature (Tleaf) measured at 8:00 h and 14:00 h on the third leaf of plantain plants D’Angola cultivar grown under different plant densities variables measured at 8:00 h and 14:00 h on the third leaf of ‘D’Angola’ plantain subjected to different Table 3. RT - Readig time (hours); QLeaf (μmol fótons m-2 s-1) - photosynthetically active solar radiation on the leaf; Ci (μmol CO2 mol-1) - internal CO2 concentration; E (mmol H2O m-2 s-1) - transpiration rate; gs (mol H2O m-2 s-1) - stomatal conductance; A (μmol CO2 m-2 s-1) - photosynthesis rate; A/Ci - CO2 carboxylation efficiency (μmol CO2 m-2 s-1/μmol CO2 mol-1); A/E (μmol CO2 m-2 s-1/mmol H2O m-2 s-1) - instantaneous water use efficiency ; A/Qleaf - photosynthesis photochemical efficiency; VPD - vapor pressure deficit (kPa); Means followed by the same letter in the columns are not different by the F test at p ≤ 0.05 Vagner A. Rodrigues Filho et al. (2007) for a solar radiation interception of 90% by the plant canopy. This denotes the feasibility of the spacing of 1.5×2.0 m, i.e., a population of 3,333 plants ha-1, to obtain a LAI of 4.34, which was confirmed by the maintenance of the fruit class for marketing, regardless of the plant density. The photosynthesis was higher at 8:00 h, and transpiration was higher at 14:00 h (Table 3), as also found by Arantes et al. (2016) for banana. The increase in temperature combined with a low air relative humidity - a high atmosphere vapor pressure deficit, which is common for the local semiarid conditions, mainly in September, October and February (Donato et al., 2015) - increases the evapotranspiration demand and affects all metabolic and physiological processes of the plant. The gas exchanges of the ‘D’Angola’ plantain fitted to an increasing linear model as a function of plant density and reading time for leaf temperature (Figure 3). The leaf temperature increased 0.001 °C when measured at 8:00 h, and 0.0007 °C when measured at 14:00 h, for each increase of plant in the population. The higher leaf temperature (Tleaf) at 14:00 h (Figure 3) affected the photosynthesis (A) by compromising the functioning of the enzymatic system, which causes stomatal closure by limiting the entry of CO2. Despite the lower stomatal conductance (gs), the transpiration (E) was higher at 14:00 h; and the instantaneous water use efficiency (A/E), photosynthesis photochemical efficiency (A/Qleaf), and carboxylation efficiency (A/Ci) were lower at 14:00 h, indicating an increase in the rubisco activity for oxygenase in detriment of carboxylation, when compared to those found in the 8:00 h readings (Donato et al., 2015; Arantes et al., 2016).h The higher leaf temperature (Tleaf) at 14:00 h (Figure 3) affected the photosynthesis (A) by compromising the functioning of the enzymatic system, which causes stomatal closure by limiting the entry of CO2. Despite the lower stomatal conductance (gs), the transpiration (E) was higher at 14:00 h; and the instantaneous water use efficiency (A/E), photosynthesis photochemical efficiency (A/Qleaf), and carboxylation efficiency (A/Ci) were lower at 14:00 h, indicating an increase in the rubisco activity for oxygenase in detriment of carboxylation, when compared to those found in the 8:00 h readings (Donato et al., 2015; Arantes et al., 2016). Vagner A. Rodrigues Filho et al. Physiological variables measured at 8:00 h and 14:00 h on the third leaf of ‘D’Angola’ plantain subjected to different plant densities RT - Readig time (hours); QLeaf (μmol fótons m-2 s-1) - photosynthetically active solar radiation on the leaf; Ci (μmol CO2 mol-1) - internal CO2 concentration; E (mmol H2O m-2 s-1) - transpiration rate; gs (mol H2O m-2 s-1) - stomatal conductance; A (μmol CO2 m-2 s-1) - photosynthesis rate; A/Ci - CO2 carboxylation efficiency (μmol CO2 m-2 s-1/μmol CO2 mol-1); A/E (μmol CO2 m-2 s-1/mmol H2O m-2 s-1) - instantaneous water use efficiency ; A/Qleaf - photosynthesis photochemical efficiency; VPD - vapor pressure deficit (kPa); Means followed by the same letter in the columns are not different by the F test at p ≤ 0.05 plant densities RT - Readig time (hours); QLeaf (μmol fótons m-2 s-1) - photosynthetically active solar radiation on the leaf; Ci (μmol CO2 mol-1) - internal CO2 concentration; E (mmol H2O m-2 s-1) - transpiration rate; gs (mol H2O m-2 s-1) - stomatal conductance; A (μmol CO2 m-2 s-1) - photosynthesis rate; A/Ci - CO2 carboxylation efficiency (μmol CO2 m-2 s-1/μmol CO2 mol-1); A/E (μmol CO2 m-2 s-1/mmol H2O m-2 s-1) - instantaneous water use efficiency ; A/Qleaf - photosynthesis photochemical efficiency; VPD - vapor pressure deficit (kPa); Means followed by the same letter in the columns are not different by the F test at p ≤ 0.05 R. Bras. Eng. Agríc. Ambiental, v.24, n.7, p.490-496, 2020. Vagner A. Rodrigues Filho et al. 496 Coelho, E. F.; Donato, S. L. R.; Oliveira, P. M. de; Cruz, A. J. S. Relações hídricas II: Evapotranspiração e coeficiente de cultura. In: Coelho, E. F. (ed). Irrigação da bananeira. Cruz das Almas: Embrapa Mandioca e Fruticultura, 2012. Cap 2. p.87‑117. The highest photochemical efficiency was found at 8:00 h. The quantum yield of photosynthesis in C3 plants is high up to approximately 30 °C, and it decreases in banana under temperatures above 34 °C (Robinson & Galán Saúco, 2012), explaining the lower quantum efficiency at 14:00 h because of the higher leaf temperatures observed in this reading time (Arantes et al., 2016; 2018). Coelho, E. F.; Oliveira, R. C. de; Pamponet, A. J. M. Necessidades hídricas de bananeira tipo Terra em condições de tabuleiros costeiros. Pesquisa Agropecuária Brasileira, v.48, p.1260-1268, 2013. https://doi.org/10.1590/S0100-204X2013000900010 Donato, S. L. R.; Coelho, E. F.; Santos, M. R. dos; Arantes, A. Literature Cited Rodríguez González, C.; Cayón Salinas, D. G.; Mira Castillo, J. J. Efecto del número de hojas funcionales a la floración sobre la producción de banano Gran Enano (Musa AAA Simmonds). Revista da Faculdad Nacional de Agronomia de Medellin, v.65, p.6585-6591, 2012. Arantes, A. de M.; Donato, S. L. R.; Silva, S. de O. e. Relação entre características morfológicas e componentes de produção em plátanos. Pesquisa Agropecuária Brasileira, v.45, p.224‑227, 2010. https://doi.org/10.1590/S0100-204X2010000200015 Arantes, A. de M.; Donato, S. L. R.; Siqueira, D. L.; Coelho, E. F. Gas exchange in ‘Pome’ banana plants grown under different irrigation systems. Engenharia Agrícola, v.38, p.197-207, 2018. https://doi. org/10.1590/1809-4430-eng.agric.v38n2p197-207/2018 Rosales, F. E.; Alvarez, J. M. Vargas, A.; Guia prática para La producción de plátano con altas densidades – Experiencias de América Latina y el Caribe. In: Rosales, F. E. (Ed). Montepellier: Bioversity Internacional. 2008. 24p. Arantes, A. de M.; Donato, S. L. R.; Siqueira, D. L.; Coelho, E. F.; Silva, T. S. Gas exchange in different varieties of banana prata in semi- arid environment. Revista Brasileira de Fruticultura, v.38, p.1-12, 2016. https://doi.org/10.1590/0100-29452016600 Santos, M. R. dos; Donato, S. L. R.; Magalhães, D. B.; Cotrim, M. P. Precocity, yield and water-use efficiency of banana plants under planting densities and irrigation depths, in semiarid region. Pesquisa Agropecuária Tropical, v. 49, p. e53036, 2019. https://doi.org/10.1590/1983-40632019v4953036 Barrera, J.; Cayón, G.; Robles, J. Influencia de la exposición de las hojas y el epicarpio de frutos sobre el desarrollo y la calidad del racimo de plátano Hartón (Musa AAB Simmonds). Agronomía Colombiana, v.27, p.73-79, 2009. Silva, J. T. A. da; Borges, A. L.; Dias, M. S. C.; Costa, E. L. da; Prudêncio, J. M. Diagnóstico nutricional da bananeira ‘Prata Anã’ para o Norte de Minas Gerais. Belo Horizonte: EPAMIG, 2002.16p. Boletim Técnico, 70 Borges, A. L.; Albuquerque, A. F. A.; Coelho, E. F.; Lima, M. B.; Donato, S. L. R.; Rodriguez, M. A. D.; Silva, S. O. E.; Cordeiro, Z. J. M. Sistema de produção: Cultivo de plátanos (Bananeiras tipo Terra). Cruz das Almas: Embrapa Mandioca e Fruticultura, 2016 (Sistema de Produção - Versão Eletrônica). Available on: <https:// www.spo.cnptia.embrapa.br/conteudo?p_p_id=conteudoportlet_ WAR_sistemasdeproducaolf6_1ga1ceportlet&p_p_ lifecycle=0&p_p_state=normal&p_p_mode=view&p_p_ col_id=column-1&p_p_col_count=1&p_r_p_-76293187_ sistemaProducaoId=8701&p_r_p_-996514994_topicoId=10001> Accessed on: Nov.2016. Silva, S. de O. e; Pires, E. T.; Pestana, R. K. N.; Alves, J. da S.; Silveira, D. de C. Avaliação de clones de banana cavendish. Ciência e Agrotecnologia, v.30, p.832-837, 2006. https://doi.org/10.1590/ S1413-70542006000500002 Taiz, L.; Zeiger, E.; Møller, I. M.; Murphy, A. Vagner A. Rodrigues Filho et al. M.; Rodrigues, M. G. V. Eficiência de uso da água em bananeira. Informe Agropecuário, v.36, p.46-59, 2015. Conclusions 1. The nutritional status of ‘D’Angola’ plantain is not dependent on plant density. 1. The nutritional status of ‘D’Angola’ plantain is not dependent on plant density. Faria, H. C. de; Donato, S. L. R.; Pereira, M. C. T.; Silva, S. de O. E. Avaliação fitotecnica de bananeiras tipo Terra sob irrigação em condições semiáridas. Ciência e Agrotecnologia, v.34, p.830-836, 2. Variations in plant height, pseudostem perimeter at the ground level, number of functional leaves, and total leaf area of ‘D’Angola’ plantain are not dependent on the plant density, whereas the leaf area index increases as the plant density is increased. 2010. https://doi.org/10.1590/S1413-70542010000400006f Israeli, Y.; Plaut, Z.; Schwartz, A. Effect of shade on banana morphology, growth and production. Scientia Horticulturae, v. 62, p. 45-56, 1995. https://doi.org/10.1016/0304-4238(95)00763-J 3. Leaf temperature of ‘D’Angola’ plantain increases as the plant density is increased, and is higher in the afternoon.h Robinson, J. C.; Galán Saúco, V. Plátanos y bananas. 2.ed. Madrid: Mundi-Prensa, 2012. 321p. t 4. The maximum estimated fruit hand weight yield of the plantain was 15.45 Mg ha-1 cycle-1 for a population of 3,386 plants ha-1, which provided a leaf area index of 4.38; whereas the highest estimated water use efficiency found was 6.07 kg ha-1 cycle-1 mm-1 for a plant density of 3,301 plants ha-1.h Rodrigues, M. G. V.; Dias, M. S. C.; Pacheco, D. D. Influência de diferentes níveis de desfolha na produção e qualidade dos frutos da bananeira “Prata-Anã”. Revista Brasileira de Fruticultura, v.31, p.755-762, 2009. https://doi.org/10.1590/S0100-29452009000300019 g y y 5. The use of 3,333 plants ha-1 with spacing of 1.5×2.0 m is recommended for ‘D’Angola’ plantain crops. 5. The use of 3,333 plants ha-1 with spacing of 1.5×2.0 m is recommended for ‘D’Angola’ plantain crops. Rodrigues, M. G. V.; Pacheco, D. D.; Natale, W.; Silva, J.T. da. Amostragem foliar da bananeira 'Prata-Anã'. Revista Brasileira de Fruticultura, v.32, p.321-325, 2010. https://doi.org/10.1590/S0100- 29452010005000039 R. Bras. Eng. Agríc. Ambiental, v.24, n.7, p.490-496, 2020. Literature Cited Fisiologia e desenvolvimento vegetal. 6.ed. Porto Alegre: Artmed, 2017. 858p. WAR_sistemasdeproducaolf6_1ga1ceportlet&p_p_ lifecycle=0&p_p_state=normal&p_p_mode=view&p_p_ col_id=column-1&p_p_col_count=1&p_r_p_-76293187_ sistemaProducaoId=8701&p_r_p_-996514994_topicoId=10001> Accessed on: Nov.2016. Turner, D. W.; Fortescue, J. A.; Thomas, D. S. Environmental physiology of the bananas (Musa spp.). Brazilian Journal of Plant Physiology, v.19, p.463‑484, 2007. https://doi.org/10.1590/S1677- 04202007000400013 Cavalcante, M. de J. B.; Andrade Neto, R. de C.; Ledo, A. da S.; Gondim, T. M. de S.; Cordeiro, Z. J. M. Manejo fitotécnico da bananeira, cultivar D'Angola (AAB), visando ao controle da Sigatoka-negra. Revista Caatinga, v.27, p.201-208, 2014. Zucoloto, M.; Lima, J. S. de S.; Coelho, R. I. Modelo matemático para estimativa da área foliar total de bananeira Prata-Anã. Revista Brasileira de Fruticultura, v.30, p.1152‑1154, 2008. https://doi. org/10.1590/S0100-29452008000400050 R. Bras. Eng. Agríc. Ambiental, v.24, n.7, p.490-496, 2020.
https://openalex.org/W2968540994	https://europepmc.org/articles/pmc6720783?pdf=render	English	null	What If Pregnancy Is Not Seventh Heaven? The Influence of Specific Life Events during Pregnancy and Delivery on the Transition of Antenatal into Postpartum Anxiety and Depression	International journal of environmental research and public health/International journal of environmental research and public health	2,019	cc-by	6,667	International Journal of Environmental Research and Public Health International Journal of Environmental Research and Public Health International Journal of Environmental Research and Public Health Judith Aris-Meijer 1,, Claudi Bockting 2,3, Ronald Stolk 1, Tjitte Verbeek 4 , Chantal Beijers 5, Mariëlle van Pampus 6 and Huibert Burger 4,5, Judith Aris-Meijer 1,, Claudi Bockting 2,3, Ronald Stolk 1, Tjitte Verbeek 4 , Chantal Beijers 5 Mariëlle van Pampus 6 and Huibert Burger 4,5, 1 Department of Epidemiology, University of Groningen, University Medical Center Groningen, 9700 RB Groningen, The Netherlands g 2 Department of Clinical Psychology, University of Groningen, 3584 CS Groningen, The Netherlands 3 D f Cli i l d H l h P h l U h U i i 3512 JE U h Th N h l d 2 Department of Clinical Psychology, University of Groningen, 3584 CS Groningen, The Netherlands 3 Department of Clinical and Health Psychology, Utrecht University, 3512 JE Utrecht, The Netherlands 4 Department of General Practice, University of Groningen, University Medical Center Groningen, 9700 RB Groningen, The Netherlands 2 Department of Clinical Psychology, University of Groningen, 3584 CS Groningen, The Netherlands 3 Department of Clinical and Health Psychology, Utrecht University, 3512 JE Utrecht, The Netherlands 4 Department of General Practice, University of Groningen, University Medical Center Groningen, 9700 RB Groningen, The Netherlands 3 Department of Clinical and Health Psychology, Utrecht University, 3512 JE Utrecht, The Netherland 4 Department of General Practice University of Groningen University Medical Center Groningen 4 Department of General Practice, University of Groningen, University Medical Center Groningen, 9700 RB Groningen, The Netherlands g 5 Interdisciplinary Center Psychopathology and Emotion Regulation, University of Groningen, University Medical Center Groningen, 9700 RB Groningen, The Netherlands y g g 6 Department of Obstetrics and Gynecology, OLVG, 1011 BM Amsterdam, The Netherlands Correspondence: j.l.aris@umcg.nl (J.A.-M.); h.burger@umcg.nl (H.B.) * Correspondence: j.l.aris@umcg.nl (J.A.-M.); h.burger@umcg.nl (H.B.) 1. Introduction Reproductive age is a period of life in which women are vulnerable to the impact of symptoms of anxiety or depression. During and outside pregnancy alike, prevalence rates of these symptoms range from 8 to 15% [1–12]. Antenatal symptoms of anxiety or depression are the most important risk factor for the occurrence of these symptoms postpartum [3,13–17], which in turn has been associated with insecure mother–child attachment [18,19]. In addition, these symptoms during pregnancy have been associated with several obstetric adverse outcomes in the child, such as preterm birth and low birth weight [20–22], as well as emotional, cognitive, and behavioral problems [13,22–26]. Well-known risk factors for antenatal anxiety or depression are a history of anxiety or depression, low partner support, lower socioeconomic status, speciﬁc personality traits, and major life events [9,27,28]. Studies among pregnant women commonly classiﬁed recent major life events as general types of events during pregnancy [14,29], whereas other studies focused on pregnant women who were facing stress due to speciﬁc conditions, that is, obstetric complications [30,31]. A few population-based studies, however, have shown that speciﬁc pregnancy-related events are likely to increase symptoms of anxiety or depression during pregnancy [11,32,33]. Childbirth itself can also be considered a major life event, especially when the delivery is complicated, for example, when the baby is delivered by an emergency caesarean section or the baby is admitted to the neonatal intensive care unit. During the past decade, there is a growing but inconclusive body of literature on the associations between delivery complications and postpartum symptoms of anxiety and depression [34–43]. A few large population-based cohort studies (n > 5000) found that experiencing obstetric events during pregnancy or events that were related to the condition of the newborn (i.e., low birth weight, preterm delivery, congenital malformations, admission to the hospital) increased the risk of symptoms of depression in the postpartum period [33,44]. However, both studies underline the consensual idea that a history of symptoms of depression is the main risk factor for symptoms of depression at a later moment in time. Nevertheless, there is no answer yet to the question to what extent the combination of antenatal symptoms of anxiety or depression and speciﬁc life events during pregnancy or delivery is associated with postpartum symptoms of anxiety or depression. Received: 17 May 2019; Accepted: 30 July 2019; Published: 9 August 2019 Received: 17 May 2019; Accepted: 30 July 2019; Published: 9 August 2019 Abstract: Introduction: Postpartum symptoms of anxiety and depression are known to have a negative impact on mother and child, and major life events constitute a major risk factor for these symptoms. Abstract: Introduction: Postpartum symptoms of anxiety and depression are known to have a negative impact on mother and child, and major life events constitute a major risk factor for these symptoms. We aimed to investigate to what extent speciﬁc life events during pregnancy, delivery complications, unfavorable obstetric outcomes, and antenatal levels of anxiety or depression symptoms were independently associated with postpartum levels of anxiety and depression symptoms. Methods: Within a prospective population-based cohort study (n = 3842) in The Netherlands, antenatal symptoms of anxiety or depression were measured at the end of the ﬁrst trimester and at ﬁve months postpartum. Antenatal life events were assessed during the third trimester, and information on delivery and obstetric outcomes was obtained from midwives and gynecologists. Linear regression analyses were performed to quantify the associations. Results: Antenatal levels of both anxiety and depression symptoms were associated with postpartum levels of anxiety and depression symptoms. Life events related to health and sickness of self or loved ones, to the relation with the partner or conﬂicts with loved ones, or to work, ﬁnance, or housing problems were signiﬁcantly associated with higher postpartum levels of anxiety symptoms (p < 0.001) and depression symptoms (p < 0.001) adjusted for antenatal levels. No statistically signiﬁcant results were observed for pregnancy-related events, delivery complications, or unfavorable obstetric outcomes. Conclusions: Women with increased antenatal levels of anxiety or depression symptoms are at increased risk of elevated levels of both postpartum depression and anxiety symptoms. Experiencing life events during pregnancy that were not related to the pregnancy was associated with higher levels of anxiety and depression in the postpartum period, as opposed to pregnancy-related events, delivery complications, or unfavorable obstetric outcomes. These results suggest that events during pregnancy but not related to the pregnancy and birth are a highly important predictor for postpartum mental health. Keywords: antenatal anxiety and depression symptoms; postpartum anxiety and depression symptoms; mode of delivery; neonatal outcomes; life events Int. J. Environ. Res. Public Health 2019, 16, 2851; doi:10.3390/ijerph16162851 www.mdpi.com/journal/ijerph Int. J. Environ. Res. Public Health 2019, 16, 2851 2 of 11 1. Introduction Using a large population-based cohort study, we investigated to what extent speciﬁc life events during pregnancy, delivery complications, unfavorable obstetric outcomes, and antenatal levels of anxiety or depression symptoms independently contribute to the risk of these symptoms in the postpartum period and whether they interact. 2.1. Sample Data were drawn from the prospective population-based Pregnancy, Anxiety and Depression (PAD) Study [11,12], which was designed to investigate symptoms of anxiety or depression and risk factors for antenatal and postpartum symptoms of anxiety or depression. Midwives and gynecologists of the collaborating primary obstetric care centers (n = 109) or hospitals (n = 7) invited pregnant women at the ﬁrst or second visit to participate. It was impossible to establish how many women were actually invited to the study, as we had no insight into the total number of pregnant women who visited the primary obstetric care center or hospital for the ﬁrst consult. The number of included women was however considerably lower than we expected based on the number of participating centers. A survey among participating midwives indicated that, for the vast majority, time pressure was the main reason to not hand out the invitations to all visiting women, and that women who were under suspicion of having symptoms of either anxiety or depression were not speciﬁcally invited. Therefore, we have no reason to believe that, with respect to characteristics relevant to the study, responders and non-responders diﬀered in any considerable way. After written informed consent was obtained, women were requested to ﬁll out online baseline questionnaires at the end of the ﬁrst 3 of 11 Int. J. Environ. Res. Public Health 2019, 16, 2851 trimester, and online follow-up assessments at the end of the second and third trimesters of pregnancy, as well as at ﬁve months postpartum. The medical ethical board of the University Medical Center Groningen approved the PAD-based study. Data used for the current study were collected between May 2010 and March 2015. Women who were at least four months postpartum were eligible to be included, as participants had the opportunity to ﬁll out the follow-up questionnaire online between four and seven months postpartum. Exclusion criteria for the current sample were study withdrawal (n = 1669), not consenting to retrieve information from their midwives (n = 1669), no data on baseline and postpartum levels of anxiety and depression symptoms (n = 827), and no data on experienced life events during pregnancy or delivery (n = 1391). This resulted in a sample of 2450 women. Of these women, 2003 (81.8%) ﬁlled out the follow-up anxiety and depression questionnaires at ﬁve months postpartum. 2.1. Sample For postpartum measures of anxiety and depression symptoms, responders were more often multiparous (p < 0.02). For postpartum measures of depression symptoms only, responders generally completed a higher education (p < 0.03). In addition, non-responders scored higher on antenatal measures of anxiety and depression compared to women who did respond to the postpartum follow-up questionnaire, although mean scores on antenatal anxiety and depression measures were below the prevailing cut-oﬀs for both responders and non-responders. Women who did not respond to the follow-up questionnaire had experienced more general life events during pregnancy compared to women who did respond (p < 0.01), although the means diﬀered less than one event for all categories. 2.2. Measurements Baseline levels of anxiety and depression symptoms measured at 12 weeks of estimated gestational age (range 5–19) and at ﬁve months postpartum (range 4–7) were analyzed. Life events during pregnancy were assessed during the third trimester. Maternal age and educational level were assessed at baseline. Educational attainment level was deﬁned as the highest completed education and divided into four categories, namely, elementary or lower tracts of secondary education, higher tracts of secondary education, higher vocational education, and university education. Socioeconomic position was calculated as the equally weighted average of the educational attainment level of the respondent, her partner, and their total income. Antenatal and postpartum symptoms of anxiety were measured using the six-item state measuring version of the Spielberger State-Trait Anxiety Inventory (STAI) [45]. Scores are on a scale from 20 to 80, with scores of ≥42 indicating an increased risk of anxiety [45]. To measure antenatal and postpartum symptoms of depression, we used the Dutch version of the ten-item Edinburgh Postnatal Depression Scale (EPDS) [46]. Scores range from 0 to 30. In line with Matthey et al. [47], we considered antenatal scores of 13 or above and postpartum scores of 10 or above to indicate risk of minor or major depression. Data on life events that were encountered during pregnancy were collected using a 46 item questionnaire, developed in the Avon Longitudinal Study of Parents and Children (ALSPAC) [48]. We divided the events into four categories, namely, (A) work, ﬁnance, or housing problems, (B) partner relation or conﬂicts with loved ones, (C) health and sickness of self or loved ones, and (D) pregnancy-related. The ﬁrst three comprise a total of 26 items on employment, illness or death of loved ones, and marital problems. The latter category includes seven items that are related to the current pregnancy, for example, undergoing tests on potential congenital anomalies of the fetus, being told that it is a twin pregnancy, ﬁnding out that the partner does not want to have the baby, or ﬁnding out that something that happened might be harmful for the fetus. Information on mode of delivery and obstetric outcome was retrieved from the midwives’ or gynecologists’ reports. We deﬁned delivery complications as instrumental vaginal delivery (i.e., forceps or vacuum extraction) or caesarean section (elective or emergency) relative to unassisted vaginal delivery. 4 of 11 Int. J. Environ. Res. 2.2. Measurements Public Health 2019, 16, 2851 Events that relate to the newborn were deﬁned in terms of unfavorable obstetric outcomes and included preterm delivery (<37 weeks gestational age) and small for gestational age (i.e., >37 weeks gestational age but <2500 grams). Gestational age was derived from midwives’ and gynecologists’ reports. They calculated this age based on last menstrual period, and then conﬁrmed it with an ultrasound. 2.3. Statistical Analyses Descriptive statistics for demographic variables, number of life events, and levels of anxiety or depressive symptoms were calculated. To allow for valid comparison of eﬀect sizes, we created z-scores for the antenatal symptoms of anxiety and depression. Subsequently, we performed a series of multivariable linear regression analyses with postpartum scores on STAI and EPDS to quantify the associations under study. The analyses quantiﬁed change in anxiety and depression symptom levels by adjusting postpartum levels for antenatal levels. In a separate analysis, potential confounders were added (i.e., socioeconomic position and nulliparity). Confounders were chosen based on their well-known association with the outcomes. To investigate associations with symptoms of anxiety or depression speciﬁcally, all analyses were additionally adjusted for depressive symptoms in the analysis of anxiety, and vice versa. Lastly, we assessed whether associations of speciﬁc life events, delivery complications, and unfavorable obstetric outcomes of the newborn with postpartum symptoms of anxiety or depression were modiﬁed by antenatal anxiety or depression symptoms. 3.1. General Descriptives Mean levels for anxiety and depression were rather stable from the antenatal to the postpartum period (Table 1), although symptoms of depression signiﬁcantly increased between the antenatal and postpartum period (mean diﬀerence = 0.36, p < 0.001). 5 of 11 Int. J. Environ. Res. Public Health 2019, 16, 2851 Table 1. Characteristics of women in the study. 3.1. General Descriptives Characteristics All Women in the Study n = 3842 No Antenatal Symptoms n = 1948 Antenatal Anxiety and Depression Symptoms n = 85 Symptoms of Antenatal Anxiety, No Antenatal Depression n = 198 Symptoms of Antenatal Depression, No Antenatal Anxiety n = 7 * Age at inclusion, mean (min–max) 30 (18–45) 30 (18–45) 30 (19–42) 31 (18–43) 32 (26–36) Nulliparity, n (%) 1431/3682 (38.9%) 768/1915 (40.1%) 28/82 (34.1%) 78/198 (39.4%) 0/5 Educational level Elementary or lower tracts secondary 154/1879 (8.1%) 93/1360 (6.8%) 12/54 (22.2%) 18/119 (15.1%) 1/5 Higher tracts secondary 567/1879 (30.0%) 403/1360 (29.6%) 15/54 (27.8%) 41/119 (34.5%) 3/5 Higher vocational or university 1158/1879 (61.6%) 864/1360 (63.5) 27/54 (50.0%) 60/119 (50.4%) 1/5 No events related to the pregnancy, n (%) 1218/2449 (49.7%) 856/1685 (50.8%) 23/65 (35.4%) 65/147 (44.2%) 2/5 median (min–max) 1 (0–5) 0 (0–5) 1 (0–3) 1 (0–4) 1 (0–3) No events related to health and sickness of self or loved ones, n (%) 1516/2407 (63.0%) 1080/1666 (64.8%) 29/64 (45.3%) 77/142 (54.2%) 2/5 median (min–max) 0 (0–8) 0 (0–8) 1 (0–6) 0 (0–4) 2 (0–5) No events related to partner relation or conﬂicts with loved ones, n (%) 1909/2480 (77.0%) 1366/1705 (80.1) 29/65 (44.6%) 91/147 (61.9%) 1/5 median (min–max) 0 (0–4) 0 (0–4) 1 (0–4) 0 (0–4) 1 (0–2) No events related to work, ﬁnance, or housing problems, n (%) 1151/2453 (46.9%) 845/1688 (50.1%) 11/64 (17.2%) 40/146 (27.4%) 1/5 median (min–max) 1 (0–9) 0 (0–8) 2 (0–8) 1 (0–7) 2 (0–6) Mode of delivery, n (%) spontaneous vaginal delivery 1737/2343 (74.1%) 1190/1613 (73.8%) 41/59 (69.5%) 107/139 (77.0%) 4/5 vacuum or forceps extraction 244/2343 (10.4%) 173/1613 (10.7%) 6/59 (10.2%) 10/139 (7.2%) 0/5 caesarean section 362/2343 (15.5%) 250/1613 (15.5%) 12/59 (20.3%) 22/139 (5.8%) 1/5 Unfavorable obstetric outcomes: preterm or low birth weight, n (%) 325/3634 (8.9%) 128/1889 (6.8%) 4/82 (4.9%) 22/196 (11.2%) 0/7 Baseline level anxiety (STAI), mean (SD) b 33 (9.20) 30 (5.97) 57 (8.90) 48 (5.07) 39 (2.62) Postpartum level anxiety (STAI), mean (SD) c 32 (10.27) 31 (8.91) 48 (14.86) 40 (11.47) 37 (4.19) Baseline level depression (EPDS), mean (SD) d 4 (3.77) 4 (2.67) 16 (2.95) 8 (2.61) 13 (0.38) Postpartum level depression (EPDS), mean (SD) e 5 (4.06) 4 (3.46) 12 (5.58) 7 (4.88) 7 (2.50) SD, standard deviation; STAI, Spielberger State-Trait Anxiety Inventory (min–max 20–80); EPDS, Edinburgh Postnatal Depression Scale (min–max 0–30). SD, standard deviation; STAI, Spielberger State-Trait Anxiety Inventory (min–max 20–80); EPDS, Edinburgh Postnatal Depression Scale (min–max 0–30). * Due to the low number of women in this group, no percentages were calculated. 3.1. General Descriptives * Due to the low number of women in this group, no percentages were calculated. Int. J. Environ. Res. Public Health 2019, 16, 2851 6 of 11 3.2. Regression Analyses of Associations between Experienced Life Events and Postpartum Levels of Anxiety or Depression 3.2. Regression Analyses of Associations between Experienced Life Events and Postpartum Levels of Anxi r Depression 3.2. Regression Analyses of Associations between Experienced Life Events and Postpartum Levels of Anxiety or Depression Antenatal symptoms of anxiety and depression were statistically signiﬁcantly associated with postpartum levels of anxiety (p < 0.001) (Table 2). Levels of postpartum anxiety increased more when women had higher levels of antenatal anxiety compared to antenatal depression; a score of one standard deviation higher on antenatal anxiety increased the postpartum anxiety scores, reaching 5.68 points, compared to the 2.78 point increase on postpartum levels of anxiety for one standard deviation higher on antenatal levels of depression. Likewise, levels of postpartum depression symptoms increased more when women had higher antenatal depression levels compared to anxiety (2.47 versus 0.57, respectively). Table 2. Associations of postpartum levels of anxiety and depression with antenatal symptoms, speciﬁc life events, and delivery complications. N = 3842. Variables Postpartum Anxiety Symptoms (N = 283) Postpartum Depression Symptoms (N = 92) B (95% CI) p-Value B (95% CI) p-Value Anxiety baseline 5.68 (5.27, 6.09) <0.001 0.57 (1.81, 2.28) <0.001 Depression baseline 2.78 (2.16, 3.40) <0.001 2.47 (2.31, 2.62) <0.001 Events—pregnancy 0.11 (−0.44, 0.66) 0.702 −0.05 (−0.26, 0.16) 0.610 Events—health and sickness of self or loved ones 1.03 (0.55, 1.52) <0.001 0.36 (0.18, 0.54) <0.001 Events—partner relation or conﬂicts with loved ones 1.73 (1.06, 2.40) <0.001 0.50 (0.24, 0.76 <0.001 Events—work, ﬁnance, or housing problems 1.01 (0.70, 1.32) <0.001 0.23 (0.10, 0.35) <0.001 Mode of delivery Spontaneous vaginal delivery vs. vacuum or forceps −0.61 (−2.02, 0.80) 0.399 0.48 (−0.07, 1.02) 0.085 Spontaneous vaginal delivery vs. caesarean section 0.01 (−1.18, 1.19) 0.992 0.23 (−0.23, 0.68) 0.326 Preterm delivery or small for gestational age 0.90 (−1.02, 2.81) 0.359 0.29 (−0.43, 1.01) 0.434 Multivariable linear regression analyses. Antenatal levels of anxiety and depression were standardized by calculating z-scores. Analyses on symptoms of postpartum anxiety were adjusted for baseline anxiety levels. Analyses on symptoms of postpartum depression were adjusted for baseline depression levels. CI, conﬁdence interval. Table 2. Associations of postpartum levels of anxiety and depression with antenatal symptoms, speciﬁc life events, and delivery complications. 3.1. General Descriptives N = 3842. Multivariable linear regression analyses. Antenatal levels of anxiety and depression were standardized by calculating z-scores. Analyses on symptoms of postpartum anxiety were adjusted for baseline anxiety levels. Analyses on symptoms of postpartum depression were adjusted for baseline depression levels. CI, conﬁdence interval. Neither the number of life events related to the pregnancy, nor the mode of delivery nor obstetric outcomes of the newborn were signiﬁcantly associated with symptoms of anxiety and depression in the postpartum period. Adding potential confounders did not notably change the associations. When adjusting for postpartum levels of anxiety, all associations between life events and postpartum depression lost their statistical signiﬁcance. For anxiety, this was only true for life events that were related to sickness and health of self or loved ones and life events related to the partner relation or a conﬂict with loved ones. 3.3. Moderation of the Associations between Experienced Life Events and Postpartum Levels of Anxiety or Depression by Antenatal Levels of Anxiety and Depression 4. Discussion The present study conﬁrmed previous research showing that antenatal symptoms of anxiety or depression during pregnancy are strongly associated with symptoms in the postpartum period. We showed that experiencing life events that were not related to the pregnancy, the mode of delivery, or the newborn were associated with elevated levels of anxiety and depression in the postpartum period. The association between postpartum levels of anxiety and events related to sickness and health of self or loved ones was found to be moderated by antenatal levels of anxiety. Standardized eﬀect sizes for levels of antenatal anxiety and depression were the highest of all predictor variables, indicating that antenatal symptomatology is the most important risk factor for having symptoms postpartum. This is in line with previous studies [3,13–15,17]. In our study, the categories of life events that related to health and sickness of self or loved ones, to the partner relation or a conﬂict with loved ones, or to work, ﬁnance, or housing problems (i.e., all categories that were not related to the pregnancy, delivery, or newborn) were associated with change in levels of anxiety and depression symptoms into the postpartum period. In the general population, housing problems have been found to increase feelings of stress [49]. Moreover, foreclosure induced a decline in mental health [50]. In addition, involuntary job loss and the past economic recession have been associated with higher suicidal rates [51,52]. However, this was not studied speciﬁcally in pregnant women and was especially found to be more prevalent in men. Experiencing a major recent life event is widely considered to be an important risk factor for depression. We therefore hypothesized that childbirth could be considered a major life event when maternal and neonatal outcomes were complicated. Surprisingly, neither the events related to the pregnancy, to the delivery, nor to the condition of the newborn were associated with change in levels of anxiety and depression symptoms. For mode of delivery, there is an inconsistent pattern of ﬁndings in the literature, which may be due to methodological limitations such as small sample sizes or the fact of not distinguishing planned and unplanned caesareans [37]. Some recent studies did ﬁnd associations between mode of delivery and depressed mood or comparable negative feelings [42,43], whereas several other studies underline our ﬁndings [34,35,39,40]. 4. Discussion Another explanation for this inconsistency in ﬁndings may be that childbirth can be perceived as highly stressful and even result in post-traumatic stress syndrome (PTSD), even after experiencing a successful birth without any complications during childbirth or adverse maternal or neonatal outcomes [53]. We did not measure perception of the childbirth or coping strategies, but this could be an important factor for future research. 3.3. Moderation of the Associations between Experienced Life Events and Postpartum Levels of Anxiety or Depression by Antenatal Levels of Anxiety and Depression The associations between number of life events and levels of postpartum anxiety symptoms were moderated by antenatal anxiety symptoms in the case of events related to sickness and health of self or loved ones (B 0.453, 95% CI 0.001–0.906, p = 0.049), and events related to work, ﬁnance, or housing problems (B 0.408, 95% CI 0.016–0.801, p = 0.041). However, after correcting for baseline levels of depression, none of the associations remained signiﬁcant (B 0.158, 95% CI −0.124 to 0.763, p = 0.158 and B 0.242, 95% CI −0.145 to 0.629, p = 0.221, respectively). The same trend was observed for the associations between antenatal life events related to work, ﬁnance, and housing problems and postpartum levels of depression symptoms (B 0.153, 95% CI 0.008–0.298, p = 0.039 versus B 0.144, 95%CI −0.001 to 0.288, p = 0.052). Int. J. Environ. Res. Public Health 2019, 16, 2851 7 of 11 7 of 11 Limitations and Strengths Some limitations must be borne in mind. First, symptoms of anxiety and depression were based on self-report questionnaires. Although both are commonly used in the identiﬁcation of symptoms and have shown to have good validity [45,46], no clinical diagnostic tools were used in the present study to establish the severity of symptoms. Second, life events were assessed using a retrospective self-report checklist, which may have been prone to recall bias through its potential link with symptoms at the time of the assessments [6]. Third, as the included midwifery practices and hospitals represent the general population in The Netherlands, and as we aimed to invite all pregnant women in their ﬁrst trimester at the clinics, the included women could represent all women in The Netherlands, and by extension most Western countries. However, the vast majority of women in our sample had a high educational level (>60% have a higher vocational or university degree), which is not a solid representation of the general population. The obtained percentages of the main outcome measures, that is, postpartum anxiety and depression symptoms, were however in line with previous studies. Finally, although the total sample was large, we encountered a relatively high percentage of missing data on the outcome measures for the postpartum levels of depression and anxiety (18%). As the statistical power to demonstrate associations with a change in levels of anxiety and depression may thus be limited, these analyses should be considered exploratory. 8 of 11 Int. J. Environ. Res. Public Health 2019, 16, 2851 However, a major strength of the present study is the large, prospective, population-based cohort (n = 2450). Additionally, to our knowledge, this study was the ﬁrst to investigate the role of speciﬁc life events that are either more general or related to the pregnancy, delivery, or newborn, in relation to the speciﬁc symptoms of anxiety or depression in the postpartum period. 5. Conclusions Our results indicate that the most important predictor for postpartum symptoms of anxiety or depression are elevated symptoms of antenatal anxiety or depression. Experiencing life events during pregnancy that were related to health and sickness of self or loved ones, to the partner relation or a conﬂict with loved ones, or to work, ﬁnance, or housing problems, was found to be associated with an increase in postpartum levels of anxiety and depression compared to antenatal levels. Midwives may play an important role in aiding women during pregnancy on coping with symptoms of anxiety or depression, and with the experience of such life events, in order to prevent postpartum levels of anxiety and depression from rising. Experiencing life events during pregnancy that were related to the pregnancy, the mode of delivery, or the newborn was found not to be associated with an increase in postpartum levels of anxiety and depression. As perception of childbirth may be an important factor here, further research in this area should focus on quantifying complicated childbirth, and should take measures of resilience, coping strategies, and perception of childbirth into account. Author Contributions: Conceptualization, H.B., C.B. (Claudi Bockting); methodology, H.B., C.B. (Claudi Bockting), R.S., J.A.-M.; validation, J.A.-M., H.B.; formal analysis, J.A.-M.; investigation, J.A.-M., T.V., C.B. (Chantal Beijers); data curation, H.B., J.A.-M., T.V., C.B. (Chantal Beijers); writing—original draft preparation, J.A.-M.; writing—review and editing, C.B. (Claudi Bockting), R.S., T.V., C.B. (Chantal Beijers), M.v.P., H.B.; supervision, R.S., C.B. (Claudi Bockting), H.B.; project administration, H.B.; funding acquisition, C.B. (Claudi Bockting), H.B. Funding: ZonMw: 120520013. Conﬂicts of Interest: The authors declare no conﬂict of interest. 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https://openalex.org/W2598562513	https://link.springer.com/content/pdf/10.1007%2Fs10910-017-0743-y.pdf	English	null	Encoded in vivo time signals from the ovary in magnetic resonance spectroscopy: poles and zeros as the cornerstone for stability of response functions of systems to external perturbations	Journal of mathematical chemistry	2,017	cc-by	34,888	J Math Chem (2017) 55:1110–1157 DOI 10.1007/s10910-017-0743-y ORIGINAL PAPER B Dževad Belki´c Dzevad.Belkic@ki.se 1 Department of Oncology-Pathology, Karolinska Institute, Building P-9, 2nd Floor, P.O. Box 260, 17176 Stockholm, Sweden 2 School of Community and Global Health, Claremont Graduate University, Claremont, CA, USA 3 Institute for Prevention Research, Keck School of Medicine, University of Southern California, Alhambra, CA, USA Encoded in vivo time signals from the ovary in magnetic resonance spectroscopy: poles and zeros as the cornerstone for stability of response functions of systems to external perturbations The overriding motivation is to improve survival for women afﬂicted with this malignancy. The reported results further hone the Padé-designed methodology for practical applications of in vivo MRS and, therefore, are anticipated to help in achieving the stated goal. six and the eleven model orders. Without spectra averaging and extrapolation, there were noticeable variances for the six and the eleven model orders with regard to all the variables under study. The present analysis and results have important implications for expediting the robust quantiﬁcation by the FPT. All the analysis herein was applied to in vivo MRS data encoded on a 3T scanner from a borderline serous cystic ovarian tumor. This clinical problem has been chosen in light of the urgent need to develop effective methods for early detection of ovarian cancer. The overriding motivation is to improve survival for women afﬂicted with this malignancy. The reported results further hone the Padé-designed methodology for practical applications of in vivo MRS and, therefore, are anticipated to help in achieving the stated goal. Keywords Magnetic resonance spectroscopy · Ovarian cancer diagnostics · Mathematical optimization · Fast Padé transform Encoded in vivo time signals from the ovary in magnetic resonance spectroscopy: poles and zeros as the cornerstone for stability of response functions of systems to external perturbations Dževad Belki´c1 · Karen Belki´c1,2,3 Received: 25 January 2017 / Accepted: 2 March 2017 / Published online: 20 March 2017 © The Author(s) 2017. This article is an open access publication Abstract In order to handle encoded data from magnetic resonance spectroscopy (MRS), advanced signal processing methods are vital. This is presently carried out using the fast Padé transform (FPT) applied to in vivo MRS time signals encoded from the ovary. We examine the essential features of the response function, namely the spec- tral poles and zeros, as the key to stability of the system to external excitations. Noise is separated from signal by reliance upon the multi-level signature of Froissart dou- blets. Our focus is upon eliminating the oversensitivity to alterations in model order K, through systematic examination of poles and zeros, as well as Padé-reconstructed total shape spectra, spectral parameters and component shape spectra. This comprehensive examination of convergence of all variables under study includes investigation of the combined role of spectra averaging and time signal extrapolation. Comparisons are made throughout between the results for six model orders (K = 575, 585, . . . , 625) with an increment of ten and eleven model orders (K = 575, 580, . . . , 625) with an increment of 5. It is demonstrated that for the reconstructed poles and zeros, as well as for magnitudes and phases, spectra averaging and Padé-based extrapolation of time signals are essential for the stability of the system and for the accurate retrieval of res- onances. Full convergence is achieved when spectra averaging and extrapolation are applied together. Spectra averaging and extrapolation are also shown to be needed to obtain stabilized results to the level of stochasticity for the component spectra for the 123 1111 J Math Chem (2017) 55:1110–1157 six and the eleven model orders. Without spectra averaging and extrapolation, there were noticeable variances for the six and the eleven model orders with regard to all the variables under study. The present analysis and results have important implications for expediting the robust quantiﬁcation by the FPT. All the analysis herein was applied to in vivo MRS data encoded on a 3T scanner from a borderline serous cystic ovarian tumor. This clinical problem has been chosen in light of the urgent need to develop effective methods for early detection of ovarian cancer. Abbreviations Ace Acetic acid AcNeu N-acetyl neuraminic acid Ala Alanine au Arbitrary units Av Average Bet Betaine BW Bandwidth Cho Choline Cit Citrate cm Centimeter Cr Creatine Crn Creatinine DFT Discrete Fourier transform DWI Diffusion weighted imaging E Ersatz FFT Fast Fourier transform FID Free induction decay FPT Fast Padé transform FWHM Full width at half maximum Glc Glucose Gln Glutamine Glu Glutamate Gly Glycine GPC Glycerophosphocholine His Histidine HLSVD Hankel–Lanczos singular value decomposition IDFT Inverse discrete Fourier transform IFFT Inverse fast Fourier transform Iso Isoleucine Ace Acetic acid AcNeu N-acetyl neuraminic acid Ala Alanine au Arbitrary units Av Average Bet Betaine BW Bandwidth Cho Choline Cit Citrate cm Centimeter Cr Creatine Crn Creatinine DFT Discrete Fourier transform DWI Diffusion weighted imaging E Ersatz FFT Fast Fourier transform FID Free induction decay FPT Fast Padé transform FWHM Full width at half maximum Glc Glucose Gln Glutamine Glu Glutamate Gly Glycine GPC Glycerophosphocholine His Histidine HLSVD Hankel–Lanczos singular value IDFT Inverse discrete Fourier transfo IFFT Inverse fast Fourier transform Iso Isoleucine 12 3 J Math Chem (2017) 55:1110–1157 1112 Lac Lactate Leu Leucine Lip Lipid Lys Lysine Mann Mannose Met Methionine m-Ins Myoinositol MR Magnetic resonance MRI Magnetic resonance imaging MRS Magnetic resonance spectroscopy NAA N-acetyl aspartate NEX Number of excitations NPV Negative predictive value PC Phosphocholine PCr Phosphocreatine ppm Parts per million PPV Positive predictive value PRESS Point resolved spectroscopy Pyr Pyruvate Rad Radian RMS Root-mean-square SNR Signal-noise ratio SNS Signal-noise separation SRI Spectral region of interest SVD Singular value decomposition TE Echo time Thr Threonine TR Repetition time TVUS Transvaginal ultrasound Tyr Tyrosine U Usual Val Valine WET Water suppression through enhanced T1 effects Lac Lactate Leu Leucine Lip Lipid Lys Lysine Mann Mannose Met Methionine m-Ins Myoinositol MR Magnetic resonance MRI Magnetic resonance imaging MRS Magnetic resonance spectroscopy NAA N-acetyl aspartate NEX Number of excitations NPV Negative predictive value PC Phosphocholine PCr Phosphocreatine ppm Parts per million PPV Positive predictive value PRESS Point resolved spectroscopy Pyr Pyruvate Rad Radian RMS Root-mean-square SNR Signal-noise ratio SNS Signal-noise separation SRI Spectral region of interest SVD Singular value decomposition TE Echo time Thr Threonine TR Repetition time TVUS Transvaginal ultrasound Tyr Tyrosine U Usual Val Valine WET Water suppression through enhanc 1 Introduction For analyzing and interpreting encoded data from magnetic resonance spectroscopy (MRS), advanced signal processing methods are of utmost importance. Detection of ovarian cancer at an early stage is an urgent public health challenge for which mathe- matical optimization of MRS holds particular promise [1–8]. In this paper, we study the crucial characteristics of the response function by robust and accurate reconstruc- tions of the spectral poles and zeros, as the prime determinants of stability of the system. Separation of signal from noise is achieved by binning two distinct groups of the reconstructed data. Such a disentangling relies upon: (i) the sign of the imag- inary frequencies or the related spin–spin relaxation times, (ii) the metric (pole-zero 123 1113 J Math Chem (2017) 55:1110–1157 distance), (iii) the inﬁnitesimal smallness of amplitudes, that are oscillation intensi- ties of time signal components, or equivalently, the strength of the poles, and (iv) the stability or resilience of magnitudes of amplitudes as well as their poles and zeros to changes in model order (amounting to alteration of the truncation level of the total acquisition time) [9–12]. The present investigation is carried out by the fast Padé transform (FPT) applied to in vivo MRS time signals encoded from the ovary. Several advantageous properties of the FPT, in particular, spectra averaging and time signal extrapolation applied in concert are further scrutinized relative to our previous studies. This is accomplished by adding the stability test of the reconstructed zeros (for the ﬁrst time) to that of the retrieved poles, and by signiﬁcantly increasing the number of model orders K. distance), (iii) the inﬁnitesimal smallness of amplitudes, that are oscillation intensi- ties of time signal components, or equivalently, the strength of the poles, and (iv) the stability or resilience of magnitudes of amplitudes as well as their poles and zeros to changes in model order (amounting to alteration of the truncation level of the total acquisition time) [9–12]. The present investigation is carried out by the fast Padé transform (FPT) applied to in vivo MRS time signals encoded from the ovary. Several advantageous properties of the FPT, in particular, spectra averaging and time signal extrapolation applied in concert are further scrutinized relative to our previous studies. This is accomplished by adding the stability test of the reconstructed zeros (for the ﬁrst time) to that of the retrieved poles, and by signiﬁcantly increasing the number of model orders K. 1 Introduction We begin with a presentation of the mathematical features of the FPT for advanced signal processing in MRS, and proceed to succinctly review the most salient features of the clinical problem, which is the need for reliable and timely detection of ovarian cancer. The progress made thus far applying advanced signal processing through the FPT–MRS for ovarian cancer diagnostics will then be summarized, setting the stage for the analysis of the present paper. 1.1 How MRS time signals can be processed 1.1.1 The fast Fourier transform: the most frequently used method for signal processing in MRS Thus far, all the available clinical magnetic resonance (MR) scanners have relied upon the fast Fourier transform (FFT) to generate the stick spectrum in the frequency domain from the encoded free induction decay (FID) digitized curves encoded in the time domain: FFT: Fm = N−1 n=0 cne−2πimn/N, 0 ≤m ≤N −1. (1a) (1a) The ﬁxed mth Fourier grid frequency is 2πm/T and this expression for Fm is a single polynomial.Thesetofcomplex-valuedtimesignalpoints{cn}representstheexpansion coefﬁcients of the Fourier polynomial (1a). The total signal length is N, whereas τ is the sampling time (dwell time, sampling rate) and the total signal duration is T (or the total acquisition time), such that T = Nτ. The bandwidth (BW) is the inverse of τ. The variables exp(±2πimn/N) are the undamped sinusoids and cosinusoids (nmτ/T = nm/N). As per t = nτ(0 ≤n ≤N −1), the continuous time variable t is discretized. With signal lengths in a composite form such as N = 2k(k = 1, 2, 3, . . .) only Nlog2N multiplications are needed, and this provides computational efﬁciency to the FFT algorithm. Insofar as N is non-composite, i.e. any positive integer, the FFT from (1a) becomes the discrete Fourier transform (DFT), in which case much larger N 2 multiplications are needed. Through the inverse Fourier transform (IFFT) for N = 2k(k = 1, 2, 3, . . .) the time signal can be retrieved from Fm by the Nlog2N computational complexity: 12 3 1114 J Math Chem (2017) 55:1110–1157 IFFT: cn = 1 N N−1 m=0 Fme2πimn/N, 0 ≤n ≤N −1. (1b) (1b) On the other hand, for N as non-composite, i.e. N ̸= 2k(k = 1, 2, 3, . . .), Eq. (1b) becomes the inverse discrete Fourier transform (IDFT) with N 2 multiplications. On the other hand, for N as non-composite, i.e. N ̸= 2k(k = 1, 2, 3, . . .), Eq. (1b p q ecomes the inverse discrete Fourier transform (IDFT) with N 2 multiplications. Because the total shape stick spectrum is produced from pre-assigned frequencies whose minimal separation is ﬁxed by the given total acquisition time T , there are no interpolation capabilities in the FFT. Signal-noise ratio (SNR) is inescapably worsened with attempts to improve resolution in the FFT, since this entails the use of longer T . 1.1 How MRS time signals can be processed However, at longer T , the physical part of the MRS time signal will have decayed and encoding would primarily bring more noise, especially in clinical MR scanners (1.5 and 3T) [13]. Further contributing to poor resolution and low SNR in the FFT is the lack of extrapolation capabilities, such that information is limited to that obtained from c0 up until the ﬁnal encoded signal point, cN−1. A “zero-ﬁlling” procedure is often done, whereby the time signal length is doubled by adding zeros to the original set {cn}(0 ≤n ≤N −1). A seeming advantage of this device may be to generate a better appearing spectrum, at the price of producing sinc-type artiﬁcial oscillations on the baseline. Essentially, however, no new information is provided by zero-ﬁlling and, thus, resolution is de facto not improved at all. Yet another reason for the poor resolution of the FFT is its linearity, due to which, noise is imported as intact from the time to the frequency domain. The most fundamental drawback is that the FFT is exclusively a non-parametric processor. Consequently, only total shape spectra, or equivalently, envelopes can be generated thereby. When post-processing through ﬁtting is subsequently done, guesses are made about the number and nature of the resonances present in the total shape spec- trum. Obviously, such a procedure is highly susceptible to error. Thus, within the FFT plus any ﬁtting approach, estimation of metabolite concentrations, i.e. the endpoint of greatest diagnostic interest, will often be inaccurate and clinically unreliable [9]. 1.1.2 The fast Padé transform: highly suitable for processing MRS time signals Therefore, without any computation, we see that the FPT is deﬁned from the onset to outperform the resolution of the FFT. It has been shown in practice [9,12] that usually: (i) the N/2 data points of the input time signal of length N sufﬁced for the FPT to match the resolution of the FFT, which used the full FID ({cn}, 0 ≤n ≤N −1), and (ii) using any ﬁxed number N ′(≤N) of the FID points, the resolving power of the FPT was doubled relative to that of the FFT, which also employed the same N ′ data entries. This resolution enhancement of the FPT is not necessarily limited to comparisons with the FFT alone. Quite the contrary, any ﬁtting technique which starts from the FFT and adjusts some free parameters to the given Fourier envelope would, at best, match the resolution of the FFT and, in turn, would be inferior to the FPT. In the FFT plus ﬁtting techniques for quantiﬁcation of spectra in MRS, the usu- ally employed ansatz is the real part of the given Fourier envelope, which is taken as being comprised of real-valued Lorentzian or Gaussian shapelines. This procedure is misleading since it can only give some incorrect estimates of spectral parameters and metabolite concentrations. The reason is that the real part of a spectral envelope reconstructed from encoded time signals is always a mixture of absorptive and disper- sive shapelines. Such mixtures are due to the interference effects caused by non-zero phases of amplitudes of the nodal oscillations. Different metabolites have different phases and, thus, no external universal phase correction, be it of the zero (ϕ0) or the ﬁrst (ϕ1) order or both applied together can simultaneously yield exclusively absorp- tive shapelines for all the components of the given envelope. On the other hand, the FPT avoids altogether the bias of favoring absorption envelopes by extracting the poly- nomial quotient directly from the complex-valued input z-transform. This means that all the reconstructed spectral parameters are affected by the non-zero phases, implying that the real and imaginary parts of spectral shapelines contain both the absorption and dispersion modes mixed together. This, in turn, would produce the metabolite concentrations that conform to the true content from the encoded time signals. There is yet another issue of critical importance for data analysis in MRS, espe- cially when it comes to the diagnostic interpretation of ﬁndings. 1.1.2 The fast Padé transform: highly suitable for processing MRS time signals 1.1.2.1 General advantages of the FPT relative to the FFT For the Maclaurin series (or equivalently, the z-transform) of a given function, the fast Padé transform, FPT, is introduced as the unique quotient of two polynomials. Uniqueness is guaranteed by requiring that the ﬁrst M terms alone of the expansion of the said polynomial ratio match exactly the ﬁrst 2M terms of the input z-transform. As such, this very deﬁnition of the FPT simultaneously provides both the error and the resolution improvement. The error itself is an explicitly given series beginning with the (M + 1)st term. Suppose that the input z-transform of length N (even) is truncated at M = N/2, while the remainder is forgotten as if it were non-existent. Then, the FPT can use just the ﬁrst available half (M = N/2) of the input data. Nevertheless, the extracted polynomial quotient in the FPT will have an expansion whose ﬁrst 2M terms would coincide with the ﬁrst N terms of the non-truncated z-transform. As such, the FPT has predicted exactly the missing second half (>N/2) of the expansion coefﬁcients in the input z-transform of length M = N/2. This faithful extrapolation/prediction amounts to 1115 J Math Chem (2017) 55:1110–1157 resolution improvement, since the N terms of a convergent power series expansion is more accurate than its truncation to N/2. Therefore, without any computation, we see that the FPT is deﬁned from the onset to outperform the resolution of the FFT. It has been shown in practice [9,12] that usually: (i) the N/2 data points of the input time signal of length N sufﬁced for the FPT to match the resolution of the FFT, which used the full FID ({cn}, 0 ≤n ≤N −1), and (ii) using any ﬁxed number N ′(≤N) of the FID points, the resolving power of the FPT was doubled relative to that of the FFT, which also employed the same N ′ data entries. This resolution enhancement of the FPT is not necessarily limited to comparisons with the FFT alone. Quite the contrary, any ﬁtting technique which starts from the FFT and adjusts some free parameters to the given Fourier envelope would, at best, match the resolution of the FFT and, in turn, would be inferior to the FPT. resolution improvement, since the N terms of a convergent power series expansion is more accurate than its truncation to N/2. 1.1.2 The fast Padé transform: highly suitable for processing MRS time signals The non-linearity of the FPT also enhances resolution and SNR by suppress- ing noise [9,12]. Moreover, the special form of the rational response function with the numerator (PK ) and denominator (QK ) polynomials, helps in cancelling noise from the Padé spectrum PK /QK . The reason is because these two polynomials PK and QK , through their expansion coefﬁcients, contain a similar amount of noise inherited from {cn}. This resembles the general experience where in the given ratio A/B, there is sub- stantial noise cancellation for observables A and B generated either experimentally by measurements or theoretically through numerical computations with ﬁnite precision. Through the parametric FPT, the spectral components can be accurately recon- structed and, hence, be of the sought clinical reliability. In this way, the number of true resonances and their spectral parameters, the fundamental frequencies {ωk} and associated amplitudes {dk}, through the set {ωk, dk} (1 ≤k ≤K) present in a given time signal {cn} (0 ≤n ≤N −1), are reconstructed by the FPT to a very high level of conﬁdence. Most importantly, from these parametric data, the computed metabolite concentrations are trustworthy [9,12,16]. Within the FPT there are two variants, the FPT(+) using z+1 ≡z and FPT(−) using z−1 with the former converging inside (\|z\| < 1) and the latter outside (\|z\| > 1) the unit circle in the complex plane of the harmonic variable z. In their complementary domains, outside and inside the unit circle, respectively, the FPT(+) and FPT(−) are convergent, as well, via the Cauchy analytical continuation. For \|z\| > 1, the FPT(−) accelerates the already convergent input series given by the Green function in the harmonic variable z−1. Through the Cauchy analytical continuation, the FPT(+) must force convergence of the input series, which diverges inside the unit circle, \|z\| < 1 [17]. The latter is a more difﬁcult task, but the FPT(+) also has advantages especially amenable to practical applications for in vivo MRS. Speciﬁcally, the FPT(+) separates noise from signal with genuine and spurious resonances fully partitioned into two oppositeregions,insideandoutsidetheunitcircle,respectively.SinceintheFPT(−),all resonances are located outside the unit circle, \|z\| > 1, spurious and genuine resonances are intermingled. Overall, the FPT(±) working with variables z±1 provide internal cross-validation. From here on, we will refer only to the FPT(+) with the understanding that the FPT(−) was also used in cross-validating tests. 1.1.2 The fast Padé transform: highly suitable for processing MRS time signals This is the matter of uniqueness of quantiﬁcation of the encoded time signals. No ﬁtting of the given Fourier envelope is unique, since different results are unavoidably obtained by changes in the conditions of adjusting the free parameters. Some examples of these condi- tion alterations are different mathematical models (mainly introduced ad hoc) for envelope shapelines, various constraints (often arbitrary) to minimizations, the user’s subjectivity, surmising the total number of metabolites, etc. For example, overﬁtting (overmodelling) or underﬁtting (undermodelling) would cause, respectively, errors of ﬁnding some ghost metabolites absent from the input FID or failing to detect cer- tain true metabolites in the scanned tissue. As to the FPT, all its reconstructed stable parameters are unique. Only the raw input FID is used with no constraints imposed onto quantiﬁcation. Moreover, the total number of metabolites is not guessed at all; rather, it is reconstructed from the input data just like the other parameters, the com- plex frequencies and complex amplitudes. This leaves no room for the FPT to either predict metabolites foreign to the input time signal, or to miss retrieving some of the actual metabolites. Such an advantageous feature of analysis of data from patients is 12 3 3 1116 J Math Chem (2017) 55:1110–1157 precisely what is needed in the clinics. The last thing the diagnostician needs is to face new dilemmas by ambiguities such as those routinely occurring with the FFT plus ﬁtting recipes. The FPT comes to the rescue with its reliability, which is the long sought goal of MRS in medicine. g g Through exhaustive studies, the FPT has been demonstrated to be the optimal method for processing MRS time signals [9,12–15]. The spectrum generated by the FPT is a non-linear response function, via the unique ratio of two polynomi- als, PK /QK , of degree K in the diagonal form. No dilemma arises whereby attempts to improve resolution worsen SNR, since with the FPT the spectrum can be com- puted at any sweep frequency ν, and not just those imposed by the Fourier grid m/T (0 ≤m ≤N −1). Hereafter, as usual, the angular or circular (ω) and linear (ν) frequencies are related by ω = 2πν. Besides interpolation, the FPT can extrapo- late beyond the total acquisition time T . This extrapolation is based on the unique polynomial quotient PK /QK , extracted directly from the encoded time signal or FID. 1.1.2 The fast Padé transform: highly suitable for processing MRS time signals With complete convergence in the FPT(±) via ω+ k ≈ω− k and d+ k ≈d− k , these parameters can jointly be denoted 123 1117 J Math Chem (2017) 55:1110–1157 as ωk and dk, respectively. Initially, we do not know the fundamental parameters {ωk, dk} from the input encoded time signal {cn}, which is modeled by the geometric progression: as ωk and dk, respectively. Initially, we do not know the fundamental parameters {ωk, dk} from the input encoded time signal {cn}, which is modeled by the geometric progression: cn = K k=1 dkein τωk, 0 ≤n ≤N −1 (Input time signal or FID). (2) (2) In physics, K represents the number of resonances, whereas in mathematics, integer K ≥1 is the model order, as well as the common degree of the polynomials PK and QK in the spectrum PK /QK , which is the diagonal form of the FPT. In physics, K represents the number of resonances, whereas in mathematics, integer K ≥1 is the model order, as well as the common degree of the polynomials PK and QK in the spectrum PK /QK , which is the diagonal form of the FPT. 1.1.2.2 The exact and truncated response functions The inﬁnite-rank Green function G(z−1) gives the exact response function. This is deﬁned as the Maclaurin series: G(z−1) = ∞ n=0 cnz−n, z = eiτω (Exact Green series), (3) (3) (3) where the time signal points {cn} (0 ≤n ≤∞) form an inﬁnite set of the expansion coefﬁcients. The total number N of available signal points {cn} is actually ﬁnite (N < ∞) in every realistic situation, such that the response function needs to be truncated. This is provided by the ﬁnite-rank Green function given as the Green polynomial G N(z−1): G N(z−1) = N−1 n=0 cnz−n (Exact Green polynomial). (4) (4) Using the terminology of discrete time series, the inﬁnite- and ﬁnite-rank Green func- tions can be termed as the inﬁnite and ﬁnite z-transform [9]. Using the terminology of discrete time series, the inﬁnite- and ﬁnite-rank Green func- tions can be termed as the inﬁnite and ﬁnite z-transform [9]. 1.1.2 The fast Padé transform: highly suitable for processing MRS time signals In the FPT(+), the input response function G N(z−1) from (4), is approximated by the causal Green–Padé function G+ K (z), as the diagonal rational polynomial in the harmonic variable z: G N(z−1) ≈G(+) K (z) ≡ K r=1 p+ r zr K s=0 q+ s zs ; FPT(+) (Causal Green–Pad´e function). (5) (5) The term “causal” indicates that the system needs to be perturbed prior to responding. As an example, insofar as an excitation begins at t0 = 0, the system’s response through the time signal {cn}(tn = nτ, τ > 0), will not appear for t < t0. Thus, cn = 0 for n < 0. If such an FID is used, the frequency response function G(+) K (z) from (5) will also be causal. Stated in another way, G(+) K (z) can be termed the advanced Green–Padé function because it is associated with time evolution of the system along the positive 12 3 1118 J Math Chem (2017) 55:1110–1157 portion of the time axis. In the FPT(−), we have G(−) K (z−1) which is termed the anti- causal (or delayed) Green–Padé function, since it is associated with time evolution of the system on the negative part of the time axis. Such a nomenclature for G(±) K (z±1) is rooted in the harmonic variables z±1. These act as operators that propagate the system at positive and negative times, respectively. Due to micro-reversibility of physical processes [9], the two descriptions by the G(±) K (z±1) are equivalent. Whereas the typical concept of time evolution is propagation in the future, i.e. at positive times, propagation at negative times can be equivalently employed in theoretical descriptions of time evolution in the past. ±1 In the theory of digital processing for discrete systems, the variables z±1 are known as the time advancing/delaying operations that advance/delay a sample by one sampling interval τ, via z±1 = exp(±iωτ), respectively. Thus, raising z±1 to the nth power via z±n will advance/delay a sample by n sampling intervals nτ as z±n = exp (±iωnτ). The FPT(+) through G(+) K (z) uses the variable z and converges inside the unit circle (\|z\| < 1). This is where the exact Green function G(z−1) diverges. The FPT(+) then induces convergence into the input divergent series from (2) via the Cauchy concept of analytical continuation, as noted. 1.1.2 The fast Padé transform: highly suitable for processing MRS time signals Subsequently, 12 123 1119 J Math Chem (2017) 55:1110–1157 the solutions of the characteristic equations, P+ K (z) = 0 and Q+ K (z) = 0, are found and these roots are denoted by z+ k,P and z+ k,Q (1 ≤k ≤K), respectively. To distinguish the roots of P+ K (z) and Q+ K (z), the second subscripts P or Q are introduced in the fundamental harmonic variables, z+ k,P and z+ k,Q, respectively. Next, the corresponding fundamental amplitudes d+ k are retrieved as the Cauchy residues of the spectrum P+ K (z)/Q+ K (z) taken at z = z+ k,Q. Non-degenerate (unequal, non-coincident) roots of Q+ K (z) represent simple poles in the spectrum P+ K /Q+ K , and their strengths are the complex amplitudes given by: d+ k = P+ K (z+ k,Q) Q+′ K (z+ k,Q) , Q+′ K (z) = d dz Q+ K (z), 1 ≤k ≤K. (6) (6) From the equivalent canonical representation of spectrum P+ K (z)/Q+ K (z) [9], the amplitudes {d+ k } are proportional to the pole-zero distance (a metric) via: (7) d+ k ∝z+ k,Q −z+ k,P. (7) This Cauchy residue reﬂects the behavior of a line integral of a meromorphic function around the kth pole. The Padé spectrum P+ K /Q+ K has its poles {z+ k,Q} as the only singularities and, consequently, represents a meromorphic function. Through these steps, the FPT(+) reconstructs the 2K complex fundamental parame- ters {ω+ k,Q, d+ k } (1 ≤k ≤K). Here, the earlier notation ω+ k is relabeled as ω+ k,Q where ω+ k,Q = [1/ (iτ)] ln z+ k,Q. The set of retrieved spectral zeros is not usually considered in any other processor used in the MRS literature. However, in the FPT(+), the zeros {z+ k,P} of the spectrum P+ K (z)/Q+ K (z) are employed in tandem with the poles {z+ k,Q} to separate signal from noise and to establish the system’s stability, as will be elaborated in this paper. A note should be made to emphasize that the sign of the imaginary frequency Im(ν+ k,Q) has both mathematical and physical meanings. Mathematical, because Im(ν+ k,Q) > 0 and Im(ν+ k,Q) < 0 correspond to the poles z+ k,Q with converging and diverging exponentials (transients), respectively. 1.1.2 The fast Padé transform: highly suitable for processing MRS time signals The convergence radii RN of G N(z−1) as N → ∞and R+ K of G+ K (z) differ markedly. The former is exactly zero, RN = 0 as N →∞ for \|z\| < 1, while the latter is non-zero, R+ K > 0 as K →∞in the same region \|z\| < 1. In this way, the FPT(+)extends the validity of the response function (spectrum) to \|z\| < 1, where the input Green series G(z−1) does not exist, due to its divergence inside the unit circle. As per the just made remark on digital processing of discrete systems, switching from the time advance to the time delay operation simply corresponds to the replace- ment of z by 1/z. However, in the FPT, care must be exercised, since its two equivalent representations FPT(±)(z±1) are not at all deducible from each other by merely replac- ing z with 1/z. The reason is in the fact that the FPT(±) do not differ from each other only in working with the time advance/delay variables z±1, respectively. Rather, as mentioned, the FPT(±)(z±1) are based upon two completely distinct concepts with two different numerical tasks for the same input G N(z−1), which is: (i) convergent for \|z\| > 1 and, thus, accelerated by the FPT(−)(z−1), and (ii) divergent for \|z\| < 1 and, hence, forced to converge by the FPT(+)(z). As such, two different systems of linear equations must be solved in the FPT(+) and FPT(−) to extract their expansion coefﬁcients of the numerator and denominator polynomials. Nevertheless, upon con- vergence of the FPT(+) and FPT(−), the results of both the parameter and shapeline (envelope, components) estimations are found to be the same. This is a veritable “check and balance” of the performance reliability of the FPT. 1.1.2.3 Extraction of the expansion coefﬁcients and solutions of the characteristic equations As per (5), the expansion coefﬁcients of the numerator and denominator polynomials, say P+ K and Q+ K are {p+ r } and {q+ s }, respectively. Note that there is no free term p+ 0 in the expansion for G(+) K (z), i.e. p+ 0 ≡0. The expansion coefﬁcients {p+ r , q+ s } of P+ K (z) and Q+ K (z) are extracted uniquely from the time signal points {cn} by solving a single system of linear equations obtained using (4) and (5). 1.1.2 The fast Padé transform: highly suitable for processing MRS time signals Physical, because Im(ν+ k,Q) > 0 and Im(ν+ k,Q) < 0 are associated with positive and negative spin–spin relaxation times T ∗+ 2k > 0 and T ∗+ 2k < 0, respectively, since T ∗+ 2k = 1/[2πIm(ν+ k,Q)]. Note that 2πIm(ν+ k,Q) is the full width at half maximum (FWHM) of the kth resonant peak. On the other hand, T ∗+ 2k as the reciprocal of the FWHM is the lifetime of the kth transient, resonant phenomenon. The former (T ∗+ 2k > 0) is physical, whereas the lat- ter (T ∗+ 2k < 0) is unphysical. The mathematical and physical meanings of the sign of Im(ν+ k,Q) are coherent. Namely, every physical phenomenon has a ﬁnite lifetime and, thus, the associated transient z+ k,Q must decay to zero at times that are inﬁnitely augmented. In other words, the harmonic z+ k,Q must be converging, which can occur only if its complex exponentials are attenuated, i.e. with Im(ν+ k,Q) > 0 and, hence, T ∗+ 2k > 0. Having Im(ν+ k,Q) > 0 is necessary, but not sufﬁcient for the kth physical 12 3 1120 J Math Chem (2017) 55:1110–1157 resonance to be genuine (i.e. one which is indeed present in the input FID as a true res- onance). To be genuine the kth resonance with the physical frequency Im(ν+ k,Q) > 0 must be stable in all four parameters Re(ν+ k,Q), Im(ν+ k,Q), d+ k and ϕ+ k as a function of e.g. the model order K. So it is the stability of complex parameters ν+ k,Q and d+ k which makes the physical kth resonance with Im(ν+ k,Q) > 0 indeed genuine. 1.1.2.4 The Usual and Ersatz modes of the component spectra There are two modes by which the component spectra can be presented. In the Usual (U) mode of component spectra, the absorption and dispersion components are mixed together. This is the case because the phases ϕ+ k are non-zero, such that the amplitudes {d+ k } (1 ≤k ≤K) are all complex-valued. The Usual mode of the component spectrum for the kth resonance is deﬁned by: P+ K (z) Q+ K (z) U k ≡ d+ k z z −z+ k,Q (Usual component k). (8) (8) The Ersatz (E) mode of component spectra is introduced by setting the reconstructed phases ϕ+ k “by hand” to zero, ϕ+ k ≡0 (1 ≤k ≤K). 123 1.1.2 The fast Padé transform: highly suitable for processing MRS time signals By so doing, interference effects among resonances are eliminated, such that purely absorptive Lorentzians are gener- ated. The Ersatz mode of the component spectrum for the kth resonance is: P+ K (z) Q+ K (z) E k ≡ d+ k z z −z+ k,Q (Ersatz component k). (9) (9) Evidently, insofar as ϕ+ k = 0 we can go from (8) to (9), by substituting d+ k ≡ d+ k exp iϕ+ k with d+ k . Here, d+ k is the magnitude (absolute value) of the complex amplitude d+ k [18]. + In the Usual and Ersatz modes, the peak positions [chemical shift, Re(ν+ k,Q)] coin- cide as long as Re(P+ K /Q+ K )U k is in the absorption mode. However, if Re(P+ K /Q+ K )U k is a dispersive component, there will be two lobes, such that the peak position Re(ν+ k,Q) for Re(P+ K /Q+ K )E k will be located between the two lobes of Re(P+ K /Q+ K )U k . This can be seen by juxtaposing the plots for Re(P+ K /Q+ K )U k and Re(P+ K /Q+ K )E k . It should be emphasized that phase ϕ+ k of the kth amplitude d+ k plays a very important role in spectral estimation. This is the case because a given value of ϕ+ k in units of radians (rad), belonging to the interval −π ≤ϕ+ k ≤+π, determines the shape of the Usual component spectrum, (P+ K /Q+ K )U k = \|d+ k \|eiϕ+ k z/(z −z+ k,Q). Thus, in a special case for ϕ+ k = 0, a clear-cut situation arises with the shapelines Re(P+ K /Q+ K )U k and Im(P+ K /Q+ K )U k being purely absorptive and dispersive, respec- tively. In fact, at ϕ+ k = 0, the Usual component becomes the Ersatz component, (P+ K /Q+ K )E k = \|d+ k \|z/(z −z+ k,Q) = {(P+ K /Q+ K )U k }ϕ+ k =0 = {(P+ K /Q+ K )U k }ϕ+ k =0. For any non-zero phase, ϕ+ k ̸= 0, absorption and dispersion shapelines are encountered in both Re(P+ K /Q+ K )U k and Im(P+ K /Q+ K )U k . 1.1.2 The fast Padé transform: highly suitable for processing MRS time signals Therefore, the physical meaning of the two 123 1121 J Math Chem (2017) 55:1110–1157 situations, ϕ+ k = 0 and ϕ+ k ̸= 0, is the lack and the presence, respectively, of interfer- ence of the absorptive and dispersive modes of the system’s vibrations. Regarding the resonance phenomenon, the true signiﬁcance of ϕ+ k is best appreciated when plotted against the sweep real-valued frequency ν (chemical shift). When a real-valued ν is away from the resonance frequency Re(ν+ k,Q), the phase ϕ+ k is a monotonic, smooth function. However, for ν in the vicinity of the resonance chemical shift, ν ≈Re(ν+ k,Q), there is a sharp jump by π/2 in ϕ+ k , in accordance with the Lewinson theorem [9]. Such an abrupt change in ϕ+ k is a sure sign that the system has undergone a resonant transition through e.g. emission of excess energy by descending from a higher to a lower energy state. The numerator (P+ K ) and denominator (Q+ K ) polynomials in (8), have the following explicit expressions, implied by (6): P+ K (z) = K r=1 p+ r zr, Q+ K (z) = K s=0 q+ s zs, p+ 0 ≡0. (10) (10) By solving the system of linear equations K s=0q+ s cs′+s = 0 deduced from (4) and (5), the expansion coefﬁcients {q+ s } for the polynomial Q+ K (z) are uniquely extracted. Subsequently, the solutions {q+ s } are reﬁned by Singular Value Decomposition (SVD). Once the set {q+ s } becomes available, the expansion coefﬁcients {p+ r } of P+ K are computed from the analytical expression (convolution) p+ r = K−r r′=0 cr′q+ r′+r The free term, q+ 0 can be set to e.g. 1 or −1. This does not affect the spectra or the spectral parameters {ω+ k,Q, d+ k }(1 ≤k ≤K) reconstructed by the FPT(+). There is a coherence between the two sets {p+ r } and {q+ s } because the former depends on the latter, through the said convolution. By solving the system of linear equations K s=0q+ s cs′+s = 0 deduced from (4) and (5), the expansion coefﬁcients {q+ s } for the polynomial Q+ K (z) are uniquely extracted. Subsequently, the solutions {q+ s } are reﬁned by Singular Value Decomposition (SVD). 1.1.2 The fast Padé transform: highly suitable for processing MRS time signals 1.1.2.7 Genuine versus spurious poles and zeros: key to stability of the system with separation of signal from noise The physical parameters of the system which gen- erated the time signals as a response to external excitations are obtained through the spectral poles and zeros. According to (7), there is also a direct relation of the ampli- tudes with the spectral poles and zeros. As stated in (6), the spectral peak amplitudes are the Cauchy residues of the system response function, P+ K /Q+ K . Thus, the system response function is driven fully by the system poles and zeros. Recall that the zeros and poles of P+ K /Q+ K are given by the roots of the characteristic equations P+ K (z) = 0 and Q+ K (z) = 0, respectively. The entire genuine information about various states of a given system is contained in the complete set of physical poles and zeros. 1.1.2.7 Genuine versus spurious poles and zeros: key to stability of the system with separation of signal from noise The physical parameters of the system which gen- erated the time signals as a response to external excitations are obtained through the spectral poles and zeros. According to (7), there is also a direct relation of the ampli- tudes with the spectral poles and zeros. As stated in (6), the spectral peak amplitudes are the Cauchy residues of the system response function, P+ K /Q+ K . Thus, the system response function is driven fully by the system poles and zeros. Recall that the zeros and poles of P+ K /Q+ K are given by the roots of the characteristic equations P+ K (z) = 0 and Q+ K (z) = 0, respectively. The entire genuine information about various states of a given system is contained in the complete set of physical poles and zeros. Further clariﬁcation can come from a complementary twofold representation in the FPT(+), one of which is denoted by zFPT(+) and is called the “zeros of the FPT(+)”. The other representation is pFPT(+) termed the “poles of the FPT(+)”. The zFPT(+) and pFPT(+) can independently generate the two sub-spectra by using exclusively either the zeros {z+ k,P} or the poles {z+ k,Q} [12]. 1.1.2 The fast Padé transform: highly suitable for processing MRS time signals As stated, the other set of roots {z+ k,P} from the character- istic equation P+ K (z) = 0 is used to separate genuine (ω+ k,P ̸= ω+ k,Q) from spurious (ω+ k,P = ω+ k,Q) resonances, where ω+ k,P = [1/(iτ)] ln z+ k,P. The characteristic poly- nomial rooting is achieved (to machine accuracy) by solving the equivalent eigenvalue problem of the extremely sparse Hessenberg or companion matrix [9]. The FPT(+) generates the set {d+ k } from the analytical formulae as the Cauchy residues given by (6). This efﬁcient procedure in the FPT(+) is contrasted with the Hankel–Lanczos sin- gular value decomposition (HLSVD). In the HLSVD, obtaining the amplitudes {dk} requires solving yet another system of linear equations via (2) by using all the found frequencies {ωk}, both true and false, with no procedure to separate one from the other. As a result, the set {dk} for the HLSVD can hardly be accurate. 1.1.2.6 Minimal numerical work with the FPT algorithms The numerical work within the FPT(+) is relatively minimal. It consists of solving a single system of linear equations for the expansion coefﬁcients {q+ s }, and generating {p+ r } from the mentioned analytical convolution formula. This is followed by rooting the characteristic polyno- mials Q+ K (z) and P+ K (z). The fundamental frequencies {ω+ k,Q} are reconstructed from the roots {z+ k,Q} of Q+ K (z). As stated, the other set of roots {z+ k,P} from the character- istic equation P+ K (z) = 0 is used to separate genuine (ω+ k,P ̸= ω+ k,Q) from spurious (ω+ k,P = ω+ k,Q) resonances, where ω+ k,P = [1/(iτ)] ln z+ k,P. The characteristic poly- nomial rooting is achieved (to machine accuracy) by solving the equivalent eigenvalue problem of the extremely sparse Hessenberg or companion matrix [9]. The FPT(+) generates the set {d+ k } from the analytical formulae as the Cauchy residues given by (6). This efﬁcient procedure in the FPT(+) is contrasted with the Hankel–Lanczos sin- gular value decomposition (HLSVD). In the HLSVD, obtaining the amplitudes {dk} requires solving yet another system of linear equations via (2) by using all the found frequencies {ωk}, both true and false, with no procedure to separate one from the other. As a result, the set {dk} for the HLSVD can hardly be accurate. 1.1.2 The fast Padé transform: highly suitable for processing MRS time signals Once the set {q+ s } becomes available, the expansion coefﬁcients {p+ r } of P+ K are computed from the analytical expression (convolution) p+ r = K−r r′=0 cr′q+ r′+r The free term, q+ 0 can be set to e.g. 1 or −1. This does not affect the spectra or the spectral parameters {ω+ k,Q, d+ k }(1 ≤k ≤K) reconstructed by the FPT(+). There is a coherence between the two sets {p+ r } and {q+ s } because the former depends on the latter, through the said convolution. 1.1.2.5 Heaviside partial fraction expansions for total shape spectra The total shape spectrum G(+) K (z) from (5) can also be computed via the Heaviside partial fraction expansion given by: P+ K (z) Q+ K (z) = K k=1 d+ k z z −z+ k,Q (Heaviside Partial Fractions). (11) (11) This is recognized as a total shape spectrum in the Usual mode as provided using (8): P+ K (z) Q+ K (z) U ≡ K k=1 P+ K (z) Q+ K (z) U k = K k=1 d+ k z z −z+ k,Q (Usual envelope). (12) (12) The lhs of (8) and (12) differ for the kth Usual component (P+ K /Q+ K )U k and the usual envelope (P+ K /Q+ K )U in that the subscript k as the summation index is omitted in the latter. The lhs of (8) and (12) differ for the kth Usual component (P+ K /Q+ K )U k and the usual envelope (P+ K /Q+ K )U in that the subscript k as the summation index is omitted in the latter. 123 12 1122 J Math Chem (2017) 55:1110–1157 1.1.2.6 Minimal numerical work with the FPT algorithms The numerical work within the FPT(+) is relatively minimal. It consists of solving a single system of linear equations for the expansion coefﬁcients {q+ s }, and generating {p+ r } from the mentioned analytical convolution formula. This is followed by rooting the characteristic polyno- mials Q+ K (z) and P+ K (z). The fundamental frequencies {ω+ k,Q} are reconstructed from the roots {z+ k,Q} of Q+ K (z). 123 1.1.2 The fast Padé transform: highly suitable for processing MRS time signals However, insofar as zeros {z+ k,P} and poles {z+ k,Q} are simultaneously used within shape spectra and/or in quantiﬁcation, the composite representation, FPT(+), is obtained through the union of the two constituent representations, the zFPT(+) and pFPT(+). Akeycharacteristicofgenuinepolesistheirstabilityvis-à-visexternalperturbation, whereas unphysical poles oscillate widely when exposed to even minimal disturbance. Furthermore, unstable poles behave like noise; they do not ever converge. They are incoherent and, as is the case for random ﬂuctuations, they cannot stabilize. The time signal cn is said to be built from the 2K stable complex pairs {ωk, dk} in the coherent sum (2), with non-zero phases (ϕk ̸= 0, k ∈[1, K]) that produce an interfer- ence effect. This is a closed, stable system. Insofar as more conﬁgurations are predicted and added beyond the saturation number K, the new collection of components in (2) will be incoherent. In other words, these are unstable components and, as such, will 123 1123 J Math Chem (2017) 55:1110–1157 have zero-valued amplitudes in (2). Note that as per quantum-mechanics, parame- ter dk is the probability amplitude of transition from one conﬁguration to another. If dk = 0, this would mean that there is zero probability for the new resonance to be incorporated into (2) beyond the K completely occupied states. The transitions in the system’s conﬁgurations (states, orbitals) with dk = 0 and dk ̸= 0 correspond to the amplitude probabilities of occurrence of the so-called forbidden (non-physical) and allowed (physical) transitions, respectively. Consequently, the phenomena of coher- ence versus incoherence and stability versus instability can be connected to the concept of genuine versus spurious resonances. Thereby, through binning the genuine and spu- rious set of reconstructions, the FPT(+) effectively suppresses the redundant degrees of freedom of the system. Physical versus unphysical resonances can also be distinguished via the direct relation between poles and zeros. Poles and zeros are distinct (z+ k,Q ̸= z+ k,P) for stable structures. Unstable resonances exhibit pole-zero conﬂuence (z+ k,Q = z+ k,P or z+ k,Q ≈z+ k,P) and these are called Froissart doublets. As per (7), genuine resonances (z+ k,Q ̸= z+ k,P) have non-zero amplitudes (d+ k ∝z+ k,Q −z+ k,P ̸= 0), while spurious structures (z+ k,Q = z+ k,P or z+ k,Q ≈z+ k,P) have zero or close to zero amplitudes (d+ k = 0 or d+ k ≈0). 1.1.2 The fast Padé transform: highly suitable for processing MRS time signals In other words, the model order K ′ in (13), or equivalently, the degree K ′ of the associated Padé rational polynomial P+ K ′/Q+ K ′ for which the reconstructed frequencies and amplitudes have stabilized, will be the exact number K of harmonics contained in the input time signal from (2). This is the process of “Signal-noise separation” (SNS), which has been compre- hensively validated for MRS time signals [10–12]. Its mechanism has also been analytically conﬁrmed [19]. Based on the special form of the rational polynomials for the Padé spectra, this stabilization via pole-zero cancellation is a unique feature of the FPT. Pole-zero coincidences can take place only in the quotients of two functions and, as a result, pole-zero cancellations occur. Identifying Froissart doublets through pole-zero conﬂuences with the subsequent stabilization of the Padé spectra is the key indication that the entire information from the input time signal has been exhausted. Thereby, the genuine parameters from all the reconstructed data {ω+ k,Q, d+ k } in the FPT(+) can be considered as the accurate approximations of the unknown fundamen- tal frequencies and amplitudes {ωk, dk}(1 ≤k ≤K) from the encoded time signal modeled by (2). 1.1.2.8 Extrapolation of in vivo encoded MRS time signals through the FPT Math- ematical modeling is of key value only when it provides prediction. The FPT does so through extrapolation in both the time and frequency domains. In the reconstructed time signal c+ n from (13), associated with spectrum P+ K ′/Q+ K ′, the running or sweep model order K ′ is the total number of resonances (genuine K and spurious KS, i.e. K ′ = K+KS)extractedfromtheencodeddata{cn}.Through(2),timesignal{cn}actu- ally contains only K resonances in total. This, in view of the relation K ′ = K +KS (i.e. K ′ > K) might suggest at ﬁrst that over-modeling had occurred in the retrieved time signal {c+ n } from (13). Nevertheless, since all the KS extra resonances are spurious with zero-valued amplitudes, the sum in (13) is, in fact, reduced to K terms alone, namely, c+ n ≡K k=1d+ k exp(in τω+ k,Q). As stated, with convergence, the set {ω+ k,Q, d+ k } can be denoted by {ωk, dk}. Thus, c+ n = cn ≡K k=1dk exp(in τωk). 1.1.2 The fast Padé transform: highly suitable for processing MRS time signals As stated, in the FPT(+), genuine and spurious resonances have positive and negative imaginary frequencies, Im(ω+ k,Q) > 0 and Im(ω+ k,Q) < 0, respectively. These correspond to T ∗+ 2k > 0 and T ∗+ 2k < 0, respectively, where T ∗+ 2k has already been introduced as the spin–spin relaxation time of the kth resonant component. Con- sequently, with increasing time nτ the exponentials in the reconstructed time signal: c+ n ≡ K ′ k=1 d+ k ein τω+ k,Q = K ′ k=1 d+ k ein τRe(ω+ k,Q)−nτIm(ω+ k,Q), (13) (13) are damped for genuine resonances and exploding for spurious resonances. Here, the former converge and the latter diverge with increasing signal number n or time nτ for a ﬁxed sampling rate τ. Thus, the diverging harmonics can be binned as the unphysical part of the retrieved time signal {c+ n }. Importantly, genuine resonances may occasionally have very small amplitudes, d+ k ≈0. However, with their feature Im(ω+ k,Q) > 0 alongside stability of all the spectral parameters, these latter resonances can be conﬁdently binned as the physical portion of the recovered FID from (13). + + After stabilization of the model order K in P+ K /Q+ K , namely, once all the physi- cal resonances have been reconstructed, computation of the Padé spectra for a higher degree polynomial, K + m(m = 1, 2, 3, . . .), yields only more non-physical reso- nances. These will show pole-zero coincidences (z+ k,Q = z+ k,P) with d+ k = 0 for k = K + m(m = 1, 2, 3, . . .). In other words, pole-zero cancellation occurs, with stabilization of the computed complex-valued total shape spectra: P+ K+m(z) Q+ K+m(z) = P+ K (z) Q+ K (z) (m = 1, 2, 3, . . .) . (14) (14) 12 123 3 1124 J Math Chem (2017) 55:1110–1157 As mentioned, with Padé reconstruction, the number of physical resonances, i.e. the number of fundamental harmonics K is treated as an unknown parameter, whose value needs also to be extracted from the input data {cn}. When the reconstructed frequencies and amplitudes have converged, K will then be ascertained. 1.2 Ovarian cancer: the need and challenge of early detection Ovarian cancer is a relatively common malignancy among women, particularly in the USA, Scandinavia, the UK, Eastern Europe and Israel. In many parts of the world, its incidence appears to be increasing [20–25]. If detected early, the prognosis for ovarian cancer is excellent: when conﬁned to a single ovary (Stage Ia), the 5-year survival rates are better than 90% [26]. Unfortunately, however, most cases are found at late stages, when the tumor has already spread outside the true pelvis [27]. Due to late detection, the case fatality rate is very high for this malignancy. In 2013 approximately 158000 women died of ovarian cancer [28]. The challenge is that for early-stage ovarian cancer there are very often no symptoms [29], and the ovary may not even be enlarged [30]. Attempts to screen for ovarian cancer have mainly entailed transvaginal ultrasound (TVUS) and serum cancer antigen (CA-125).1 Although there is some recent evidence that may indicate the contrary [31], most of the ﬁndings from large-scale random- ized trials show that the use of CA-125 and TVUS to screen for ovarian cancer in asymptomatic women does not diminish mortality nor help in earlier ovarian cancer detection [32,33]. Moreover, with this strategy, there are many false positive ﬁndings, and these can have harmful consequences. Most notably, women will often undergo surgical removal of benign ovarian lesions [34]. Consequently, for women who are not at clearly high risk for ovarian cancer, the harms of routine screening for ovarian cancer are considered to override the beneﬁts [35]. Screening with CA-125 and TVUS is often carried out among women at high ovarian cancer risk. However, there is no prospective evidence that this strategy contributes to early ovarian cancer detection [36,37]. Biomarkers other than CA-125 have been examined [38,39], but none have been found to improve diagnostic accuracy sufﬁciently to be recommend for routine ovarian cancer screening [40]. Currently, the most effective means of reducing ovarian cancer risk in women who are carriers of harmful mutations of BRCA1 or BRCA2 genes is salpingo- oophorectomy: surgical removal of the fallopian tubes and ovaries. Salpingo- oophorectomy is recommended by the National Comprehensive Cancer Network for women 35–40years of age, who have completed childbearing [41]. Although cancer risk is reduced thereby, serious issues arise, associated with “mutilation of a healthy organ, termination of fertility, self-wounding, and castration” [42]. 1 Serum cancer antigen, CA-125 is a protein whose presence is often associated with ovarian cancer. However, it has poor sensitivity for early stage ovarian cancer and is also non-speciﬁc, being present in other malignancies as well as in a number of non-cancerous conditions, including pregnancy. 1.1.2 The fast Padé transform: highly suitable for processing MRS time signals In other words, {c+ n } accurately reconstructs the input data {cn} from (2), so that {c+ n } = {cn} for the ﬁrst N points (0 ≤n ≤N −1). However, the total length of the reconstructed FID does not need to stop at N, i.e. {c+ n }(n = 0, 1, . . . , N −1, N, N + 1, . . .). Any additional data point for n ≥N in the full set {c+ n } relative to {cn} are the Padé-based extrapolations that would have been available had the encoding continued after cN−1, beyond the total acquisition time T , i.e. at times nτ > T . It is therefore shown that through its extrapolation capabilities in the time domain, the FPT(+) can indeed pro- vide reliable prediction. This Padé-generated extrapolation of the input time signal is reliable because it is based upon the converged set of reconstructed genuine pairs {ω+ k,Q, d+ k }(1 ≤k ≤K) whose total number K is also retrieved by the “stability test” for the fundamental parameters and spectra; for the latter, see (14). These extrapolation features of the FPT(+) have been found to be of particular value for processing MRS 12 123 1125 J Math Chem (2017) 55:1110–1157 time signals encoded in vivo from the ovary [8], as will be summarized in Sect. 1.3.2. We now proceed to a brief presentation of the clinical issues concerning ovarian cancer detection. time signals encoded in vivo from the ovary [8], as will be summarized in Sect. 1.3.2. We now proceed to a brief presentation of the clinical issues concerning ovarian cancer detection. 1.2 Ovarian cancer: the need and challenge of early detection Attempts have also been made to use magnetic resonance imaging (MRI) for non- invasive detection of ovarian cancer. In some cases, the high spatial resolution of MRI can help clarify the nature of ovarian lesions that are indeterminate on TVUS [43–45]. However, even with MRI, nearly 25% of benign ovarian lesions were considered to be 12 3 3 1126 J Math Chem (2017) 55:1110–1157 malignant when MRI was used as a second imaging technique, after TVUS [44]. The main problem with MRI is that despite its high sensitivity, many non-malignant lesions willbeincorrectlydiagnosedascancerous.Althoughsomefurtherimprovementwithin MR has been offered by e.g. diffusion-weighted imaging (DWI), an unacceptably large percentage of false positive ﬁndings for adnexal lesions have also been reported with DWI [46,47]. Via magnetic resonance spectroscopy, MRS, the metabolic features of tissue or organs can be assessed, such that the molecular changes characterizing the cancer process, namely, the “hallmarks of cancer” [48] may be detected [49]. It has long been noted that MRS could greatly contribute to early ovarian cancer detection [36,50]. However, with conventional FFT plus ﬁtting type of analyses of MRS time signals from the ovary, the diagnostic yield has been limited, as we now describe in brief. 1.3 Results to date applying MRS to time signals encoded from the ovary 1.3.1 Conventional Fourier analysis of MRS time signals encoded from the ovary 1.3.1 Conventional Fourier analysis of MRS time signals encoded from the ovary 1.3.1 Conventional Fourier analysis of MRS time signals encoded from the ovary In our meta-analysis [7], we examined the published studies that employed in vivo MRS to a total of 134 cancerous and 114 benign ovarian lesions as well as three “borderline” ovarian lesions, with encoding performed using clinical MR scanners (1.5 or 3T). All these studies applied the FFT to the MRS time signals, and post- processing through ﬁtting was sometimes also performed. In these investigations, a very small number of resonances were identiﬁed. Among these were lipid (Lip) res- onating at ∼1.3ppm (parts per million) and lactate (Lac) doublet peak also appearing at a resonant frequency of ∼1.3ppm, as an indicator of anaerobic glycolysis. The Lac doublet is J-modulated and appears as inverted for echo times (TE) of 136ms. Also found fairly often were choline (Cho) at 3.2ppm or total Cho from 3.14 to 3.34ppm. Choline is an indicator of membrane damage, cellular proliferation and cell density, reﬂecting phospholipid metabolism of cell membranes. Further, creatine (Cr) at 3.0ppm, has frequently been detected as a marker of energy metabolism. A peak resonating at ∼2.0ppm was also sometimes reported. Based on in vitro analysis, this latter composite peak is comprised of N-acetyl aspartate (NAA) and N-acetyl groups from glycoproteins and/or glycolipids [51]. The metabolic information was primarily described qualitatively (presence or absence of a given resonance), and these are the data that were pooled for meta-analysis [7]. The only two metabolites signiﬁcantly more often found in cancerous lesions were Cho and Lac. However, relying on Cho detection alone, some 50 benign lesions would be incorrectly classiﬁed as cancerous, i.e. as false positive results, such that the positive predictive value (PPV) was com- puted to be 66%. Moreover, twenty malignant ovarian lesions would be incorrectly classiﬁed as benign according to lack of detected Cho. The latter are the false negative results, such that the negative predictive value (NPV) was 57.4%. A stronger model for Cho was obtained when age and magnetic ﬁeld strength, B0, were included. However, due to missing data, the latter model included much fewer patients. An unadjusted model with Lac alone generated better prediction, but there were data for only 25% of patients. The best PPV, NPV and overall accuracy were achieved with an adjusted J Math Chem (2017) 55:1110–1157 1127 model including both Lac and Cho among a total of 50 patients. 1.3.1 Conventional Fourier analysis of MRS time signals encoded from the ovary Yet, 4 of 24 patients with ovarian cancer were predicted to have benign lesions and 4 of 26 patients with benign ovarian lesions were predicted to have ovarian cancer. The overall conclusion from this meta-analysis is that in vivo MRS with conventional Fourier-based process- ing did not adequately distinguish malignant from benign ovarian lesions [7]. Via in vitro MRS, employing analytical chemistry methods and using much stronger static magnetic ﬁelds (e.g. 14.1T), greater metabolic insight can potentially be obtained for distinguishing cancerous from benign ovarian lesions [52]. A comparison of ﬂuid analyzed in vitro from 12 malignant and 23 benign ovarian cysts revealed signiﬁcantly higher concentrations of a number of metabolites in cancerous than in non-malignant cyst ﬂuid [52]. These metabolites were Cho and Lac, as well as isoleucine (Iso) (1.02ppm), valine (Val) (1.04ppm), threonine (Thr) (1.33 ppm), alanine (Ala) (1.51 ppm), lysine (Lys) (1.67–1.78ppm), methionine (Met) (2.13ppm) and glutamine (Gln) (2.42–2.52ppm). In ovarian serous cystadenocarcinomas, N-acetyl aspartate, NAA, was found in high concentrations, associated with water accumulation, according to a study employing gas chromatography–mass spectrometry of ovarian cyst ﬂuid [53]. When human epithelial ovarian carcinoma cell lines were compared to normal or immortalized ovarian epithelial cells, phosphocholine (PC) at ∼3.225ppm was found to be three to eight times higher in the malignant relative to the normal cells [54]. It should be noted that PC has been identiﬁed as a biomarker of malignant transforma- tion [55], possibly mediated, at least in part, by a loss of the tumor suppressor p53 function [56]. Taken as a whole, as reviewed in Ref. [7], there are quite extensive in vitro data indicating that a number of MR-observable compounds can distinguish can- cerous versus benign ovarian lesions. However, these metabolites need to be reliably quantiﬁed, a task for which the FFT with or without ﬁtting is inadequate. Therefore, systematic investigations were deemed justiﬁed using the advanced processing capa- bilities of the fast Padé transform, FPT, as will now be summarized. 1.3.2 The fast Padé transform applied to synthesized MRS time signals and to those encoded from the ovary Ontheother hand, therewereresonances that exhibited marked instability with even the slightest change in partial signal length or noise level σ, and these were classiﬁed as spurious. Thereby, all the genuine metabolic information was retained in the denoised spectrum, with the spurious part discarded. Later, a comparative study [6] of the capabilities of the FPT(+) and FPT(−) was carried out using synthesized noise-corrupted benign ovarian cyst time signals similar to those encoded in Ref. [52]. Both FPT variants, the FPT(+)and FPT(−), unequivocally identiﬁed all the genuine resonances at short total signal lengths and the metabolite concentrations were accurately computed. Notably, it was the FPT(+) which offered the most effective Signal-noise separation, SNS. This was due to the separation of the genuine and spurious resonances inside and outside the unit circle, respectively in the complex z-plane. Stated equivalently, the FPT(+) distinctly separates genuine and spurious resonances in the complex frequency plane, since they have Im(ω+ k,Q) > 0 and Im(ω+ k,Q) < 0, respectively. Another advantageous feature of the FPT(+) was that the pole-zero coincidences of spurious resonances remained complete at high noise levels. It was deemed likely that these capabilities of the FPT(+) could be particularly useful for processing MRS time signals encoded in vivo from the ovary. 1.3.2.2 The fast Padé transform applied to MRS time signals encoded in vivo from a borderline serous cystic ovary tumor In the ﬁrst study [7] applying the FPT to MRS time signals encoded in vivo from a borderline serous cystic ovarian tumor on a 3T MR scanner, at a quite short partial signal length of NP = 800 (K = 400) the FPT-generated total shape spectrum was shown to be better resolved compared to that produced from the FFT. Thus, there is further conﬁrmation of the high resolution capa- bilities of the FPT also for in vivo MRS data encoded from the ovary, as has previously been shown in the proof-of-principle studies on the corresponding synthesized MRS time signals associated with the ovary [1–6]. A spectra averaging procedure [19] was applied and shown to be able to stabilize the non-parametric shape estimation in face of a marked sensitivity to alteration in model order K. The problem of noise stem- ming from the encoding itself, is further exacerbated by the emergence of unphysical resonances from reconstruction by any processor. As a consequence, noise-like spikes appear. 1.3.2 The fast Padé transform applied to synthesized MRS time signals and to those encoded from the ovary 1.3.2 The fast Padé transform applied to synthesized MRS time signals and to those encoded from the ovary 1.3.2.1 Applications to synthesized MRS time signals associated with the ovary: proof-of-principle studies The ﬁrst studies [1,2] were carried out via the FPT(−) on synthesized noiseless time signals associated with MRS data from Ref. [52] for benign and malignant ovarian cyst ﬂuid. For each of the input 12 true metabolites, all the spectral parameters were accurately reconstructed and the metabolite concentrations correctly computed with a very small number of signal points (64) of the chosen full time signal with N = 1024. These results remained stable at longer partial signal lengths all the way up to N [1,2]. We compared the performance of the FPT(−) with that of the FFT. At the partial signal length NP = 64, the FFT yielded only rudimen- tary, uninterpretable spectra. The FFT needed some formidable 512 times more signal points, i.e. 32768, in order to generate the converged absorption total shape spec- tra for the noiseless data corresponding to benign and cancerous ovary [1,2]. Thus, the FPT(−) was shown to have clearly superior resolving capability for processing noiseless MRS data associated with the ovary. 12 3 1128 J Math Chem (2017) 55:1110–1157 In the subsequent studies, the FPT(−) was applied to simulated MRS time signals reminiscent of those from ovarian cancer, with the addition of increasing levels of noise. At lower noise levels, σ = 0.01156 RMS, where RMS is the root-mean- square of the noise-free time signal, some 128 signal points were needed to accurately reconstruct the spectral parameters for the input 12 physical resonances. The remaining 52 reconstructed resonances were spurious, and at this lower level of added noise, such spurious resonances could all be identiﬁed by their pole-zero conﬂuences as well as by the associated zero-valued amplitudes [3]. However, at higher levels of noise (σ = 0.1156 RMS, σ = 0.1296 RMS and σ = 0.2890 RMS), the pole-zero coincidences in the FPT(−) were not always complete and near-zero amplitudes were found for some of the spurious resonances [4,5]. The stability test then became crucial, such that when varying the partial signal length NP, and/or also by adding yet more noise, a set of resonances, including those with very small amplitudes, was identiﬁed bytheir constancyandbinnedas genuine. 1.3.2 The fast Padé transform applied to synthesized MRS time signals and to those encoded from the ovary By taking the arithmetic average of some 11 complex envelopes, an average 12 123 1129 J Math Chem (2017) 55:1110–1157 complex envelope was generated in which these spikes were greatly attenuated or van- ishedaltogether[7].DuetotheencodingatashortTEof30ms,thetotalshapespectrum was extremely dense, as many short-lived metabolites had not yet decayed. The com- plex average envelope was inverted to produce a new complex FID. Using the latter reconstructed time signal, subsequent parametric analysis through the FPT(+) recov- ered dense component spectra in the two modes described in Sect. 1.1.2.4. Namely, one was the Usual mode where the absorption and dispersion components are mixed. The other was the Ersatz mode where only the absorptive Lorentzian components exist, where the reconstructed phases are set to zero to artiﬁcially eliminate interference effects (for visual purposes alone). A large number of metabolites, including potential cancer biomarkers, were identiﬁed and quantiﬁed thereby. Among these were Cho, PC, NAA, Ala, Iso, Val, Lip, Lac, Lys, N-acetylneuraminic acid (AcNeu), glutamine (Glu) and myoinositol (m-Ins), etc. Many of these resonances were not detected with Fourier plus ﬁtting of in vivo MRS data from the ovary. complex envelope was generated in which these spikes were greatly attenuated or van- ishedaltogether[7].DuetotheencodingatashortTEof30ms,thetotalshapespectrum was extremely dense, as many short-lived metabolites had not yet decayed. The com- plex average envelope was inverted to produce a new complex FID. Using the latter reconstructed time signal, subsequent parametric analysis through the FPT(+) recov- ered dense component spectra in the two modes described in Sect. 1.1.2.4. Namely, one was the Usual mode where the absorption and dispersion components are mixed. The other was the Ersatz mode where only the absorptive Lorentzian components exist, where the reconstructed phases are set to zero to artiﬁcially eliminate interference effects (for visual purposes alone). A large number of metabolites, including potential cancer biomarkers, were identiﬁed and quantiﬁed thereby. Among these were Cho, PC, NAA, Ala, Iso, Val, Lip, Lac, Lys, N-acetylneuraminic acid (AcNeu), glutamine (Glu) and myoinositol (m-Ins), etc. Many of these resonances were not detected with Fourier plus ﬁtting of in vivo MRS data from the ovary. In the follow-up study [8], the FPT was further optimized for encoded in vivo MRS timesignalsfromaborderlineserouscysticovariantumor.Thiswasachievedbyacom- bination of spectra averaging and time signal extrapolation. In particular, as described in Sect. 1.1.2.8, the Padé-based extrapolation capability, through a rational function provides salient information beyond the last encoded signal point cN−1(t > T ). 1.3.2 The fast Padé transform applied to synthesized MRS time signals and to those encoded from the ovary Con- vergence of reconstructions was assessed for a sequence of six successive values of K. Variances were markedly diminished for the reconstructed parameters (complex frequencies and complex amplitudes) when spectra averaging and extrapolation were carried out in combination. In that study [8], it became clear that applications of the FPT for analysis of in vivo encoded MRS time signals would be brought to the point of practical implementation insofar as the system’s stability was examined most thoroughly. This would require in-depth scrutiny of the poles and zeros which, as mentioned, are the key to separating genuine from spurious content. In other words, insofar as the challenge of eliminating the oversensitivity to alterations in model order K were to be effectively surmounted, a more comprehensive investigation was deemed necessary. This would incorporate further study of the role of spectra averaging and time signal extrapolation for the Padé-reconstructed envelopes, spectral parameters and component shape spectra, with a particular focus upon density distributions of the constituent poles and zeros. Of critical importance is to systematically examine convergence of all the variables under study for a larger number of values of K than in Ref. [8]. Such a comprehensive inquiry is our present goal. 2.1 In vivo acquisition of MRS time signals from a borderline serous cystic ovarian tumor 2.1 In vivo acquisition of MRS time signals from a borderline serous cystic ovarian tumor We applied the FPT(+) to encoded FID data from a 56year-old patient with an enlarged left ovary, as detected on TVUS. The patient was included in the in vivo MRS study of ovarian cyst ﬂuid as per Ref. [51]. Our colleagues from the Department of Obstetrics 123 1130 J Math Chem (2017) 55:1110–1157 and Gynecology, Radboud University Nijmegen Medical Center in the Netherlands kindly provided us with these data. A 3T Magnetom Tim Trio, Siemens MR clinical scanner was used to encode the MRS time signals. The bandwidth, BW, was 1200Hz, and the Larmor frequency was νL = 127.732MHz corresponding to the magnetic ﬁeld strength B0 = 3T. The sampling time τ was 0.833 ms (τ = 1/BW ≈0.833ms). The voxel of interest (3cm×3cm×3cm) was in the inferior cystic portion of the tumor. A point-resolved spectroscopy sequence (PRESS) was used, with repetition time (TR) of 2000ms and two echo times, TE=30 and 136ms. In the present study, we examine only the FID data encoded at 30ms. Partial suppression of the giant water peak was achieved through encoding via WET (water suppression through enhanced T1 effects). A total of 64 FIDs were encoded and then averaged to improve SNR, such that the so-called number of excitations (NEX) was 64. Each of the encoded 64 time signals contained 1024 data points. Subsequent to the in vivo MRS encoding, the tumor was surgically excised and subjected to histopathologic analysis, which revealed a borderline serous cystic ovarian lesion [51]. 2.2 Reconstructions by the FPT(+) using the in vivo MRS time signals The encoded FID of length N = 1024 was not corrected for the zero-order phase ϕ0. In encoded MRS time signals, the phases ϕk of the amplitudes dk are typically non-zero (ϕk ̸= 0), because of dephasing which occurs during encoding, as described in Sect. 1.1.2.4. The expansion coefﬁcients of the polynomials P+ K and Q+ K in the FPT(+) were calculated directly from the input time signal {cn} using the deﬁnition (5) and following the outlines in Sect. 1.1.2.3. A system of linear equations was solved for the expansion coefﬁcients {q+ s } of the denominator polynomial Q+ K (z). The expansion coefﬁcients {p+ r } of the numerator polynomial P+ K (z) are deduced from the analytical expression given in Sect. 1.1.2. With the set {p+ r , q+ s } at hand, the characteristic polynomials P+ K (z) and Q+ K (z) were rooted via P+ K (z) = 0 and Q+ K (z) = 0. Reconstruction of the frequencies {ω+ k,Q} and {ω+ k,P} was achieved through the roots {z+ k,Q} and {z+ k,P} of Q+ K (z) and P+ K (z), respectively. The amplitudes {d+ k }(1 ≤k ≤K) were deduced from the Cauchy analytical formula for the residues as given by (6). The total shape spectra were parametrically computed via the Heaviside partial fraction expansion from Eq. (11). Reconstruction of the component spectra is performed in the Usual, U, and Ersatz, E, modes, according to Eqs. (8) and (9), respectively. All the components and envelopes will be plotted at 1024 real-valued sweep frequencies, recalling that the full length N of the encoded FID is also 1024. 2.3 Padé-based spectra averaging As noted, spectra averaging is a strategy that can diminish the over-sensitivity of the spectral parameters as well as of spectra to changes in model order K [7,8,19]. In the current study, prior to averaging, we shall use the parametric FPT(+) to gen- erate a number of envelopes for a range of model orders K. Using the encoded 123 1131 J Math Chem (2017) 55:1110–1157 time signal, the complex Usual envelopes (P+ K /Q+ K )U will be computed for K = 575, 580, . . . , 625 (in constant increment K = 5) and for K = 575, 585, . . . , 625 (in constant increment K = 10). The arithmetic average is subsequently taken separately for each of the two groups of K, with the result denoted by {FPT(+)}U Av, where the subscript Av denotes average. For brevity, and in accordance with the conventions in mathematics, we shall from here on refer to “K = 575(5)625” instead of “K = 575, 580, . . . , 625 (K = 5)” and “K = 575(10)625” in lieu of “K = 575, 585, . . . , 625 (K = 10)”. 2.4 The extrapolation procedure within the FPT Both the extrapolation and interpolation capabilities exist within the FPT, as discussed in Sect. 1.1.2. In contrast to the FFT, whereby only the length of the encoded FID determines the number of Fourier grid frequencies νF m ≡m/T (0 ≤m ≤N −1), in the FPT, the Padé spectrum P+ K /Q+ K can be computed at arbitrary sweep fre- quency ν between any two adjacent values of νF m and for ν > νF mmax. Consequently, both interpolation and extrapolation can be implemented. We compute the complex envelopes (P+ K /Q+ K )U at 5N = 5 × 1024(5120) equidistant sweep frequencies ν for K = 575(5)625. The arithmetic average of these spectra then yields the complex average envelope {FPT(+)}U Av at the same 5N frequencies ν. Inverting {FPT(+)}U Av by the IDFT generates the reconstructed FID of length 5N. This reconstructed FID is then truncated to 2N (2048) before quantiﬁcation, for convenience. Thereby, Padé-based extrapolation and interpolation of the encoded FID are carried out, with extension of the input time signal to twice the original T (N = 1024 vs. 2048, T vs. 2T ). Further, the reconstructed time signal from the complex average envelope {FPT(+)}U Av is quan- tiﬁed by the parametric FPT(+) for 6 and 11 values of model order K = 575(10)625 and K = 575(5)625, respectively. The ensuing sets of spectral parameters are com- pared with their counterparts for K = 575(10)625 and K = 575(5)625 retrieved from the encoded time signal supplemented with 2K −1024 points of zero amplitudes. For the latter six and eleven sets of spectral parameters, there is no spectra averaging nor Padé-based extrapolation. 3.2 Averaging of envelopes through the FPT(+) In the top two panels of Fig. 1 are the two parts of the MRS time signal encoded from a borderline serous cystic ovarian lesion, with a total number of 1024 data points. The real part of the encoded time signal is displayed on the upper left panel (a), with the imaginary part on the upper right panel (b). To guide the eye, a magenta line is drawn across the abscissae of panels (a, b). Thereby, it can be noted that below ∼300ms, the FID waveforms are asymmetric around the abscissae. This is because the residual water peak is still much more abundant (about 7 times) compared to all the other metabolites. Above 300 ms, the time signal exhibits nearly symmetrical oscillations around the abscissa. In panel (c) of Fig. 1 within the chosen spectral region of interest (SRI) between 0.75 and 3.75ppm, the real parts of 11 Usual envelopes Re P+ K /Q+ K U are shown. These envelopes, displayed in green, were generated by the parametric FPT(+) for K = 575(5)625. Since NP/2 = K, the corresponding partial signal lengths are NP = 1150(10)1250. Here, each NP is longer than the total length 1024 of the encoded FID, such that the missing 2K −1024 data were the added time signal points with zero amplitudes. The most prominent structure in panel (c) is a spike close to 3.4ppm, and there are many other spikes interspersed throughout the entire SRI. (+) U In the top two panels of Fig. 1 are the two parts of the MRS time signal encoded from a borderline serous cystic ovarian lesion, with a total number of 1024 data points. The real part of the encoded time signal is displayed on the upper left panel (a), with the imaginary part on the upper right panel (b). To guide the eye, a magenta line is drawn across the abscissae of panels (a, b). Thereby, it can be noted that below ∼300ms, the FID waveforms are asymmetric around the abscissae. This is because the residual water peak is still much more abundant (about 7 times) compared to all the other metabolites. Above 300 ms, the time signal exhibits nearly symmetrical oscillations around the abscissa. In panel (c) of Fig. 3.1 Conventions We begin by brieﬂy describing the conventions used to present the results. Each ﬁgure in this paper was designed to be entirely self-contained, with the complete, detailed information included therein. In addition to a summarizing principal title at the top of the ﬁgure, the speciﬁcs of each panel are also described, so that the reader is not necessarily obliged to refer back to the main text. The relevant formulae are displayed for each panel, and the ordinates and abscissae are completely labeled. The partial signal lengths, NP, employed will always be even, so that K is an integer in relation NP/2 = K and the diagonal form P+ K (z)/Q+ K (z) of the spectrum in the FPT(+) will be used throughout Sect. 3. As mentioned, the increment K will be speciﬁed within small parentheses located between the lower and upper values of the model order 12 3 1132 J Math Chem (2017) 55:1110–1157 K. As was the case for the equations presented in Sect. 1.1.2.4, in the ﬁgures too, the superscript U denotes Usual and E indicates Ersatz. The subscript Av denotes average. The standard conventions Re and Im, to indicate the real and imaginary parts of complex quantities, respectively, will be used throughout. 3.2 Averaging of envelopes through the FPT(+) 1 within the chosen spectral region of interest (SRI) between 0.75 and 3.75ppm, the real parts of 11 Usual envelopes Re P+ K /Q+ K U are shown. These envelopes, displayed in green, were generated by the parametric FPT(+) for K = 575(5)625. Since NP/2 = K, the corresponding partial signal lengths are NP = 1150(10)1250. Here, each NP is longer than the total length 1024 of the encoded FID, such that the missing 2K −1024 data were the added time signal points with zero amplitudes. The most prominent structure in panel (c) is a spike close to 3.4ppm, and there are many other spikes interspersed throughout the entire SRI. Panel (d) shows, in blue, the real part Re{FPT(+)}U Av of the complex arithmetic aver- age envelope for the mentioned 11 envelopes. No spikes are noted therein, such that an apparently clean total shape spectrum is generated, and a large number of metabolites can be identiﬁed. The latter are denoted by abbreviations above the corresponding peaks, with assignments based upon Refs. [51,52,57,58]. The largest resonances are observed in the chemical shift region between 2.0 and 2.1ppm. Therein, two peaks with a deep split between them, correspond to acNeu (2.06ppm), as the taller narrower resonance, while the shorter and broader peak is assigned to NAA (2.03ppm). All the metabolite abbreviations are deﬁned in the list at the beginning of the present paper. Next, the reconstructed FID is produced by the IDFT inversion of the Padé- generated complex average envelope of length 5N = 5120 which is afterwards truncated to 2N = 2048, as stated. The real and imaginary parts of the reconstructed time signal are displayed, respectively, on panels (e, f) of Fig. 1. This reconstructed FID is seen to be regularized, i.e. it is now symmetric around the abscissae throughout the entire displayed time, i.e. from zero to 1024ms. Note that the reconstructed FID with 2N = 2048 was provisionally truncated to N, and only the ﬁrst half, i.e. 1024 time signal points are displayed in panels (e, f). This is done for the purpose of having a direct comparison of the waveforms of the reconstructed time signal with that of the input FID data of length 1024 from panels (a, b), respectively. Figure 2 deals with convergence of average envelopes. It provides a more in depth examination of the oversensitivity of reconstructions to changes in model order K. 123 3.2 Averaging of envelopes through the FPT(+) 1 The real and imaginary parts (a, b) of the FID encoded in vivo with a 3T MR scanner from a borderline serous cystic ovarian lesion. Water partially suppressed via the WET procedure in the course of encoding. Horizontal magenta lines guide the eye through departures from the level of the zero-valued amplitudes in the oscillations of the FID. Encoded FID data courtesy of the group from Ref. [51]. The real parts of 11 Usual envelopes, Re(P+ K /Q+ K )U, marked in green, for K = 575(5)625 wherein many large noise-like spikes are seen (c). Eleven complex envelopes are averaged; the real part of the result is denoted by Re{FPT(+)}U Av where a “clean” appearing spectrum is generated, and shown in blue (d). Metabolite assignments are presented in (d), with full names given in the list of abbreviations. The real and imaginary parts of the reconstructed FID produced by the IDFT inversion of the complex average envelope are displayed on (e, f), respectively. This reconstructed FID is symmetric around the abscissae throughout the entire time, i.e. 3.2 Averaging of envelopes through the FPT(+) from 0 to 1023ms (Color online) In Vivo MRS for Ovarian Tumor : Encoded & Reconstructed Time Signals (Free Induction Decays, FID) Parametric FPT (+) : Envelopes for Varying Model Orders K and the Arithmetic Average Envelope 0 200 400 600 800 1000 −0.05 0 0.05 0.1 0.15 0.2 0.25 (a) Real Part of the Encoded FID 1024 Real FID Data Points No Correction for Phase φ0 Time (ms) or Signal Number, n 10−3 × Re(FID) (au) 0 200 400 600 800 1000 −0.25 −0.2 −0.15 −0.1 −0.05 0 0.05 (b) Imaginary Part of the Encoded FID 1024 Imaginary FID Data Points No Correction for Phase φ0 Time (ms) or Signal Number, n 10−3 × Im(FID) (au) 1 1.5 2 2.5 3 3.5 −1 −0.5 0 0.5 1 1.5 2 2.5 Real Parts of Envelopes for 11 Model Orders K : Re(P + K /Q+ K )U ≡ Re{Σk=1 K d+ kz/(z − z+ k,Q )} (c) NAA acNeu FPT(+) : 11 Envelopes for Model Orders K = 575(5)625 Use of the Encoded FID Chemical Shift (ppm) 10−3 × Re(P + K /Q+ K )U (au) 1 1.5 2 2.5 3 3.5 −1 −0.5 0 0.5 1 1.5 2 Real Part of the Average Envelope: Re{FPT (+)}U Av ≡ Re{(1/11)ΣK=575 625(5)(P + K /Q+ K )U } (d) Re{FPT (+)}U Av ≡ Re{(1/11)ΣK=575 625(5)Σk=1 K d+ kz/(z − z+ k,Q ) } FPT(+) : Use of the Encoded FID Average Envelope Gly m−Ins Glc m−Ins Mann His Bet GPC PC Cho Crn Tyr Cit PCr Cr NAA Gln Bet m−Ins Cit Glu Gln Pyr Gly Met Gln NAA acNeu Lys Ace Lys Leu Ala Lys Iso Lac Lip Thr Lip Gly Val Iso Leu Chemical Shift (ppm) 10−3 × Re{FPT (+)}U Av (au) 0 200 400 600 800 1000 −0.2 −0.1 0 0.1 0.2 (e) Real Part of the Reconstructed FID 1024 Real FID Data Points No Correction for Phase φ0 From the Inverted Complex Average Envelope Time (ms) or Signal Number, n 10−3 × Re(FID) (au) 0 200 400 600 800 1000 −0.2 −0.1 0 0.1 0.2 (f) Imaginary Part of the Reconstructed FID 1024 Imaginary FID Data Points No Correction for Phase φ0 From the Inverted Complex Average Envelope Time (ms) or Signal Number, n 10−3 × Im(FID) (au) In Vivo MRS for Ovarian Tumor : Encoded & Reconstructed Time Signals (Free Induction Decays, FID) Parametric FPT (+) : Envelopes for Varying Model Orders K and the Arithmetic Average Envelope Fig. 3.2 Averaging of envelopes through the FPT(+) from 0 to 1023ms (Color online) Parametric FPT (+) : Envelopes for Varying Model Orders K and the Arithmetic Average Envelope 0 200 400 600 800 1000 −0.25 −0.2 −0.15 −0.1 −0.05 0 0.05 (b) Imaginary Part of the Encoded FID 1024 Imaginary FID Data Points No Correction for Phase φ0 Time (ms) or Signal Number, n 10−3 × Im(FID) (au) U K Model Orders K and the Arithmetic Average Envelope 0 200 400 600 800 1000 −0.05 0 0.05 0.1 0.15 0.2 0.25 (a) Real Part of the Encoded FID 1024 Real FID Data Points No Correction for Phase φ0 Time (ms) or Signal Number, n 10−3 × Re(FID) (au) Imaginary Part of the Encoded FID (c) Real Parts of Envelopes for 11 Model Orders K : Re(P + K /Q+ K )U ≡ Re{Σk=1 K d+ kz/(z − z+ k,Q )} 1 1.5 2 2.5 3 3.5 −1 −0.5 0 0.5 1 1.5 2 2.5 Real Parts of Envelopes for 11 Model Orders K : Re(P + K /Q+ K )U ≡ Re{Σk=1 K d+ kz/(z − z+ k,Q )} (c) NAA acNeu FPT(+) : 11 Envelopes for Model Orders K = 575(5)625 Use of the Encoded FID Chemical Shift (ppm) 10−3 × Re(P + K /Q+ K )U (au) Real Part of the Average Envelope: Re{FPT (+)}U ≡Re{(1/11)Σ625(5)(P + /Q+ )U } (d) 0 200 400 600 800 1000 −0.2 −0.1 0 0.1 0.2 (e) Real Part of the Reconstructed FID 1024 Real FID Data Points No Correction for Phase φ0 From the Inverted Complex Average Envelope Time (ms) or Signal Number, n 10−3 × Re(FID) (au) 0 200 400 600 800 1000 −0.2 −0.1 0 0.1 0.2 (f) Imaginary Part of the Reconstructed FID 1024 Imaginary FID Data Points No Correction for Phase φ0 From the Inverted Complex Average Envelope Time (ms) or Signal Number, n 10−3 × Im(FID) (au) Fig. 1 The real and imaginary parts (a, b) of the FID encoded in vivo with a 3T MR scanner from a borderline serous cystic ovarian lesion. Water partially suppressed via the WET procedure in the course of encoding. Horizontal magenta lines guide the eye through departures from the level of the zero-valued amplitudes in the oscillations of the FID. Encoded FID data courtesy of the group from Ref. [51]. 3.2 Averaging of envelopes through the FPT(+) 1 The real and imaginary parts (a, b) of the FID encoded in vivo with a 3T MR scanner from a borderline serous cystic ovarian lesion. Water partially suppressed via the WET procedure in the course of encoding. Horizontal magenta lines guide the eye through departures from the level of the zero-valued amplitudes in the oscillations of the FID. Encoded FID data courtesy of the group from Ref. [51]. The real parts of 11 Usual envelopes, Re(P+ K /Q+ K )U, marked in green, for K = 575(5)625 wherein many large noise-like spikes are seen (c). Eleven complex envelopes are averaged; the real part of the result is denoted by Re{FPT(+)}U Av where a “clean” appearing spectrum is generated, and shown in blue (d). Metabolite assignments are presented in (d), with full names given in the list of abbreviations. The real and imaginary parts of the reconstructed FID produced by the IDFT inversion of the complex average envelope are displayed on (e, f), respectively. This reconstructed FID is symmetric around the abscissae throughout the entire time, i.e. 3.2 Averaging of envelopes through the FPT(+) In panel (a), the real parts of six individual envelopes reconstructed for model orders K = 123 1133 J Math Chem (2017) 55:1110–1157 0 200 400 600 800 1000 −0.05 0 0.05 0.1 0.15 0.2 0.25 (a) Real Part of the Encoded FID 1024 Real FID Data Points No Correction for Phase φ0 Time (ms) or Signal Number, n 10−3 × Re(FID) (au) 0 200 400 600 800 1000 −0.25 −0.2 −0.15 −0.1 −0.05 0 0.05 (b) Imaginary Part of the Encoded FID 1024 Imaginary FID Data Points No Correction for Phase φ0 Time (ms) or Signal Number, n 10−3 × Im(FID) (au) Real Parts of Envelopes for 11 Model Orders K : Re(P + K /Q+ K )U ≡ Re{Σk=1 K d+ kz/(z − z+ k Q )} (c) In Vivo MRS for Ovarian Tumor : Encoded & Reconstructed Time Signals (Free Induction Decays, FID) Parametric FPT (+) : Envelopes for Varying Model Orders K and the Arithmetic Average Envelope In Vivo MRS for Ovarian Tumor : Encoded & Reconstructed Time Signals (Free Induction Decays, FID) In Vivo MRS for Ovarian Tumor : Encoded & Reconstructed Time Signals (Free Induction Decays, FID) 0 200 400 600 800 1000 −0.05 0 0.05 0.1 0.15 0.2 0.25 (a) Real Part of the Encoded FID 1024 Real FID Data Points No Correction for Phase φ0 Time (ms) or Signal Number, n 10−3 × Re(FID) (au) 0 200 400 600 800 1000 −0.25 −0.2 −0.15 −0.1 −0.05 0 0.05 (b) Imaginary Part of the Encoded FID 1024 Imaginary FID Data Points No Correction for Phase φ0 Time (ms) or Signal Number, n 10−3 × Im(FID) (au) 1 1.5 2 2.5 3 3.5 −1 −0.5 0 0.5 1 1.5 2 2.5 Real Parts of Envelopes for 11 Model Orders K : Re(P + K /Q+ K )U ≡ Re{Σk=1 K d+ kz/(z − z+ k,Q )} (c) NAA acNeu FPT(+) : 11 Envelopes for Model Orders K = 575(5)625 Use of the Encoded FID Chemical Shift (ppm) 10−3 × Re(P + K /Q+ K )U (au) 1 1.5 2 2.5 3 3.5 −1 −0.5 0 0.5 1 1.5 2 Real Part of the Average Envelope: Re{FPT (+)}U Av ≡ Re{(1/11)ΣK=575 625(5)(P + K /Q+ K )U } (d) Re{FPT (+)}U Av ≡ Re{(1/11)ΣK=575 625(5)Σk=1 K d+ kz/(z − z+ k,Q ) } FPT(+) : Use of the Encoded FID Average Envelope Gly m−Ins Glc m−Ins Mann His Bet GPC PC Cho Crn Tyr Cit PCr Cr NAA Gln Bet m−Ins Cit Glu Gln Pyr Gly Met Gln NAA acNeu Lys Ace Lys Leu Ala Lys Iso Lac Lip Thr Lip Gly Val Iso Leu Chemical Shift (ppm) 10−3 × Re{FPT (+)}U Av (au) 0 200 400 600 800 1000 −0.2 −0.1 0 0.1 0.2 (e) Real Part of the Reconstructed FID 1024 Real FID Data Points No Correction for Phase φ0 From the Inverted Complex Average Envelope Time (ms) or Signal Number, n 10−3 × Re(FID) (au) 0 200 400 600 800 1000 −0.2 −0.1 0 0.1 0.2 (f) Imaginary Part of the Reconstructed FID 1024 Imaginary FID Data Points No Correction for Phase φ0 From the Inverted Complex Average Envelope Time (ms) or Signal Number, n 10−3 × Im(FID) (au) In Vivo MRS for Ovarian Tumor : Encoded & Reconstructed Time Signals (Free Induction Decays, FID) Parametric FPT (+) : Envelopes for Varying Model Orders K and the Arithmetic Average Envelope Fig. 3.2 Averaging of envelopes through the FPT(+) The real parts of 11 Usual envelopes, Re(P+ K /Q+ K )U, marked in green, for K = 575(5)625 wherein many large noise-like spikes are seen (c). Eleven complex envelopes are averaged; the real part of the result is denoted by Re{FPT(+)}U Av where a “clean” appearing spectrum is generated, and shown in blue (d). Metabolite assignments are presented in (d), with full names given in the list of abbreviations. The real and imaginary parts of the reconstructed FID produced by the IDFT inversion of the complex average envelope are displayed on (e, f), respectively. This reconstructed FID is symmetric around the abscissae throughout the entire time, i.e. 3.2 Averaging of envelopes through the FPT(+) from 0 to 1023ms (Color online) 12 3 1134 J Math Chem (2017) 55:1110–1157 Spectra Averaging for Mitigation of Oversensitivity of Reconstructions to Alterations in Model Order K Convergence Rate of the Average Envelope for 2 Overlapping Sets of 6 and 11 Individual Envelopes Spectra Averaging for Mitigation of Oversensitivity of Reconstructions to Alterations in Model Order K Convergence Rate of the Average Envelope for 2 Overlapping Sets of 6 and 11 Individual Envelopes 1 1.5 2 2.5 3 3.5 −1 −0.5 0 0.5 1 1.5 2 2.5 Real Parts of Envelopes for 6 Model Orders K : Re(P + K /Q+ K )U ≡ Re{Σk=1 K d+ kz/(z − z+ k,Q )} (a) NAA acNeu FPT(+) : 6 Envelopes for Model Orders K = 575(10)625 Use of the Encoded FID Chemical Shift (ppm) 10−3 × Re(P + K /Q+ K )U (au) 1 1.5 2 2.5 3 3.5 −1 −0.5 0 0.5 1 1.5 2 Real Part of the Average Envelope: Re{FPT (+)}U Av ≡ Re{(1/6)ΣK=575 625(10)(P + K /Q+ K )U } (b) Re{FPT (+)}U Av ≡ Re{(1/6)ΣK=575 625(10)Σk=1 K d+ kz/(z − z+ k,Q )} FPT(+) : Use of the Encoded FID Average Envelope Gly m−Ins Glc m−Ins Mann His Bet GPC PC Cho Crn Tyr Cit PCr Cr NAA Gln Bet m−Ins Cit Glu Gln Pyr Gly Met Gln NAA acNeu Lys Ace Lys Leu Ala Lys Iso Lac Lip Thr Lip Gly Val Iso Leu Chemical Shift (ppm) 10−3 × Re{FPT (+)}U Av (au) 1 1.5 2 2.5 3 3.5 −1 −0.5 0 0.5 1 1.5 2 2.5 Real Parts of Envelopes for 11 Model Orders K : Re(P + K /Q+ K )U ≡ Re{Σk=1 K d+ kz/(z − z+ k,Q )} (c) NAA acNeu FPT(+) : 11 Envelopes for Model Orders K = 575(5)625 Use of the Encoded FID Chemical Shift (ppm) 10−3 × Re(P + K /Q+ K )U (au) 1 1.5 2 2.5 3 3.5 −1 −0.5 0 0.5 1 1.5 2 Real Part of the Average Envelope: Re{FPT (+)}U Av ≡ Re{(1/11)ΣK=575 625(5)(P + K /Q+ K )U } (d) Re{FPT (+)}U Av ≡ Re{(1/11)ΣK=575 625(5)Σk=1 K d+ kz/(z − z+ k,Q )} FPT(+) : Use of the Encoded FID Average Envelope Gly m−Ins Glc m−Ins Mann His Bet GPC PC Cho Crn Tyr Cit PCr Cr NAA Gln Bet m−Ins Cit Glu Gln Pyr Gly Met Gln NAA acNeu Lys Ace Lys Leu Ala Lys Iso Lac Lip Thr Lip Gly Val Iso Leu Chemical Shift (ppm) 10−3 × Re{FPT (+)}U Av (au) Convergence Rate of the Average Envelope for 2 Overlapping Sets of 6 and 11 Individual Envelopes Fig. 3.2 Averaging of envelopes through the FPT(+) 2 The real parts of six Usual envelopes, Re(P+ K /Q+ K )U, marked in black, green, cyan, red, magenta and blue for K = 575(10)625, with a number of noise-like spikes (a). The real part of the corresponding average envelope Re{FPT(+)}U Av (b). The real parts of 11 Usual envelopes, Re(P+ K /Q+ K )U, for K = 575(5)625 exhibiting even larger noise-like spikes (c). The real part of the corresponding average envelope Re{FPT(+)}U Av (d) (Color online) 1 1.5 2 2.5 3 3.5 −1 −0.5 0 0.5 1 1.5 2 2.5 Real Parts of Envelopes for 11 Model Orders K : Re(P + K /Q+ K )U ≡ Re{Σk=1 K d+ kz/(z − z+ k,Q )} (c) NAA acNeu FPT(+) : 11 Envelopes for Model Orders K = 575(5)625 Use of the Encoded FID Chemical Shift (ppm) 10−3 × Re(P + K /Q+ K )U (au) (c) 1 1.5 2 2.5 3 3.5 −1 −0.5 0 0.5 Use of the Encoded FID Chemical Shift (ppm) 10−3 × Re(P K 1 1.5 2 2.5 3 3.5 −1 −0.5 0 0.5 1 1.5 2 Real Part of the Average Envelope: Re{FPT (+)}U Av ≡ Re{(1/11)ΣK=575 625(5)(P + K /Q+ K )U } (d) Re{FPT (+)}U Av ≡ Re{(1/11)ΣK=575 625(5)Σk=1 K d+ kz/(z − z+ k,Q )} FPT(+) : Use of the Encoded FID Average Envelope Gly m−Ins Glc m−Ins Mann His Bet GPC PC Cho Crn Tyr Cit PCr Cr NAA Gln Bet m−Ins Cit Glu Gln Pyr Gly Met Gln NAA acNeu Lys Ace Lys Leu Ala Lys Iso Lac Lip Thr Lip Gly Val Iso Leu Chemical Shift (ppm) 10−3 × Re{FPT (+)}U Av (au) 1 1.5 2 2.5 3 3.5 −1 −0.5 0 0.5 1 1.5 2 Real Part of the Average Envelope: Re{FPT (+)}U Av ≡ Re{(1/11)ΣK=575 625(5)(P + K /Q+ K )U } (d) Re{FPT (+)}U Av ≡ Re{(1/11)ΣK=575 625(5)Σk=1 K d+ kz/(z − z+ k,Q )} FPT(+) : Use of the Encoded FID Average Envelope Gly m−Ins Glc m−Ins Mann His Bet GPC PC Cho Crn Tyr Cit PCr Cr NAA Gln Bet m−Ins Cit Glu Gln Pyr Gly Met Gln NAA acNeu Lys Ace Lys Leu Ala Lys Iso Lac Lip Thr Lip Gly Val Iso Leu Chemical Shift (ppm) 10−3 × Re{FPT (+)}U Av (au) Fig. 3.2 Averaging of envelopes through the FPT(+) 2 The real parts of six Usual envelopes, Re(P+ K /Q+ K )U, marked in black, green, cyan, red, magenta and blue for K = 575(10)625, with a number of noise-like spikes (a). The real part of the corresponding average envelope Re{FPT(+)}U Av (b). The real parts of 11 Usual envelopes, Re(P+ K /Q+ K )U, for K = 575(5)625 exhibiting even larger noise-like spikes (c). 3.2 Averaging of envelopes through the FPT(+) The real part of the corresponding average envelope Re{FPT(+)}U Av (d) (Color online) 1 1.5 2 2.5 3 3.5 −1 −0.5 0 0.5 1 1.5 2 2.5 Real Parts of Envelopes for 6 Model Orders K : Re(P + K /Q+ K )U ≡ Re{Σk=1 K d+ kz/(z − z+ k,Q )} (a) NAA acNeu FPT(+) : 6 Envelopes for Model Orders K = 575(10)625 Use of the Encoded FID Chemical Shift (ppm) 10−3 × Re(P + K /Q+ K )U (au) (+) U 625(10) + + U ( ) g g p pp g p Real Parts of Envelopes for 6 Model Orders K : Re(P + K /Q+ K )U ≡ Re{Σk=1 K d+ kz/(z − z+ k,Q )} Real Parts of Envelopes for 6 Model Orders K : Re(P + K /Q+ K )U ≡ Re{Σk=1 K d+ kz/(z − z+ k,Q )} 1 1.5 2 2.5 3 3.5 −1 −0.5 0 0.5 1 1.5 2 Real Part of the Average Envelope: Re{FPT (+)}U Av ≡ Re{(1/6)ΣK=575 625(10)(P + K /Q+ K )U } (b) Re{FPT (+)}U Av ≡ Re{(1/6)ΣK=575 625(10)Σk=1 K d+ kz/(z − z+ k,Q )} FPT(+) : Use of the Encoded FID Average Envelope Gly m−Ins Glc m−Ins Mann His Bet GPC PC Cho Crn Tyr Cit PCr Cr NAA Gln Bet m−Ins Cit Glu Gln Pyr Gly Met Gln NAA acNeu Lys Ace Lys Leu Ala Lys Iso Lac Lip Thr Lip Gly Val Iso Leu Chemical Shift (ppm) 10−3 × Re{FPT (+)}U Av (au) Real Part of the Average Envelope: Re{FPT (+)}U Av ≡ Re{(1/6)ΣK=575 625(10)(P + K /Q+ K )U } Chemical Shift (ppm) 1 1.5 2 2.5 3 3.5 −1 −0.5 0 0.5 1 1.5 2 2.5 Real Parts of Envelopes for 11 Model Orders K : Re(P + K /Q+ K )U ≡ Re{Σk=1 K d+ kz/(z − z+ k,Q )} (c) NAA acNeu FPT(+) : 11 Envelopes for Model Orders K = 575(5)625 Use of the Encoded FID Chemical Shift (ppm) 10−3 × Re(P + K /Q+ K )U (au) 1 1.5 2 2.5 3 3.5 −1 −0.5 0 0.5 1 1.5 2 Real Part of the Average Envelope: Re{FPT (+)}U Av ≡ Re{(1/11)ΣK=575 625(5)(P + K /Q+ K )U } (d) Re{FPT (+)}U Av ≡ Re{(1/11)ΣK=575 625(5)Σk=1 K d+ kz/(z − z+ k,Q )} FPT(+) : Use of the Encoded FID Average Envelope Gly m−Ins Glc m−Ins Mann His Bet GPC PC Cho Crn Tyr Cit PCr Cr NAA Gln Bet m−Ins Cit Glu Gln Pyr Gly Met Gln NAA acNeu Lys Ace Lys Leu Ala Lys Iso Lac Lip Thr Lip Gly Val Iso Leu Chemical Shift (ppm) 10−3 × Re{FPT (+)}U Av (au) Fig. 3.2 Averaging of envelopes through the FPT(+) 2 The real parts of six Usual envelopes, Re(P+ K /Q+ K )U, marked in black, green, cyan, red, magenta and blue for K = 575(10)625, with a number of noise-like spikes (a). The real part of the corresponding average envelope Re{FPT(+)}U Av (b). The real parts of 11 Usual envelopes, Re(P+ K /Q+ K )U, for K = 575(5)625 exhibiting even larger noise-like spikes (c). The real part of the corresponding average envelope Re{FPT(+)}U Av (d) (Color online) 123 1135 J Math Chem (2017) 55:1110–1157 575(10)625 are depicted with the colors ordered as black, green, cyan, red, magenta and blue, respectively. Therein, the discrepancy among these six envelopes can be clearly seen. Particularly notable are the magenta and green spikes albeit of fairly small heights at about 3.4ppm, as well as a somewhat larger cyan spike at about 1.55ppm. With averaging of these six envelopes, none of the spikes are visualized any longer in panel (b) of Fig. 2, such that the real part of the average envelope of the six envelopes appears to be entirely in blue. We proceed in panel (c) of Fig. 2 to show the real parts of 11 individual envelopes reconstructed for model orders K = 575(5)625, with the color-coding black (K = 575), green (K = 585), cyan (K = 595), red (K = 605), magenta (K = 615), blue (K = 625), i.e. the same as for K = 575(10)625, plus green (K = 580), cyan (K = 590), red (K = 600), magenta (K = 610) and blue (K = 620) for the remaining K = 580(10)620. Compared to panel (a) of Fig. 2, a much taller magenta-colored spike (K = 610) near 3.4ppm appears. Scattered throughout the SRI are several more discrepancies among the model orders, seen as spikes. This ﬁnding indicates that from the additional ﬁve model orders (K = 580, 590, 600, 610, 620) relative to K = 575, 585, 595, 605, 615, 625 from panel (a), further discrepancies occur, most notably at about 3.4ppm, where the magenta spike is the largest structure in the entire SRI. However, in panel (d), the real part of the average envelope for these 11 model orders is almost entirely identical to panel (b). Thus, convergence of the average envelopes has evidently been achieved. Regarding convergence, we do not stop with total shape spectra. 3.2 Averaging of envelopes through the FPT(+) Rather, in the remainder of this presentation, we shall extend the analysis to a much more stringent test of convergence which refers to spectral parameters (Figs. 3–10) and component shape spectra (Figs. 11, 12). For consistency, similarly to Fig. 2 for total shape spec- tra, Figs. 3–12 will also refer to two groups of model orders, K = 575(10)625 and K = 575(5)625 with 6 and 11 values of K, respectively. Moreover, we will stratify the convergence mechanism in order to determine its main pathway. To this end, we shall compare the reconstructed spectral parameters for the two distinct cases, with and without “spectra averaging” and “time signal extrapolation”. The needed data are gen- erated through four steps. First (i), the encoded FID is used to parametrically compute a sequence of envelopes for a set of values of K. Second (ii), the arithmetic average envelope is created using the envelopes from the 1st step. Third (iii), the complex average envelope from the 2nd step is inverted by the IDFT to yield the reconstructed time signal. Fourth (iv), the reconstructed FID from the 3rd step is subjected to quan- tiﬁcation by the FPT(+) to give the triples of parameters {ω+ k,Q, d+ k , ω+ k,P} from which the Usual and Ersatz component spectra are predicted. 3.3 Poles and zeros reconstructed by the FPT(+) with spectra averaging and time signal extrapolation Here, we examine the poles and zeros retrieved by the FPT(+) from the recon- structed FID data stemming from the combined effect of spectra averaging and time signal extrapolation. Panel (a) of Fig. 3 presents six sets of reconstructed poles in the Argand diagram depicting the imaginary, Im(ν+ k,Q), versus real, Re(ν+ k,Q), fre- quencies. These are again color-coded as black, green, cyan, red, magenta and blue, 12 12 3 3 1136 J Math Chem (2017) 55:1110–1157 1 1.5 2 2.5 3 3.5 −0.06 −0.04 −0.02 0 0.02 0.04 (a) FPT(+) ; All Poles (Circles) : ν+ k,Q = [1/(2πiτ)]ln(z+ k,Q ) , { z+ k,Q : Roots of Characteristic Equation Q+ K(z) = 0 } NAA acNeu 6 Argand Diagrams of All Poles From the Reconstructed FID Re(ν+ k,Q ) (ppm) Im(ν+ k,Q ) (ppm) Poles & Zeros in Component Spectra {P+ K(z)/Q+ K(z)}U k at K = 575(10)625 (Averaging & Extrapolation) Genuine: Stable & Physical Im( ν+ k,Q ) > 0 and Spurious: Unstable & Unphysical Im( ν+ k,Q ) < 0 1 1.5 2 2.5 3 3.5 −0.06 −0.04 −0.02 0 0.02 0.04 (a) FPT(+) ; All Poles (Circles) : ν+ k,Q = [1/(2πiτ)]ln(z+ k,Q ) , { z+ k,Q : Roots of Characteristic Equation Q+ K(z) = 0 } NAA acNeu 6 Argand Diagrams of All Poles From the Reconstructed FID Re(ν+ k,Q ) (ppm) Im(ν+ k,Q ) (ppm) 1 1.5 2 2.5 3 3.5 −0.06 −0.04 −0.02 0 0.02 0.04 (b) FPT(+) ; All Zeros (Circles) : ν+ k,P = [1/(2πiτ)]ln(z+ k,P ) , { z+ k,P : Roots of Characteristic Equation P+ K(z) = 0 } 6 Argand Diagrams of All Zeros From the Reconstructed FID Re(ν+ k,P ) (ppm) Im(ν+ k,P ) (ppm) 1 1.5 2 2.5 3 3.5 −0.06 −0.04 −0.02 0 0.02 0.04 (c) FPT(+) ; All Poles (Circles) : ν+ k,Q = [1/(2πiτ)]ln(z+ k,Q ) and All Zeros (Dots): ν+ k,P = [1/(2πiτ)]ln(z+ k,P ) NAA acNeu 6 Argand Diagrams of All Poles & All Zeros From the Reconstructed FID Re(ν+ k,X ) (ppm) ; X = P, Q Im(ν+ k,X ) (ppm) ; X = P, Q Poles & Zeros in Component Spectra {P+ K(z)/Q+ K(z)}U k at K = 575(10)625 (Averaging & Extrapolation) Genuine: Stable & Physical Im( ν+ k,Q ) > 0 and Spurious: Unstable & Unphysical Im( ν+ k,Q ) < 0 Fig. 3.3 Poles and zeros reconstructed by the FPT(+) with spectra averaging and time signal extrapolation 3 Quantiﬁcation of the reconstructed time signal (with spectra averaging and FID extrapolation) for K = 575(10)625 yielding: six sets of Argand plots of Im(ν+ k,Q) versus Re(ν+ k,Q) for six sets of poles (a), Im(ν+ k,P) versus Re(ν+ k,P) for 6 sets of zeros (b) and the poles plus zeros Im(ν+ k,X) versus Re(ν+ k,X ) with X = P, Q (c). Genuine poles and zeros are located at Im(ν+ k,X ) > 0, and spurious at Im(ν+ k,X ) < 0, where X = P, Q (Color online) 1 1.5 2 2.5 3 3.5 −0.06 −0.04 −0.02 0 0.02 0.04 (b) FPT(+) ; All Zeros (Circles) : ν+ k,P = [1/(2πiτ)]ln(z+ k,P ) , { z+ k,P : Roots of Characteristic Equation P+ K(z) = 0 } 6 Argand Diagrams of All Zeros From the Reconstructed FID Re(ν+ k,P ) (ppm) Im(ν+ k,P ) (ppm) (b) 1 1.5 2 2.5 3 3.5 −0.06 −0.04 −0.02 0 0.02 0.04 (c) FPT(+) ; All Poles (Circles) : ν+ k,Q = [1/(2πiτ)]ln(z+ k,Q ) and All Zeros (Dots): ν+ k,P = [1/(2πiτ)]ln(z+ k,P ) NAA acNeu 6 Argand Diagrams of All Poles & All Zeros From the Reconstructed FID Re(ν+ k,X ) (ppm) ; X = P, Q Im(ν+ k,X ) (ppm) ; X = P, Q (c) Fig. 3 Quantiﬁcation of the reconstructed time signal (with spectra averaging and FID extrapolation) for K = 575(10)625 yielding: six sets of Argand plots of Im(ν+ k,Q) versus Re(ν+ k,Q) for six sets of poles (a), Im(ν+ k,P) versus Re(ν+ k,P) for 6 sets of zeros (b) and the poles plus zeros Im(ν+ k,X) versus Re(ν+ k,X ) with X = P, Q (c). 3.3 Poles and zeros reconstructed by the FPT(+) with spectra averaging and time signal extrapolation Genuine poles and zeros are located at Im(ν+ k,X ) > 0, and spurious at Im(ν+ k,X ) < 0, where X = P, Q (Color online) 123 12 123 1137 J Math Chem (2017) 55:1110–1157 1 1.5 2 2.5 3 3.5 −0.06 −0.04 −0.02 0 0.02 0.04 (a) FPT(+) ; All Poles (Circles) : ν+ k,Q = [1/(2πiτ)]ln(z+ k,Q ) , { z+ k,Q : Roots of Characteristic Equation Q+ K(z) = 0 } NAA acNeu 11 Argand Diagrams of All Poles From the Reconstructed FID Re(ν+ k,Q ) (ppm) Im(ν+ k,Q ) (ppm) Poles & Zeros in Component Spectra {P+ K(z)/Q+ K(z)}U k at K = 575(5)625 (Averaging & Extrapolation) Genuine: Stable & Physical Im( ν+ k,Q ) > 0 and Spurious: Unstable & Unphysical Im( ν+ k,Q ) < 0 1 1.5 2 2.5 3 3.5 −0.06 −0.04 −0.02 0 0.02 0.04 (a) FPT(+) ; All Poles (Circles) : ν+ k,Q = [1/(2πiτ)]ln(z+ k,Q ) , { z+ k,Q : Roots of Characteristic Equation Q+ K(z) = 0 } NAA acNeu 11 Argand Diagrams of All Poles From the Reconstructed FID Re(ν+ k,Q ) (ppm) Im(ν+ k,Q ) (ppm) 1 1.5 2 2.5 3 3.5 −0.06 −0.04 −0.02 0 0.02 0.04 (b) FPT(+) ; All Zeros (Circles) : ν+ k,P = [1/(2πiτ)]ln(z+ k,P ) , { z+ k,P : Roots of Characteristic Equation P+ K(z) = 0 } 11 Argand Diagrams of All Zeros From the Reconstructed FID Re(ν+ k,P ) (ppm) Im(ν+ k,P ) (ppm) 1 1.5 2 2.5 3 3.5 −0.06 −0.04 −0.02 0 0.02 0.04 (c) FPT(+) ; All Poles (Circles) : ν+ k,Q = [1/(2πiτ)]ln(z+ k,Q ) and All Zeros (Dots): ν+ k,P = [1/(2πiτ)]ln(z+ k,P ) NAA acNeu 11 Argand Diagrams of All Poles & All Zeros From the Reconstructed FID Re(ν+ k,X ) (ppm) ; X = P, Q Im(ν+ k,X ) (ppm) ; X = P, Q Poles & Zeros in Component Spectra {P+ K(z)/Q+ K(z)}U k at K = 575(5)625 (Averaging & Extrapolation) Genuine: Stable & Physical Im( ν+ k,Q ) > 0 and Spurious: Unstable & Unphysical Im( ν+ k,Q ) < 0 Fig. 3.3 Poles and zeros reconstructed by the FPT(+) with spectra averaging and time signal extrapolation Due to such stabil- ity, these poles are all considered as genuine. In sharp contrast, in the region of Im(ν+ k,Q) < 0, there is no agreement whatsoever among the poles retrieved at dif- ferent model order K. Because of such instability, all the poles with Im(ν+ k,Q) < 0 are categorized as spurious. Visually, this instability is manifested via distinct circles in each of the six colors for Im(ν+ k,Q) < 0 throughout the entire SRI for chemical shifts Re(ν+ k,Q) ∈[0.75, 3.75]ppm. A very similar pattern is observed for the Argand plot of the Padé-reconstructed zeros, shown in panel (b) of Fig. 3 as Im(ν+ k,P) ver- sus Re(ν+ k,P). Namely, in the region of Im(ν+ k,P) > 0, there is concordance to the level of stochasticity among the displayed six color-coded sets of reconstructed gen- uine zeros for K = 575 (black) , 585 (green) , 595 (cyan) , 605 (red) , 615 (magenta) and 625 (blue). In other words, at Im(ν+ k,Q) > 0, nearly all the circles from the ﬁrst ﬁve of the mentioned colors are hidden underneath the last plotted blue-coded circle (K = 625). The remaining zeros, distributed in the region Im(ν+ k,P) < 0, are seen as distinct circles with all the six colors almost throughout the entire SRI, indicating instability with any change in model order K. Therefore, these latter roots of P+ K (z) are spurious zeros. Panel (c) of Fig. 3 combines the results of panels (a, b). Namely, both the reconstructed poles and zeros for K = 575, 585, 595, 605, 615 and 625 are displayed together in the Argand plot of Im(ν+ k,X) versus Re(ν+ k,X), with X = P and X = Q. The poles are illustrated in the same way as in panel (a), namely as color- coded circles, whereas the zeros are depicted as color-coded dots. Thereby, it can be clearly seen that the genuine poles and zeros, at Im(ν+ k,X) > 0, all in blue color are pre- dominantly non-coincident, and only sometimes lying at close distances. In contrast, nearly all poles and zeros are coincident at Im(ν+ k,Q) < 0 and Im(ν+ k,P) < 0, such that color-concordant dots and circles, i.e. Froissart doublets as spurious resonances, are seen as being distributed throughout the SRI. 3.3 Poles and zeros reconstructed by the FPT(+) with spectra averaging and time signal extrapolation with association to K = 575, 585, 595, 605, 615 and 625, corresponding to the par- tial signal lengths NP = 1150, 1170, 1190, 1210, 1230 and 1250, respectively. In the region of Im(ν+ k,Q) > 0, there is complete agreement to the level of stochas- ticity among the six sets of reconstructed poles, which almost always appear as blue circles (the last plotted curve is in blue for K = 625). Due to such stabil- ity, these poles are all considered as genuine. In sharp contrast, in the region of Im(ν+ k,Q) < 0, there is no agreement whatsoever among the poles retrieved at dif- ferent model order K. Because of such instability, all the poles with Im(ν+ k,Q) < 0 are categorized as spurious. Visually, this instability is manifested via distinct circles in each of the six colors for Im(ν+ k,Q) < 0 throughout the entire SRI for chemical shifts Re(ν+ k,Q) ∈[0.75, 3.75]ppm. A very similar pattern is observed for the Argand plot of the Padé-reconstructed zeros, shown in panel (b) of Fig. 3 as Im(ν+ k,P) ver- sus Re(ν+ k,P). Namely, in the region of Im(ν+ k,P) > 0, there is concordance to the level of stochasticity among the displayed six color-coded sets of reconstructed gen- uine zeros for K = 575 (black) , 585 (green) , 595 (cyan) , 605 (red) , 615 (magenta) and 625 (blue). In other words, at Im(ν+ k,Q) > 0, nearly all the circles from the ﬁrst ﬁve of the mentioned colors are hidden underneath the last plotted blue-coded circle (K = 625). The remaining zeros, distributed in the region Im(ν+ k,P) < 0, are seen as distinct circles with all the six colors almost throughout the entire SRI, indicating instability with any change in model order K. Therefore, these latter roots of P+ K (z) are spurious zeros. Panel (c) of Fig. 3 combines the results of panels (a, b). Namely, both the reconstructed poles and zeros for K = 575, 585, 595, 605, 615 and 625 are displayed together in the Argand plot of Im(ν+ k,X) versus Re(ν+ k,X), with X = P and X = Q. The poles are illustrated in the same way as in panel (a), namely as color- coded circles, whereas the zeros are depicted as color-coded dots. 3.3 Poles and zeros reconstructed by the FPT(+) with spectra averaging and time signal extrapolation 4 Quantiﬁcation of the reconstructed time signal (with spectra averaging and FID extrapola- tion) for K = 575(5)625 yielding: 11 sets of Argand plots Im(ν+ k,Q) versus Re(ν+ k,Q) for 11 sets of poles (a), Im(ν+ k,P) versus Re(ν+ k,P) for 11 sets of zeros (b) and the poles plus zeros Im(ν+ k,X ) versus Re(ν+ k,X ) with X = P, Q (c). Genuine poles and zeros are located at Im(ν+ k,X ) > 0, and spurious at Im(ν+ k,X ) < 0, where X = P, Q (Color online) 1 1.5 2 2.5 3 3.5 −0.06 −0.04 −0.02 0 0.02 0.04 (b) FPT(+) ; All Zeros (Circles) : ν+ k,P = [1/(2πiτ)]ln(z+ k,P ) , { z+ k,P : Roots of Characteristic Equation P+ K(z) = 0 } 11 Argand Diagrams of All Zeros From the Reconstructed FID Re(ν+ k,P ) (ppm) Im(ν+ k,P ) (ppm) (b) 1 1.5 2 2.5 3 3.5 −0.06 −0.04 −0.02 0 0.02 0.04 (c) FPT(+) ; All Poles (Circles) : ν+ k,Q = [1/(2πiτ)]ln(z+ k,Q ) and All Zeros (Dots): ν+ k,P = [1/(2πiτ)]ln(z+ k,P ) NAA acNeu 11 Argand Diagrams of All Poles & All Zeros From the Reconstructed FID Re(ν+ k,X ) (ppm) ; X = P, Q Im(ν+ k,X ) (ppm) ; X = P, Q (c) Fig. 4 Quantiﬁcation of the reconstructed time signal (with spectra averaging and FID extrapola- tion) for K = 575(5)625 yielding: 11 sets of Argand plots Im(ν+ k,Q) versus Re(ν+ k,Q) for 11 sets of poles (a), Im(ν+ k,P) versus Re(ν+ k,P) for 11 sets of zeros (b) and the poles plus zeros Im(ν+ k,X ) versus Re(ν+ k,X ) with X = P, Q (c). Genuine poles and zeros are located at Im(ν+ k,X ) > 0, and spurious at Im(ν+ k,X ) < 0, where X = P, Q (Color online) 12 3 1138 J Math Chem (2017) 55:1110–1157 with association to K = 575, 585, 595, 605, 615 and 625, corresponding to the par- tial signal lengths NP = 1150, 1170, 1190, 1210, 1230 and 1250, respectively. In the region of Im(ν+ k,Q) > 0, there is complete agreement to the level of stochas- ticity among the six sets of reconstructed poles, which almost always appear as blue circles (the last plotted curve is in blue for K = 625). 3.3 Poles and zeros reconstructed by the FPT(+) with spectra averaging and time signal extrapolation Thereby, it can be clearly seen that the genuine poles and zeros, at Im(ν+ k,X) > 0, all in blue color are pre- dominantly non-coincident, and only sometimes lying at close distances. In contrast, nearly all poles and zeros are coincident at Im(ν+ k,Q) < 0 and Im(ν+ k,P) < 0, such that color-concordant dots and circles, i.e. Froissart doublets as spurious resonances, are seen as being distributed throughout the SRI. In Fig. 4, we examine the reconstructed poles and zeros for all 11 model orders, K = 575(5)625 using the procedure as in Fig. 3 to identify genuine and spurious ﬁndings. Here, the color-coding of symbols is the same as for the curves in Fig. 2, i.e. K = 575 (black) , 585 (green) , 595 (cyan) , 605 (red) , 615 (magenta) and 625 (blue) for K = 575(10)625, plus the addendum as green (K = 580), cyan (K = 590), red (K = 600), magenta (K = 610) and blue (K = 620) for K = 580(10)620. In the region of Im(ν+ k,Q) > 0 of panel (a) of Fig. 4 within the level of stochasticity, the assembly of the reconstructed poles for the 11 model orders is practically identical to their counterparts from panel (a) of Fig. 3 for the six model orders. Thus, for two such overlapping groups of values of K, the stability of the reconstructed poles with Im(ν+ k,Q) > 0 is demonstrated, indicating a converged result which, hence, is binned as genuine. In contradistinction, however, for the region of Im(ν+ k,Q) < 0, the distribution of a complementary collection of the reconstructed poles appears to be entirely different from those in the same region Im(ν+ k,Q) < 0 of panel (a) of Fig. 3. This instability points to the lack of convergence and, therefore, these poles are 12 123 1139 J Math Chem (2017) 55:1110–1157 spurious. Since there are nearly twice as many model orders in Fig. 4a, as expected, the spurious poles are much more densely packed compared to their counterparts on Fig. 3a. The only similarity between Figs. 3a and 4a for the region Im(ν+ k,Q) < 0 is that all six colors of circles are discernable. The values of the reconstructed zeros for the 11 model orders displayed in panel (b) of Fig. 4 are fully consistent with those for the reconstructed poles. 3.3 Poles and zeros reconstructed by the FPT(+) with spectra averaging and time signal extrapolation Namely, in the region of Im(ν+ k,P) > 0, the distribution of the genuine reconstructed zeros in Fig. 4b is identiﬁed by being virtually the same to the level of stochasticity as those for the six model orders of Fig. 3b. Again, stability of thesereconstructedzerosforIm(ν+ k,P) > 0 andtheirconvergenceisdemonstrated,thus qualifying them as genuine. For Im(ν+ k,P) < 0, the reconstructed unstable spurious zeros on Fig. 4b are more numerous, as well as distinctly identiﬁable vis-à-vis model order and of a different distribution than for the six model orders on Fig. 3b. Consistent with the results of panels (a) and (b) of Figs. 3 and 4, the pattern of genuine poles and zeros all in the region Im(ν+ k,Q) > 0 and Im(ν+ k,P) > 0, respectively, appears to be identical for the six and eleven model orders. There are almost exclusively pole-zero coincidences in Im(ν+ k,P) < 0 and Im(ν+ k,Q) < 0 of panel (c) of Fig. 4, with the only notable difference from panel (c) of Fig. 3 being that the Froissart doublets are substantially denser in the former. 3.4 Magnitudes and phases reconstructed by the FPT(+) with spectra averaging and time signal extrapolation Figure 5 presents the magnitudes and phases retrieved by the FPT(+) using the recon- structed FID data resulting from the IDFT inversion of the complex average envelope alongside extrapolation. In panel (a), six sets of reconstructed magnitudes d+ k versus chemical shift are shown for K = 575(10)625. Most notably, at physical frequencies Im(ν+ k,Q) > 0, with the exception of very slight discrepancies around 3.4 and 3.6 ppm, nearly all the other reconstructed magnitudes appear as purely blue circles (the last plotted at K = 625), indicating full agreement among the six sets of the reconstructed magnitudes. Hence, these are genuine magnitudes. Although some of these genuine magnitudes are very small, they are still non-zero. The diagram of unphysical, i.e. zero-valued magnitudes at Im(ν+ k,Q) < 0 is presented in panel (b). Therein, although densely packed, circles of all the six colors can visibly be identiﬁed, indicating that the magnitudes for Im(ν+ k,Q) < 0 are unstable with change in model order K and, thus, they are binned as spurious. The plot of the retrieved phases ϕ+ k versus chemical shift is shown in panel (c). Therein, at Im(ν+ k,Q) > 0, there is close agreement among the six sets of reconstructed phases throughout the SRI, except for a few small discordances near 2.6ppm and from 3.3 to 3.6ppm. Dramatically contrasted to panel (c) are the reconstructed unphysical phases for Im(ν+ k,Q) < 0 shown in panel (d) of Fig. 5. In this latter negative imaginary frequency region, there is no concordance whatsoever among the six sets of reconstructed spurious phases, such that circles of all six colors appear throughout the SRI. Therefore, these phases at Im(ν+ k,Q) < 0 are spurious. k,Q Proceeding to Fig. 6, the ﬁndings for the 11 model orders K = 575(5)625 are displayed for the reconstructed magnitudes and phases. Therein, in panel (a) of Fig. 3.4 Magnitudes and phases reconstructed by the FPT(+) with spectra averaging and time signal extrapolation 6 for Im(ν+ k,Q) > 0, the genuine magnitudes are identiﬁed by being essentially 12 3 1140 J Math Chem (2017) 55:1110–1157 1 1.5 2 2.5 3 3.5 0 0.01 0.02 0.03 0.04 NAA acNeu (a) FPT(+) ; Physical Magnitudes (Circles) at Im( ν+ k,Q ) > 0 : \|d + k \| = \| P+ K(z+ k,Q ) / {[(d/dz)Q+ K(z)]z=z + k,Q }\| 6 Diagrams of Physical Magnitudes From the Reconstructed FID Chemical Shift (ppm) 10−3 × \|d+ k \| (au) 1 1.5 2 2.5 3 3.5 0 0.01 0.02 0.03 0.04 (b) FPT(+) ; Unphysical Magnitudes (Circles) at Im( ν+ k,Q ) < 0 : \|d + k \| = \| P+ K(z+ k,Q ) / {[(d/dz)Q+ K(z)]z=z + k,Q }\| 6 Diagrams of Unphysical Magnitudes From the Reconstructed FID Chemical Shift (ppm) 10−3 × \|d+ k \| (au) 1 1.5 2 2.5 3 3.5 −4 −2 0 2 4 NAA acNeu (c) −π +π FPT(+) ; Physical Phases (Circles) at Im( ν+ k,Q ) > 0 : φ+ k = Arg(d+ k) = Arctan({Im(d+ k)}/{Re(d+ k)}) 6 Diagrams of Physical Phases From the Reconstructed FID Chemical Shift (ppm) φ+ k (rad) 1 1.5 2 2.5 3 3.5 −4 −2 0 2 4 (d) −π +π FPT(+) ; Unphysical Phases (Circles) at Im( ν+ k,Q ) < 0 : φ+ k = Arg(d+ k) = Arctan({Im(d+ k)}/{Re(d+ k)}) 6 Diagrams of Unphysical Phases From the Reconstructed FID Chemical Shift (ppm) φ+ k (rad) Amplitudes in Component Spectra {P+ K(z)/Q+ K(z)}U k at K = 575(10)625 (Averaging & Extrapolation) Negligible Variances of Magnitudes & Phases at Re( ν+ k,Q ) Corresponding to Physical Im( ν+ k,Q ) > 0 Fig. 5 Quantiﬁcation of the reconstructed time signal (with spectra averaging and FID extrapolation) for K = 575(10)625 yielding: six sets of magnitude \|d+ k \| versus chemical shift at Im(ν+ k,Q) > 0 (a) and at Im(ν+ k,Q) < 0 (b), as well as phase ϕ+ k versus chemical shift at Im(ν+ k,Q) > 0 (c) and Im(ν+ k,Q) < 0 (d). 3.4 Magnitudes and phases reconstructed by the FPT(+) with spectra averaging and time signal extrapolation Here, Im(ν+ k,Q) > 0 and Im(ν+ k,Q) < 0 are physical and unphysical frequencies, respectively (Color online) Amplitudes in Component Spectra {P+ K(z)/Q+ K(z)}U k at K = 575(10)625 (Averaging & Extrapolation) Negligible Variances of Magnitudes & Phases at Re( ν+ k,Q ) Corresponding to Physical Im( ν+ k,Q ) > 0 1 1.5 2 2.5 3 3.5 0 0.01 0.02 0.03 0.04 NAA acNeu (a) FPT(+) ; Physical Magnitudes (Circles) at Im( ν+ k,Q ) > 0 : \|d + k \| = \| P+ K(z+ k,Q ) / {[(d/dz)Q+ K(z)]z=z + k,Q }\| 6 Diagrams of Physical Magnitudes From the Reconstructed FID Chemical Shift (ppm) 10−3 × \|d+ k \| (au) Amplitudes in Component Spectra {P+ K(z)/Q+ K(z)}U k at K = 575(10)625 (Averaging & Extrapolation) Negligible Variances of Magnitudes & Phases at Re( ν+ k,Q ) Corresponding to Physical Im( ν+ k,Q ) > 0 1 1.5 2 2.5 3 3.5 0 0.01 0.02 0.03 0.04 NAA acNeu (a) FPT(+) ; Physical Magnitudes (Circles) at Im( ν+ k,Q ) > 0 : \|d + k \| = \| P+ K(z+ k,Q ) / {[(d/dz)Q+ K(z)]z=z + k,Q }\| 6 Diagrams of Physical Magnitudes From the Reconstructed FID Chemical Shift (ppm) 10−3 × \|d+ k \| (au) 1 1.5 2 2.5 3 3.5 0 0.01 0.02 0.03 0.04 (b) FPT(+) ; Unphysical Magnitudes (Circles) at Im( ν+ k,Q ) < 0 : \|d + k \| = \| P+ K(z+ k,Q ) / {[(d/dz)Q+ K(z)]z=z + k,Q }\| 6 Diagrams of Unphysical Magnitudes From the Reconstructed FID Chemical Shift (ppm) 10−3 × \|d+ k \| (au) Amplitudes in Component Spectra {P+ K(z)/Q+ K(z)}U k at K = 575(10)625 (Averaging & Extrapolation) Negligible Variances of Magnitudes & Phases at Re( ν+ k,Q ) Corresponding to Physical Im( ν+ k,Q ) > 0 Chemical Shift (ppm) 1 1.5 2 2.5 3 3.5 0 0.01 0.02 0.03 0.04 (b) FPT(+) ; Unphysical Magnitudes (Circles) at Im( ν+ k,Q ) < 0 : \|d + k \| = \| P+ K(z+ k,Q ) / {[(d/dz)Q+ K(z)]z=z + k,Q }\| 6 Diagrams of Unphysical Magnitudes From the Reconstructed FID Chemical Shift (ppm) 10−3 × \|d+ k \| (au) 1 1.5 2 2.5 3 3.5 0 0.01 Chemical Shift (ppm) 10−3 × \|d 1 1.5 2 2.5 3 3.5 −4 −2 0 2 4 NAA acNeu (c) −π +π FPT(+) ; Physical Phases (Circles) at Im( ν+ k,Q ) > 0 : φ+ k = Arg(d+ k) = Arctan({Im(d+ k)}/{Re(d+ k)}) 6 Diagrams of Physical Phases From the Reconstructed FID Chemical Shift (ppm) φ+ k (rad) 1 1.5 2 2.5 3 3.5 −4 −2 0 2 4 NAA acNeu (c) −π +π FPT(+) ; Physical Phases (Circles) at Im( ν+ k,Q ) > 0 : φ+ k = Arg(d+ k) = Arctan({Im(d+ k)}/{Re(d+ k)}) 6 Diagrams of Physical Phases From the Reconstructed FID Chemical Shift (ppm) φ+ k (rad) (c) 1 1.5 2 2.5 3 3.5 −4 −2 0 2 4 (d) −π +π FPT(+) ; Unphysical Phases (Circles) at Im( ν+ k,Q ) < 0 : φ+ k = Arg(d+ k) = Arctan({Im(d+ k)}/{Re(d+ k)}) 6 Diagrams of Unphysical Phases From the Reconstructed FID Chemical Shift (ppm) φ+ k (rad) (d) Fig. 3.4 Magnitudes and phases reconstructed by the FPT(+) with spectra averaging and time signal extrapolation 5 Quantiﬁcation of the reconstructed time signal (with spectra averaging and FID extrapolation) for K = 575(10)625 yielding: six sets of magnitude \|d+ k \| versus chemical shift at Im(ν+ k,Q) > 0 (a) and at Im(ν+ k,Q) < 0 (b), as well as phase ϕ+ k versus chemical shift at Im(ν+ k,Q) > 0 (c) and Im(ν+ k,Q) < 0 (d). 3.4 Magnitudes and phases reconstructed by the FPT(+) with spectra averaging and time signal extrapolation Here, Im(ν+ k,Q) > 0 and Im(ν+ k,Q) < 0 are physical and unphysical frequencies, respectively (Color online) 12 1141 J Math Chem (2017) 55:1110–1157 1 1.5 2 2.5 3 3.5 0 0.01 0.02 0.03 0.04 NAA acNeu (a) FPT(+) ; Physical Magnitudes (Circles) at Im( ν+ k,Q ) > 0 : \|d + k \| = \| P+ K(z+ k,Q ) / {[(d/dz)Q+ K(z)]z=z + k,Q }\| 11 Diagrams of Physical Magnitudes From the Reconstructed FID Chemical Shift (ppm) 10−3 × \|d+ k \| (au) Amplitudes in Component Spectra {P+ K(z)/Q+ K(z)}U k at K = 575(5)625 (Averaging & Extrapolation) Negligible Variances of Magnitudes & Phases at Re( ν+ k,Q ) Corresponding to Physical Im( ν+ k,Q ) > 0 Amplitudes in Component Spectra {P+ K(z)/Q+ K(z)}U k at K = 575(5)625 (Averaging & Extrapolation) 1 1.5 2 2.5 3 3.5 0 0.01 0.02 0.03 0.04 NAA acNeu (a) FPT(+) ; Physical Magnitudes (Circles) at Im( ν+ k,Q ) > 0 : \|d + k \| = \| P+ K(z+ k,Q ) / {[(d/dz)Q+ K(z)]z=z + k,Q }\| 11 Diagrams of Physical Magnitudes From the Reconstructed FID Chemical Shift (ppm) 10−3 × \|d+ k \| (au) 1 1.5 2 2.5 3 3.5 0 0.01 0.02 0.03 0.04 (b) FPT(+) ; Unphysical Magnitudes (Circles) at Im( ν+ k,Q ) < 0 : \|d + k \| = \| P+ K(z+ k,Q ) / {[(d/dz)Q+ K(z)]z=z + k,Q }\| 11 Diagrams of Unphysical Magnitudes From the Reconstructed FID Chemical Shift (ppm) 10−3 × \|d+ k \| (au) 1 1.5 2 2.5 3 3.5 −4 −2 0 2 4 NAA acNeu (c) −π +π FPT(+) ; Physical Phases (Circles) at Im( ν+ k,Q ) > 0 : φ+ k = Arg(d+ k) = Arctan({Im(d+ k)}/{Re(d+ k)}) 11 Diagrams of Physical Phases From the Reconstructed FID Chemical Shift (ppm) φ+ k (rad) 1 1.5 2 2.5 3 3.5 −4 −2 0 2 4 (d) −π +π FPT(+) ; Unphysical Phases (Circles) at Im( ν+ k,Q ) < 0 : φ+ k = Arg(d+ k) = Arctan({Im(d+ k)}/{Re(d+ k)}) 11 Diagrams of Unphysical Phases From the Reconstructed FID Chemical Shift (ppm) φ+ k (rad) Amplitudes in Component Spectra {P+ K(z)/Q+ K(z)}U k at K = 575(5)625 (Averaging & Extrapolation) Negligible Variances of Magnitudes & Phases at Re( ν+ k,Q ) Corresponding to Physical Im( ν+ k,Q ) > 0 Fig. 3.4 Magnitudes and phases reconstructed by the FPT(+) with spectra averaging and time signal extrapolation Here, Im(ν+ k,Q) > 0 and Im(ν+ k,Q) < 0 are physical and unphysical frequencies, respectively (Color online) Amplitudes in Component Spectra {P+ K(z)/Q+ K(z)}U k at K = 575(5)625 (Averaging & Extrapolation) Negligible Variances of Magnitudes & Phases at Re( ν+ k,Q ) Corresponding to Physical Im( ν+ k,Q ) > 0 1 1.5 2 2.5 3 3.5 0 0.01 0.02 0.03 0.04 NAA acNeu (a) FPT(+) ; Physical Magnitudes (Circles) at Im( ν+ k,Q ) > 0 : \|d + k \| = \| P+ K(z+ k,Q ) / {[(d/dz)Q+ K(z)]z=z + k,Q }\| 11 Diagrams of Physical Magnitudes From the Reconstructed FID Chemical Shift (ppm) 10−3 × \|d+ k \| (au) 1 1.5 2 2.5 3 3.5 0 0.01 0.02 0.03 0.04 (b) FPT(+) ; Unphysical Magnitudes (Circles) at Im( ν+ k,Q ) < 0 : \|d + k \| = \| P+ K(z+ k,Q ) / {[(d/dz)Q+ K(z)]z=z + k,Q }\| 11 Diagrams of Unphysical Magnitudes From the Reconstructed FID Chemical Shift (ppm) 10−3 × \|d+ k \| (au) Amplitudes in Component Spectra {P+ K(z)/Q+ K(z)}U k at K = 575(5)625 (Averaging & Extrapolation) Negligible Variances of Magnitudes & Phases at Re( ν+ k,Q ) Corresponding to Physical Im( ν+ k,Q ) > 0 K K k Negligible Variances of Magnitudes & Phases at Re( ν+ k,Q ) Corresponding to Physical Im( ν+ k,Q ) > 0 FPT(+) ; Physical Magnitudes (Circles) at Im( ν+ k,Q ) > 0 : \|d + k \| = \| P+ K(z+ k,Q ) / {[(d/dz)Q+ K(z)]z=z + k,Q }\| Chemical Shift (ppm) 1 1.5 2 2.5 3 3.5 0 0.01 0.02 0.03 0.04 (b) FPT(+) ; Unphysical Magnitudes (Circles) at Im( ν+ k,Q ) < 0 : \|d + k \| = \| P+ K(z+ k,Q ) / {[(d/dz)Q+ K(z)]z=z + k,Q }\| 11 Diagrams of Unphysical Magnitudes From the Reconstructed FID Chemical Shift (ppm) 10−3 × \|d+ k \| (au) 1 1.5 2 2.5 3 3.5 0 0.01 Chemical Shift (ppm) 10−3 × 1 1.5 2 2.5 3 3.5 −4 −2 0 2 4 NAA acNeu (c) −π +π FPT(+) ; Physical Phases (Circles) at Im( ν+ k,Q ) > 0 : φ+ k = Arg(d+ k) = Arctan({Im(d+ k)}/{Re(d+ k)}) 11 Diagrams of Physical Phases From the Reconstructed FID Chemical Shift (ppm) φ+ k (rad) 1 1.5 2 2.5 3 3.5 −4 −2 0 2 4 NAA acNeu (c) −π +π FPT(+) ; Physical Phases (Circles) at Im( ν+ k,Q ) > 0 : φ+ k = Arg(d+ k) = Arctan({Im(d+ k)}/{Re(d+ k)}) 11 Diagrams of Physical Phases From the Reconstructed FID Chemical Shift (ppm) φ+ k (rad) (c) 1 1.5 2 2.5 3 3.5 −4 −2 0 2 4 (d) −π +π FPT(+) ; Unphysical Phases (Circles) at Im( ν+ k,Q ) < 0 : φ+ k = Arg(d+ k) = Arctan({Im(d+ k)}/{Re(d+ k)}) 11 Diagrams of Unphysical Phases From the Reconstructed FID Chemical Shift (ppm) φ+ k (rad) (d) Fig. 3.4 Magnitudes and phases reconstructed by the FPT(+) with spectra averaging and time signal extrapolation 6 Quantiﬁcation of the reconstructed time signal (with spectra averaging and FID extrapolation) for K = 575(5)625 yielding: 11 sets of magnitude \|d+ k \| versus chemical shift at Im(ν+ k,Q) > 0 (a) and at Im(ν+ k,Q) < 0 (b), as well as phase ϕ+ k versus chemical shift Im(ν+ k,Q) > 0 (c) and Im(ν+ k,Q) < 0 (d). 3.4 Magnitudes and phases reconstructed by the FPT(+) with spectra averaging and time signal extrapolation 6 Quantiﬁcation of the reconstructed time signal (with spectra averaging and FID extrapolation) for K = 575(5)625 yielding: 11 sets of magnitude \|d+ k \| versus chemical shift at Im(ν+ k,Q) > 0 (a) and at Im(ν+ k,Q) < 0 (b), as well as phase ϕ+ k versus chemical shift Im(ν+ k,Q) > 0 (c) and Im(ν+ k,Q) < 0 (d). Here, Im(ν+ k,Q) > 0 and Im(ν+ k,Q) < 0 are physical and unphysical frequencies, respectively (Color online) 12 3 1142 J Math Chem (2017) 55:1110–1157 indistinguishable from their counterparts on panel (a) of Fig. 5. Here, the circles at Im(ν+ k,Q) > 0 are practically all blue, with a very few slight discordances that are at the level of stochasticity. As expected, the zero-valued reconstructed magnitudes for Im(ν+ k,Q) < 0 in panel b of Fig. 6 for all 11 model orders are more fully packed than was the case for the six model orders in Fig. 5b. Moreover, the instability can be discerned here too via the circles of distinguishable colors. Thus, these non-converged magnitudes for unphysical frequencies Im(ν+ k,Q) < 0 are all spurious. Panel (c) of Figs. 5 and 6 for the reconstructed phases ϕ+ k versus chemical shift appear to be iden- tical at Im(ν+ k,Q) > 0. Therefore, such stable converged reconstructions are genuine phases. In panel (d) of Fig. 6 the unphysical phases for Im(ν+ k,Q) < 0 are much more abundant and, moreover, throughout the SRI, they are distinct for different model orders. Being manifestly unstable and non-converging, these phases are characterized as spurious. Thus, overall, when spectra averaging and time signal extrapolation have been applied together, the reconstructed genuine magnitudes and phases determined for Im(ν+ k,Q) > 0 areclassiﬁedasgenuineafterconvergencewasattainedwhencomparing the six and eleven model orders in Figs. 5 and 6, respectively. On the other hand, for Im(ν+ k,Q) < 0, the lack of stabilization of the zero-valued, unphysical magnitudes and of the unphysical phases for six and eleven model orders is also apparent. Then, such magnitudes and phases are considered as spurious. 3.5 Poles and zeros reconstructed by the FPT(+) with no spectra averaging nor time signal extrapolation Next, we reconstruct the poles and zeros by directly using the encoded FID to which the FPT(+) is applied for the six model orders, K = 575(10)625. No averaging is performed, and no interpolation nor extrapolation by the Padé rational function is carried out, such that the encoded 1024 FID data points are used with additional 2K −1024 zeros. Figures 7a presents the Argand plot as the imaginary, Im(ν+ k,Q), versus real, Re(ν+ k,Q), frequencies for six sets of poles reconstructed by the parametric FPT(+) from the six FIDs, with the common 1024 encoded time signal points and 2K −1024 zeros. The real and imaginary parts of the encoded 1024 FID data points are those from Fig. 1a and b, respectively. The poles from panel (a) of Fig. 7 are displayed for the interval of K = 575(10)625 and, as previously, color-coded as black, green, cyan, red, magenta and blue, respectively. In contradistinction to the results with aver- aging and extrapolation, here, in the region of physical frequencies Im(ν+ k,Q) > 0, there is appreciable variance among the six sets of reconstructed poles, throughout the chemical shift region. As such, many of the reconstructed poles, even for physical frequencies Im(ν+ k,Q) > 0, at a given chemical shift, Re(ν+ k,Q), can be distinguished. This is most notable at about 3.6ppm where individual poles are seen in all six col- ors. At about 2.05ppm, there are also several individual poles reconstructed around the resonant frequencies of NAA and acNeu. 3.5 Poles and zeros reconstructed by the FPT(+) with no spectra averaging nor time signal extrapolation In the region of unphysical frequencies Im(ν+ k,Q) < 0, the reconstructed poles are visibly unstable, showing enhanced sensi- 123 12 1143 J Math Chem (2017) 55:1110–1157 1 1.5 2 2.5 3 3.5 −0.06 −0.04 −0.02 0 0.02 0.04 (a) FPT(+) ; All Poles (Circles) : ν+ k,Q = [1/(2πiτ)]ln(z+ k,Q ) , { z+ k,Q : Roots of Characteristic Equation Q+ K(z) = 0 } NAA acNeu 6 Argand Diagrams of All Poles From the Encoded FID Re(ν+ k,Q ) (ppm) Im(ν+ k,Q ) (ppm) Poles & Zeros in Components {P+ K(z)/Q+ K(z)}U k at K = 575(10)625 (No Averaging & No Extrapolation) Increased Variances of Poles & Zeros at Re( ν+ k,Q ) Corresponding to Physical Im( ν+ k,Q ) > 0 1 1.5 2 2.5 3 3.5 −0.06 −0.04 −0.02 0 0.02 0.04 (a) FPT(+) ; All Poles (Circles) : ν+ k,Q = [1/(2πiτ)]ln(z+ k,Q ) , { z+ k,Q : Roots of Characteristic Equation Q+ K(z) = 0 } NAA acNeu 6 Argand Diagrams of All Poles From the Encoded FID Re(ν+ k,Q ) (ppm) Im(ν+ k,Q ) (ppm) 1 1.5 2 2.5 3 3.5 −0.06 −0.04 −0.02 0 0.02 0.04 (b) FPT(+) ; All Zeros (Circles) : ν+ k,P = [1/(2πiτ)]ln(z+ k,P ) , { z+ k,P : Roots of Characteristic Equation P+ K(z) = 0 } 6 Argand Diagrams of All Zeros From the Encoded FID Re(ν+ k,P ) (ppm) Im(ν+ k,P ) (ppm) 1 1.5 2 2.5 3 3.5 −0.06 −0.04 −0.02 0 0.02 0.04 (c) FPT(+) ; All Poles (Circles) : ν+ k,Q = [1/(2πiτ)]ln(z+ k,Q ) and All Zeros (Dots): ν+ k,P = [1/(2πiτ)]ln(z+ k,P ) NAA acNeu 6 Argand Diagrams of All Poles & All Zeros From the Encoded FID Re(ν+ k,X ) (ppm) ; X = P, Q Im(ν+ k,X ) (ppm) ; X = P, Q Poles & Zeros in Components {P+ K(z)/Q+ K(z)}U k at K = 575(10)625 (No Averaging & No Extrapolation) Increased Variances of Poles & Zeros at Re( ν+ k,Q ) Corresponding to Physical Im( ν+ k,Q ) > 0 Fig. 3.5 Poles and zeros reconstructed by the FPT(+) with no spectra averaging nor time signal extrapolation 7 Quantiﬁcation of the encoded time signal (without spectra averaging or FID extrapolation) of length 1024, supplemented with 2K −1024 zeros for K = 575(10)625 yielding: six sets of Argand plots of Im(ν+ k,Q) versus Re(ν+ k,Q), for six sets of poles (a), Im(ν+ k,P) versus Re(ν+ k,P) for 6 sets of zeros (b), and the poles plus zeros Im(ν+ k,X ) versus Re(ν+ k,X ) with X = P, Q (c). Genuine poles and zeros are located at Im(ν+ k,X ) > 0, and spurious at Im(ν+ k,X) < 0, where X = P, Q (Color online) 1 1.5 2 2.5 3 3.5 −0.06 −0.04 −0.02 0 0.02 0.04 (b) FPT(+) ; All Zeros (Circles) : ν+ k,P = [1/(2πiτ)]ln(z+ k,P ) , { z+ k,P : Roots of Characteristic Equation P+ K(z) = 0 } 6 Argand Diagrams of All Zeros From the Encoded FID Re(ν+ k,P ) (ppm) Im(ν+ k,P ) (ppm) FPT(+) ; All Zeros (Circles) : ν+ k,P = [1/(2πiτ)]ln(z+ k,P ) , { z+ k,P : Roots of Characteristic Equation P+ K(z) = 0 } (b) 1 1.5 2 2.5 3 3.5 −0.06 −0.04 −0.02 0 0.02 0.04 (c) FPT(+) ; All Poles (Circles) : ν+ k,Q = [1/(2πiτ)]ln(z+ k,Q ) and All Zeros (Dots): ν+ k,P = [1/(2πiτ)]ln(z+ k,P ) NAA acNeu 6 Argand Diagrams of All Poles & All Zeros From the Encoded FID Re(ν+ k,X ) (ppm) ; X = P, Q Im(ν+ k,X ) (ppm) ; X = P, Q (c) Fig. 7 Quantiﬁcation of the encoded time signal (without spectra averaging or FID extrapolation) of length 1024, supplemented with 2K −1024 zeros for K = 575(10)625 yielding: six sets of Argand plots of Im(ν+ k,Q) versus Re(ν+ k,Q), for six sets of poles (a), Im(ν+ k,P) versus Re(ν+ k,P) for 6 sets of zeros (b), and the poles plus zeros Im(ν+ k,X ) versus Re(ν+ k,X ) with X = P, Q (c). 3.5 Poles and zeros reconstructed by the FPT(+) with no spectra averaging nor time signal extrapolation Genuine poles and zeros are located at Im(ν+ k,X ) > 0, and spurious at Im(ν+ k,X) < 0, where X = P, Q (Color online) 12 3 1144 J Math Chem (2017) 55:1110–1157 1 1.5 2 2.5 3 3.5 −0.06 −0.04 −0.02 0 0.02 0.04 (a) FPT(+) ; All Poles (Circles) : ν+ k,Q = [1/(2πiτ)]ln(z+ k,Q ) , { z+ k,Q : Roots of Characteristic Equation Q+ K(z) = 0 } NAA acNeu 11 Argand Diagrams of All Poles From the Encoded FID Re(ν+ k,Q ) (ppm) Im(ν+ k,Q ) (ppm) Poles & Zeros in Components {P+ K(z)/Q+ K(z)}U k at K = 575(5)625 (No Averaging & No Extrapolation) Increased Variances of Poles & Zeros at Re( ν+ k,Q ) Corresponding to Physical Im( ν+ k,Q ) > 0 1 1.5 2 2.5 3 3.5 −0.06 −0.04 −0.02 0 0.02 0.04 (a) FPT(+) ; All Poles (Circles) : ν+ k,Q = [1/(2πiτ)]ln(z+ k,Q ) , { z+ k,Q : Roots of Characteristic Equation Q+ K(z) = 0 } NAA acNeu 11 Argand Diagrams of All Poles From the Encoded FID Re(ν+ k,Q ) (ppm) Im(ν+ k,Q ) (ppm) 1 1.5 2 2.5 3 3.5 −0.06 −0.04 −0.02 0 0.02 0.04 (b) FPT(+) ; All Zeros (Circles) : ν+ k,P = [1/(2πiτ)]ln(z+ k,P ) , { z+ k,P : Roots of Characteristic Equation P+ K(z) = 0 } 11 Argand Diagrams of All Zeros From the Encoded FID Re(ν+ k,P ) (ppm) Im(ν+ k,P ) (ppm) 1 1.5 2 2.5 3 3.5 −0.06 −0.04 −0.02 0 0.02 0.04 (c) FPT(+) ; All Poles (Circles) : ν+ k,Q = [1/(2πiτ)]ln(z+ k,Q ) and All Zeros (Dots): ν+ k,P = [1/(2πiτ)]ln(z+ k,P ) NAA acNeu 11 Argand Diagrams of All Poles & All Zeros From the Encoded FID Re(ν+ k,X ) (ppm) ; X = P, Q Im(ν+ k,X ) (ppm) ; X = P, Q Poles & Zeros in Components {P+ K(z)/Q+ K(z)}U k at K = 575(5)625 (No Averaging & No Extrapolation) Increased Variances of Poles & Zeros at Re( ν+ k,Q ) Corresponding to Physical Im( ν+ k,Q ) > 0 Fig. 3.5 Poles and zeros reconstructed by the FPT(+) with no spectra averaging nor time signal extrapolation 8 Quantiﬁcation of the encoded time signal (without spectra averaging or FID extrapolation) of length 1024, supplemented with 2K −1024 zeros for K = 575(5)625 yielding: 11 sets of Argand plots Im(ν+ k,Q) versus Re(ν+ k,Q) for 11 sets of poles (a), Im(ν+ k,P) versus Re(ν+ k,P) for 11 sets of zeros (b), and the poles plus zeros Im(ν+ k,X) versus Re(ν+ k,X ) with X = P, Q respectively (c). Genuine poles and zeros are located at Im(ν+ k,X ) > 0, and spurious at Im(ν+ k,X) < 0, where X = P, Q (Color online) 1 1.5 2 2.5 3 3.5 −0.06 −0.04 −0.02 0 0.02 0.04 (b) FPT(+) ; All Zeros (Circles) : ν+ k,P = [1/(2πiτ)]ln(z+ k,P ) , { z+ k,P : Roots of Characteristic Equation P+ K(z) = 0 } 11 Argand Diagrams of All Zeros From the Encoded FID Re(ν+ k,P ) (ppm) Im(ν+ k,P ) (ppm) (b) 1 1.5 2 2.5 3 3.5 −0.06 −0.04 −0.02 0 0.02 0.04 (c) FPT(+) ; All Poles (Circles) : ν+ k,Q = [1/(2πiτ)]ln(z+ k,Q ) and All Zeros (Dots): ν+ k,P = [1/(2πiτ)]ln(z+ k,P ) NAA acNeu 11 Argand Diagrams of All Poles & All Zeros From the Encoded FID Re(ν+ k,X ) (ppm) ; X = P, Q Im(ν+ k,X ) (ppm) ; X = P, Q (c) Fig. 8 Quantiﬁcation of the encoded time signal (without spectra averaging or FID extrapolation) of length 1024, supplemented with 2K −1024 zeros for K = 575(5)625 yielding: 11 sets of Argand plots Im(ν+ k,Q) versus Re(ν+ k,Q) for 11 sets of poles (a), Im(ν+ k,P) versus Re(ν+ k,P) for 11 sets of zeros (b), and the poles plus zeros Im(ν+ k,X) versus Re(ν+ k,X ) with X = P, Q respectively (c). Genuine poles and zeros are located at Im(ν+ k,X ) > 0, and spurious at Im(ν+ k,X) < 0, where X = P, Q (Color online) 123 12 1145 J Math Chem (2017) 55:1110–1157 tivity to model order K, with distinct circles in each of the six colors throughout the entire SRI. A somewhat discrepant pattern is also seen in panel (b) in the region of Im(ν+ k,P) > 0, where many reconstructed zeros of various colors are identiﬁed, most notably at around 1.8ppm. 3.5 Poles and zeros reconstructed by the FPT(+) with no spectra averaging nor time signal extrapolation In the region of Im(ν+ k,P) < 0, almost all the retrieved zeros appear as unstable, exhibiting marked sensitivity at varying K, as apparent through distinct circles in each of the six colored symbols throughout the entire SRI. Panel (c) shows six sets for both the reconstructed poles and zeros for K = 575(10)625 in the Argand diagram of Im(ν+ k,Q) versus Re(ν+ k,Q), as well as Im(ν+ k,P) versus Re(ν+ k,P), with the poles as color-coded circles and zeros as color-coded dots. There is clear coincidence of nearly all the poles and zeros at Im(ν+ k,Q) < 0 and Im(ν+ k,P) < 0, such that these spurious resonances, i.e. Froissart doublets are evident throughout the SRI. By contrast, at Im(ν+ k,Q) > 0 and Im(ν+ k,P) > 0, the reconstructed physical poles and zeros are mostly non-coincident, albeit multi-colored, signaling some ﬂuctuation with alteration of K. Proceeding to Fig. 8, the Argand plot is shown as the imaginary, Im(ν+ k,Q), versus real, Re(ν+ k,Q) frequencies for 11 sets of poles reconstructed by the parametric FPT(+) from 11 FIDs for K = 575(5)625. There is some variance at Im(ν+ k,Q) > 0 among the 11 sets of these reconstructed physical poles throughout the chemical shift region, with the main feature being the greater density due to using the larger number of model orders compared to Fig. 7a. There is also greater density at Im(ν+ k,Q) < 0 for the unphysical poles in Fig. 8a than what is seen in Fig. 7a for six model orders K = 575(10)625.Withoutaveragingandextrapolation,the11setsofreconstructedphysical zeros in Fig. 8b show more compactness for Im(ν+ k,P) > 0, but not appreciably better concordancethanwasthecasewiththesixsetsofphysicalzerosinFig.7b.Theunphys- ical zeros Im(ν+ k,P) < 0, as expected, are also denser and differently distributed in Fig. 8b compared to Fig. 7b. These results, as clearly summarized in panel (c) of Fig. 8 withthedenser andnon-concordant dots andcircles of thevarious colors at Im(ν+ k,Q) > 0 and Im(ν+ k,P) > 0, further indicate that without averaging and extrapolation the reconstructed physical poles and zeros exhibit noticeable variance among the 11 model orders. Froissart doublets with the underlying pole-zero coincidence for Im(ν+ k,Q) < 0 and Im(ν+ k,P) < 0 are seen on Fig. 8c as being more densely packed than in Fig. 7c. 3.6 Magnitudes and phases reconstructed by the FPT(+) with no spectra averaging nor extrapolation In Fig. 9, the magnitudes and phases of amplitudes d+ k are plotted, as reconstructed by the FPT(+) for six sets of time signals (the encoded FID supplemented with 2K −1024 zeros), with no averaging and no extrapolation. In panel (a) of Fig. 9 at Im(ν+ k,Q) > 0, the reconstructed physical magnitudes \|d+ k \| versus chemical shifts are shown for = 575(10)625. These reconstructed physical magnitudes are non-zero, although some are quite small. There are several chemical shift regions in which some discrepancies are noted in relation to model order K. Most pronounced are those at about 1.3, 2.1, 3.4 and 3.6ppm, where several of the color-coded circles can be distinguished. The magnitude diagram of unphysical frequencies Im(ν+ k,Q) < 0 in panel (b) of Fig. 3.6 Magnitudes and phases reconstructed by the FPT(+) with no spectra averaging nor extrapolation 9 shows the instability occurring with change in model order K; in fact, these are all 12 3 1146 J Math Chem (2017) 55:1110–1157 Amplitudes in Components {P+ K(z)/Q+ K(z)}U k at K = 575(10)625 (No Averaging & No Extrapolation) Increased Variances of Magnitudes & Phases at Re( ν+ k,Q ) Corresponding to Physical Im( ν+ k,Q ) > 0 1 1.5 2 2.5 3 3.5 0 0.01 0.02 0.03 0.04 NAA acNeu (a) FPT(+) ; Physical Magnitudes (Circles) at Im( ν+ k,Q ) > 0 : \|d + k \| = \| P+ K(z+ k,Q ) / {[(d/dz)Q+ K(z)]z=z + k,Q }\| 6 Diagrams of Physical Magnitudes From the Encoded FID Chemical Shift (ppm) 10−3 × \|d+ k \| (au) 1 1.5 2 2.5 3 3.5 0 0.01 0.02 0.03 0.04 (b) FPT(+) ; Unphysical Magnitudes (Circles) at Im( ν+ k,Q ) < 0 : \|d + k \| = \| P+ K(z+ k,Q ) / {[(d/dz)Q+ K(z)]z=z + k,Q }\| 6 Diagrams of Unphysical Magnitudes From the Encoded FID Chemical Shift (ppm) 10−3 × \|d+ k \| (au) 1 1.5 2 2.5 3 3.5 −4 −2 0 2 4 NAA acNeu (c) −π +π FPT(+) ; Physical Phases (Circles) at Im( ν+ k,Q ) > 0 : φ+ k = Arg(d+ k) = Arctan({Im(d+ k)}/{Re(d+ k)}) 6 Diagrams of Physical Phases From the Encoded FID Chemical Shift (ppm) φ+ k (rad) 1 1.5 2 2.5 3 3.5 −4 −2 0 2 4 (d) −π +π FPT(+) ; Unphysical Phases (Circles) at Im( ν+ k,Q ) < 0 : φ+ k = Arg(d+ k) = Arctan({Im(d+ k)}/{Re(d+ k)}) 6 Diagrams of Unphysical Phases From the Encoded FID Chemical Shift (ppm) φ+ k (rad) Amplitudes in Components {P+ K(z)/Q+ K(z)}U k at K = 575(10)625 (No Averaging & No Extrapolation) Increased Variances of Magnitudes & Phases at Re( ν+ k,Q ) Corresponding to Physical Im( ν+ k,Q ) > 0 Fig. 9 Quantiﬁcation of the encoded time signal (without spectra averaging or FID extrapolation) of length 1024, supplemented with 2K −1024 zeros for K = 575(10)625 yielding six sets of magnitude \|d+ k \| versus chemical shift at Im(ν+ k,Q) > 0 (a) and at Im(ν+ k,Q) < 0 (b), as well as phase ϕ+ k versus chemical shift Im(ν+ k,Q) > 0 (c) and Im(ν+ k,Q) < 0 (d). 3.6 Magnitudes and phases reconstructed by the FPT(+) with no spectra averaging nor extrapolation Here, Im(ν+ k,Q) > 0 and Im(ν+ k,Q) < 0 are physical and unphysical frequencies, respectively (Color online) 1 1.5 2 2.5 3 3.5 0 0.01 0.02 0.03 0.04 NAA acNeu (a) FPT(+) ; Physical Magnitudes (Circles) at Im( ν+ k,Q ) > 0 : \|d + k \| = \| P+ K(z+ k,Q ) / {[(d/dz)Q+ K(z)]z=z + k,Q }\| 6 Diagrams of Physical Magnitudes From the Encoded FID Chemical Shift (ppm) 10−3 × \|d+ k \| (au) Amplitudes in Components {P+ K(z)/Q+ K(z)}U k at K = 575(10)625 (No Averaging & No Extrapolation) Increased Variances of Magnitudes & Phases at Re( ν+ k,Q ) Corresponding to Physical Im( ν+ k,Q ) > 0 1 1.5 2 2.5 3 3.5 0 0.01 0.02 0.03 NAA acNeu 6 Diagrams of Physical Magnitudes From the Encoded FID Chemical Shift (ppm) 10−3 × \|d+ k \| (au) 1 1.5 2 2.5 3 3.5 0 0.01 0.02 0.03 0.04 (b) FPT(+) ; Unphysical Magnitudes (Circles) at Im( ν+ k,Q ) < 0 : \|d + k \| = \| P+ K(z+ k,Q ) / {[(d/dz)Q+ K(z)]z=z + k,Q }\| 6 Diagrams of Unphysical Magnitudes From the Encoded FID Chemical Shift (ppm) 10−3 × \|d+ k \| (au) Chemical Shift (ppm) 1 1.5 2 2.5 3 3.5 0 0.01 0.02 0.03 0.04 (b) FPT(+) ; Unphysical Magnitudes (Circles) at Im( ν+ k,Q ) < 0 : \|d + k \| = \| P+ K(z+ k,Q ) / {[(d/dz)Q+ K(z)]z=z + k,Q }\| 6 Diagrams of Unphysical Magnitudes From the Encoded FID Chemical Shift (ppm) 10−3 × \|d+ k \| (au) (b) 1 1.5 2 2.5 3 3.5 −4 −2 0 2 4 NAA acNeu (c) −π +π FPT(+) ; Physical Phases (Circles) at Im( ν+ k,Q ) > 0 : φ+ k = Arg(d+ k) = Arctan({Im(d+ k)}/{Re(d+ k)}) 6 Diagrams of Physical Phases From the Encoded FID Chemical Shift (ppm) φ+ k (rad) (c) 1 1.5 2 2.5 3 3.5 −4 −2 0 2 4 (d) −π +π FPT(+) ; Unphysical Phases (Circles) at Im( ν+ k,Q ) < 0 : φ+ k = Arg(d+ k) = Arctan({Im(d+ k)}/{Re(d+ k)}) 6 Diagrams of Unphysical Phases From the Encoded FID Chemical Shift (ppm) φ+ k (rad) (d) Fig. 3.6 Magnitudes and phases reconstructed by the FPT(+) with no spectra averaging nor extrapolation 9 Quantiﬁcation of the encoded time signal (without spectra averaging or FID extrapolation) of length 1024, supplemented with 2K −1024 zeros for K = 575(10)625 yielding six sets of magnitude \|d+ k \| versus chemical shift at Im(ν+ k,Q) > 0 (a) and at Im(ν+ k,Q) < 0 (b), as well as phase ϕ+ k versus chemical shift Im(ν+ k,Q) > 0 (c) and Im(ν+ k,Q) < 0 (d). 3.6 Magnitudes and phases reconstructed by the FPT(+) with no spectra averaging nor extrapolation Here, Im(ν+ k,Q) > 0 and Im(ν+ k,Q) < 0 are physical and unphysical frequencies, respectively (Color online) 123 12 123 1147 J Math Chem (2017) 55:1110–1157 1147 1 1.5 2 2.5 3 3.5 0 0.01 0.02 0.03 0.04 NAA acNeu (a) FPT(+) ; Physical Magnitudes (Circles) at Im( ν+ k,Q ) > 0 : \|d + k \| = \| P+ K(z+ k,Q ) / {[(d/dz)Q+ K(z)]z=z + k,Q }\| 11 Diagrams of Physical Magnitudes From the Encoded FID Chemical Shift (ppm) 10−3 × \|d+ k \| (au) 1 1.5 2 2.5 3 3.5 0 0.01 0.02 0.03 0.04 (b) FPT(+) ; Unphysical Magnitudes (Circles) at Im( ν+ k,Q ) < 0 : \|d + k \| = \| P+ K(z+ k,Q ) / {[(d/dz)Q+ K(z)]z=z + k,Q }\| 11 Diagrams of Unphysical Magnitudes From the Encoded FID Chemical Shift (ppm) 10−3 × \|d+ k \| (au) 1 1.5 2 2.5 3 3.5 −4 −2 0 2 4 NAA acNeu (c) −π +π FPT(+) ; Physical Phases (Circles) at Im( ν+ k,Q ) > 0 : φ+ k = Arg(d+ k) = Arctan({Im(d+ k)}/{Re(d+ k)}) 11 Diagrams of Physical Phases From the Encoded FID Chemical Shift (ppm) φ+ k (rad) 4 (d) +π FPT(+) ; Unphysical Phases (Circles) at Im( ν+ k,Q ) < 0 : φ+ k = Arg(d+ k) = Arctan({Im(d+ k)}/{Re(d+ k)}) 11 Diagrams of Unphysical Phases From the Encoded FID Amplitudes in Components {P+ K(z)/Q+ K(z)}U k at K = 575(5)625 (No Averaging & No Extrapolation) Increased Variances of Magnitudes & Phases at Re( ν+ k,Q ) Corresponding to Physical Im( ν+ k,Q ) > 0 J Math Chem (2017) 55:1110–1157 1 1.5 2 2.5 3 3.5 0 0.01 0.02 0.03 0.04 NAA acNeu (a) FPT(+) ; Physical Magnitudes (Circles) at Im( ν+ k,Q ) > 0 : \|d + k \| = \| P+ K(z+ k,Q ) / {[(d/dz)Q+ K(z)]z=z + k,Q }\| 11 Diagrams of Physical Magnitudes From the Encoded FID Chemical Shift (ppm) 10−3 × \|d+ k \| (au) Amplitudes in Components {P+ K(z)/Q+ K(z)}U k at K = 575(5)625 (No Averaging & No Extrapolation) Increased Variances of Magnitudes & Phases at Re( ν+ k,Q ) Corresponding to Physical Im( ν+ k,Q ) > 0 Amplitudes in Components {P+ K(z)/Q+ K(z)}U k at K = 575(5)625 (No Averaging & No Extrapolation) Increased Variances of Magnitudes & Phases at Re( ν+ k,Q ) Corresponding to Physical Im( ν+ k,Q ) > 0 1 1.5 2 2.5 3 3.5 0 0.01 0.02 0.03 0.04 NAA acNeu (a) FPT(+) ; Physical Magnitudes (Circles) at Im( ν+ k,Q ) > 0 : \|d + k \| = \| P+ K(z+ k,Q ) / {[(d/dz)Q+ K(z)]z=z + k,Q }\| 11 Diagrams of Physical Magnitudes From the Encoded FID Chemical Shift (ppm) 10−3 × \|d+ k \| (au) 1 1.5 2 2.5 3 3.5 0 0.01 0.02 0.03 0.04 (b) FPT(+) ; Unphysical Magnitudes (Circles) at Im( ν+ k,Q ) < 0 : \|d + k \| = \| P+ K(z+ k,Q ) / {[(d/dz)Q+ K(z)]z=z + k,Q }\| 11 Diagrams of Unphysical Magnitudes From the Encoded FID Chemical Shift (ppm) 10−3 × \|d+ k \| (au) 1 1.5 2 2.5 3 3.5 −4 −2 0 2 4 NAA acNeu (c) −π +π FPT(+) ; Physical Phases (Circles) at Im( ν+ k,Q ) > 0 : φ+ k = Arg(d+ k) = Arctan({Im(d+ k)}/{Re(d+ k)}) 11 Diagrams of Physical Phases From the Encoded FID Chemical Shift (ppm) φ+ k (rad) 1 1.5 2 2.5 3 3.5 −4 −2 0 2 4 (d) −π +π FPT(+) ; Unphysical Phases (Circles) at Im( ν+ k,Q ) < 0 : φ+ k = Arg(d+ k) = Arctan({Im(d+ k)}/{Re(d+ k)}) 11 Diagrams of Unphysical Phases From the Encoded FID Chemical Shift (ppm) φ+ k (rad) k,Q k,Q Fig. 3.6 Magnitudes and phases reconstructed by the FPT(+) with no spectra averaging nor extrapolation 10 Quantiﬁcation of the encoded time signal (without spectra averaging or FID extrapolation) of length 1024, supplemented with 2K −1024 zeros for K = 575(5)625 yielding: 11 sets of magnitude \|d+ k \| versus chemical shift at Im(ν+ k,Q) > 0 (a) and at Im(ν+ k,Q) < 0 (b), as well as phase ϕ+ k versus chemical shift Im(ν+ k,Q) > 0 (c) and Im(ν+ k,Q) < 0 (d). Here, Im(ν+ k,Q) > 0 and Im(ν+ k,Q) < 0 are physical and unphysical frequencies, respectively (Color online) FPT(+) ; Physical Magnitudes (Circles) at Im( ν+ k,Q ) > 0 : \|d + k \| = \| P+ K(z+ k,Q ) / {[(d/dz)Q+ K(z)]z=z + k,Q }\| 1 1.5 2 2.5 3 3.5 0 0.01 acNeu Chemical Shift (ppm) 10−3 × \| 1 1.5 2 2.5 3 3.5 0 0.01 0.02 0.03 0.04 (b) FPT(+) ; Unphysical Magnitudes (Circles) at Im( ν+ k,Q ) < 0 : \|d + k \| = \| P+ K(z+ k,Q ) / {[(d/dz)Q+ K(z)]z=z + k,Q }\| 11 Diagrams of Unphysical Magnitudes From the Encoded FID Chemical Shift (ppm) 10−3 × \|d+ k \| (au) Chemical Shift (ppm) 1 1.5 2 2.5 3 3.5 0 0.01 0.02 0.03 0.04 (b) FPT(+) ; Unphysical Magnitudes (Circles) at Im( ν+ k,Q ) < 0 : \|d + k \| = \| P+ K(z+ k,Q ) / {[(d/dz)Q+ K(z)]z=z + k,Q }\| 11 Diagrams of Unphysical Magnitudes From the Encoded FID Chemical Shift (ppm) 10−3 × \|d+ k \| (au) (b) 1 1.5 2 2.5 3 3.5 −4 −2 0 2 4 NAA acNeu (c) −π +π FPT(+) ; Physical Phases (Circles) at Im( ν+ k,Q ) > 0 : φ+ k = Arg(d+ k) = Arctan({Im(d+ k)}/{Re(d+ k)}) 11 Diagrams of Physical Phases From the Encoded FID Chemical Shift (ppm) φ+ k (rad) (c) 1 1.5 2 2.5 3 3.5 −4 −2 0 2 4 (d) −π +π FPT(+) ; Unphysical Phases (Circles) at Im( ν+ k,Q ) < 0 : φ+ k = Arg(d+ k) = Arctan({Im(d+ k)}/{Re(d+ k)}) 11 Diagrams of Unphysical Phases From the Encoded FID Chemical Shift (ppm) φ+ k (rad) (d) Fig. 3.6 Magnitudes and phases reconstructed by the FPT(+) with no spectra averaging nor extrapolation 10 Quantiﬁcation of the encoded time signal (without spectra averaging or FID extrapolation) of length 1024, supplemented with 2K −1024 zeros for K = 575(5)625 yielding: 11 sets of magnitude \|d+ k \| versus chemical shift at Im(ν+ k,Q) > 0 (a) and at Im(ν+ k,Q) < 0 (b), as well as phase ϕ+ k versus chemical shift Im(ν+ k,Q) > 0 (c) and Im(ν+ k,Q) < 0 (d). Here, Im(ν+ k,Q) > 0 and Im(ν+ k,Q) < 0 are physical and unphysical frequencies, respectively (Color online) 12 3 1148 J Math Chem (2017) 55:1110–1157 zero-valued amplitudes. In panel (c), which is the plot of phases ϕ+ k versus chemical shift, variance is observed at Im(ν+ k,Q) > 0 among the six sets of the reconstructed physical phases, throughout the SRI. The phases at Im(ν+ k,Q) < 0 shown in panel (d) are completely discordant for the six sets of these unphysical reconstructions. We now present in Fig. 10, the 11 sets of Padé-reconstructed magnitudes and phases for K = 575(5)625, without averaging and extrapolation. In panel (a) of Fig. 10, for the found magnitudes at physical frequencies Im(ν+ k,Q) > 0, full stabilization has not occurred, such that several chemical shift regions show discrepancies with alteration in model order K. As was the case for the six model orders K shown in Fig. 9, these discrepancies for the 11 model orders K are most clearly seen at about 1.3, 2.1, 3.4 and 3.6ppm. The distinct physical magnitudes at Im(ν+ k,Q) > 0 are noted to be more tightly packed for the 11 model orders in Fig. 10a for K = 575(5)625 than the six model orders in Fig. 9a for K = 575(10)625. This is also the case for the unphysical zero-valued magnitudes from Fig. 10b with Im(ν+ k,Q) < 0, that are again more densely distributed, but still distinguishable. The reconstructed physical phases at Im(ν+ k,Q) > 0 presented in panel (c) of Fig. 10 are also more numerous and discernable than their counterparts in Fig. 9c. Further, the unphysical phases at Im(ν+ k,Q) < 0 on Fig. 10d are not only denser than in panel (d) of Fig. 9, but also show an entirely different distribution, reﬂective of the instability of non-physical resonances in the face of different model orders K. In summary, regarding Figs. 3–10, it follows that without time signal extrapolation and spectra averaging (Figs. 3.6 Magnitudes and phases reconstructed by the FPT(+) with no spectra averaging nor extrapolation 7–10), a new and opposing feature emerges relative to the case with time signal extrapolation and spectra averaging (Figs. 3–6), indicating that the complex frequencies and complex amplitudes, even for physical frequencies, Im(ν+ k,Q) > 0 do not completely stabilize. 123 3.7 Component spectra In both Figs. 11 and 12, we examine the convergence of the component spectra built from the reconstructed parameters at K = 575(10)625 and K = 575(5)625 for the six and eleven model orders K, with and without spectra averaging and extrapolation. Beginning with the panel (a) of Fig. 11, the Usual components were built from the six model orders with spectra averaging and extrapolation. Therein, practically full convergence to the level of stochasticity was attained throughout the SRI. Thus, most of the components appear totally as blue (the last plotted curve for K = 625 is in blue). The only minimal exception is at about 3.4ppm, where very slight green and cyan are seen to top the up-going peaks, and an even smaller green down-going peak can be discerned. Further, in panel (b) for all the 11 model orders with spectra averaging and extrapolation,theﬁndingsfortheUsualcomponentsareessentiallyidenticaltothosein panel (a) of Fig. 11. On the other hand, without spectra averaging and extrapolation, both for the six model orders (panel c) and for the 11 model orders (panel d), the sets of Usual components are seen as discrepant. Speciﬁcally, all six colors can be distinguished, such that full stabilization has not been achieved. These ﬁndings are quite concordant for the Ersatz component spectra shown in Fig. 12. 3.7 Component spectra Here, (a, c) are for 6 model orders K = 575(10)625 and (b, d) for 11 model orders K = 575(5)625 (Color online) Convergence Rate of Component Spectra for 2 Overlapping Sets of 6 and 11 Values of Model Orders K Usual Components (Phases of Amplitudes Kept) With and Without Prior Averaging and Extrapolation 1 1.5 2 2.5 3 3.5 −1 −0.5 0 0.5 1 1.5 Real Parts of 6 Usual Components: Re(P + K /Q+ K )U k ≡ Re{d+ kz/(z − z+ k,Q )} , k ∈ [1,K ] , K = 575(10)625 (a) NAA acNeu Lip 6 Sets of Usual Components Using the Reconstructed FID FPT(+) Averaging & Extrapolation Chemical Shift (ppm) 10−3 × Re(P+ K /Q+ K )U k (au) Convergence Rate of Component Spectra for 2 Overlapping Sets of 6 and 11 Values of Model Orders K Usual Components (Phases of Amplitudes Kept) With and Without Prior Averaging and Extrapolation (b) Chemical Shift (ppm) 1 1.5 2 2.5 3 3.5 −1 −0.5 0 0.5 1 1.5 Real Parts of 6 Usual Components: Re(P + K /Q+ K )U k ≡ Re{d+ kz/(z − z+ k,Q )} , k ∈ [1,K ] , K = 575(10)625 (c) NAA acNeu Lip 6 Sets of Usual Components Using the Encoded FID FPT(+) No Averaging & No Extrapolation Chemical Shift (ppm) 10−3 × Re(P+ K /Q+ K )U k (au) 1 1.5 2 2.5 3 3.5 −1 −0.5 0 0.5 1 1.5 Real Parts of 11 Usual Components: Re(P + K /Q+ K )U k ≡ Re{d+ kz/(z − z+ k,Q )} , k ∈ [1,K ] , K = 575(5)625 (d) NAA acNeu Lip 11 Sets of Usual Components Using the Encoded FID FPT(+) No Averaging & No Extrapolation Chemical Shift (ppm) 10−3 × Re(P+ K /Q+ K )U k (au) Fig. 11 Usual component shape spectra using the FIDs that are either reconstructed (with spectra averaging and FID extrapolation) (a, b) or encoded (c, d) (without spectra averaging or FID extrapolation). 3.7 Component spectra Here, in panel (a) with spectra averaging and extrapolation carried out at 123 1149 J Math Chem (2017) 55:1110–1157 1 1.5 2 2.5 3 3.5 −1 −0.5 0 0.5 1 1.5 Real Parts of 6 Usual Components: Re(P + K /Q+ K )U k ≡ Re{d+ kz/(z − z+ k,Q )} , k ∈ [1,K ] , K = 575(10)625 (a) NAA acNeu Lip 6 Sets of Usual Components Using the Reconstructed FID FPT(+) Averaging & Extrapolation Chemical Shift (ppm) 10−3 × Re(P+ K /Q+ K )U k (au) 1 1.5 2 2.5 3 3.5 −1 −0.5 0 0.5 1 1.5 Real Parts of 11 Usual Components: Re(P + K /Q+ K )U k ≡ Re{d+ kz/(z − z+ k,Q )} , k ∈ [1,K ] , K = 575(5)625 (b) NAA acNeu Lip 11 Sets of Usual Components Using the Reconstructed FID FPT(+) Averaging & Extrapolation Chemical Shift (ppm) 10−3 × Re(P+ K /Q+ K )U k (au) 1 1.5 2 2.5 3 3.5 −1 −0.5 0 0.5 1 1.5 Real Parts of 6 Usual Components: Re(P + K /Q+ K )U k ≡ Re{d+ kz/(z − z+ k,Q )} , k ∈ [1,K ] , K = 575(10)625 (c) NAA acNeu Lip 6 Sets of Usual Components Using the Encoded FID FPT(+) No Averaging & No Extrapolation Chemical Shift (ppm) 10−3 × Re(P+ K /Q+ K )U k (au) 1 1.5 2 2.5 3 3.5 −1 −0.5 0 0.5 1 1.5 Real Parts of 11 Usual Components: Re(P + K /Q+ K )U k ≡ Re{d+ kz/(z − z+ k,Q )} , k ∈ [1,K ] , K = 575(5)625 (d) NAA acNeu Lip 11 Sets of Usual Components Using the Encoded FID FPT(+) No Averaging & No Extrapolation Chemical Shift (ppm) 10−3 × Re(P+ K /Q+ K )U k (au) Convergence Rate of Component Spectra for 2 Overlapping Sets of 6 and 11 Values of Model Orders K Usual Components (Phases of Amplitudes Kept) With and Without Prior Averaging and Extrapolation Fig. 11 Usual component shape spectra using the FIDs that are either reconstructed (with spectra averaging and FID extrapolation) (a, b) or encoded (c, d) (without spectra averaging or FID extrapolation). 3.7 Component spectra Here, (a, c) are for 6 model orders K = 575(10)625 and (b, d) for 11 model orders K = 575(5)625 (Color online) 1 1.5 2 2.5 3 3.5 −1 −0.5 0 0.5 1 1.5 Real Parts of 11 Usual Components: Re(P + K /Q+ K )U k ≡ Re{d+ kz/(z − z+ k,Q )} , k ∈ [1,K ] , K = 575(5)625 (d) NAA acNeu Lip 11 Sets of Usual Components Using the Encoded FID FPT(+) No Averaging & No Extrapolation Chemical Shift (ppm) 10−3 × Re(P+ K /Q+ K )U k (au) (d) Fig. 11 Usual component shape spectra using the FIDs that are either reconstructed (with spectra averaging and FID extrapolation) (a, b) or encoded (c, d) (without spectra averaging or FID extrapolation). 3.7 Component spectra 12 Ersatz component shape spectra using the FIDs that are either reconstructed (with spectra averaging Chemical Shift (ppm) 1 1.5 2 2.5 3 3.5 0 0.5 1 1.5 Real Parts of 11 Ersatz Components: Re(P + K /Q+ K )E k ≡ Re{\|d+ k\|z/(z − z+ k,Q )} , k ∈ [1,K ] , K = 575(5)625 (b) NAA acNeu Lip 11 Sets of Ersatz Components Using the Reconstructed FID FPT(+) : Averaging & Extrapolation Chemical Shift (ppm) 10−3 × Re(P+ K /Q+ K )E k (au) (pp ) 1 1.5 2 2.5 3 3.5 0 0.5 1 1.5 Real Parts of 11 Ersatz Components: Re(P + K /Q+ K )E k ≡ Re{\|d+ k\|z/(z − z+ k,Q )} , k ∈ [1,K ] , K = 575(5)625 (b) NAA acNeu Lip 11 Sets of Ersatz Components Using the Reconstructed FID FPT(+) : Averaging & Extrapolation Chemical Shift (ppm) 10−3 × Re(P+ K /Q+ K )E k (au) 1 1.5 2 2.5 3 3.5 0 0.5 1 1.5 Real Parts of 6 Ersatz Components: Re(P + K /Q+ K )E k ≡ Re{\|d+ k\|z/(z − z+ k,Q )} , k ∈ [1,K ] , K = 575(10)625 (c) NAA acNeu Lip 6 Sets of Ersatz Components Using the Encoded FID FPT(+) : No Averaging & No Extrapolation Chemical Shift (ppm) 10−3 × Re(P+ K /Q+ K )E k (au) 0.5 1 1.5 Real Parts of 11 Ersatz Components: Re(P + K /Q+ K )E k ≡ Re{\|d+ k\|z/(z − z+ k,Q )} , k ∈ [1,K ] , K = 575(5)625 (d) NAA acNeu Lip FPT(+) : No Averaging & No Extrapolation × Re(P+ K /Q+ K )E k (au) (b) 1 1.5 2 2.5 3 3.5 0 0.5 1 1.5 Real Parts of 6 Ersatz Components: Re(P + K /Q+ K )E k ≡ Re{\|d+ k\|z/(z − z+ k,Q )} , k ∈ [1,K ] , K = 575(10)625 (c) NAA acNeu Lip 6 Sets of Ersatz Components Using the Encoded FID FPT(+) : No Averaging & No Extrapolation Chemical Shift (ppm) 10−3 × Re(P+ K /Q+ K )E k (au) (c) 1 1.5 2 2.5 3 3.5 0 0.5 1 1.5 Real Parts of 11 Ersatz Components: Re(P + K /Q+ K )E k ≡ Re{\|d+ k\|z/(z − z+ k,Q )} , k ∈ [1,K ] , K = 575(5)625 (d) NAA acNeu Lip 11 Sets of Ersatz Components Using the Encoded FID FPT(+) : No Averaging & No Extrapolation Chemical Shift (ppm) 10−3 × Re(P+ K /Q+ K )E k (au) (d) Fig. 3.7 Component spectra Here, (a, c) are for 6 model orders K = 575(10)625 and (b, d) for 11 model orders K = 575(5)625 (Color online) 12 123 1150 J Math Chem (2017) 55:1110–1157 1 1.5 2 2.5 3 3.5 0 0.5 1 1.5 Real Parts of 6 Ersatz Components: Re(P + K /Q+ K )E k ≡ Re{\|d+ k\|z/(z − z+ k,Q )} , k ∈ [1,K ] , K = 575(10)625 (a) NAA acNeu Lip 6 Sets of Ersatz Components Using the Reconstructed FID FPT(+) : Averaging & Extrapolation Chemical Shift (ppm) 10−3 × Re(P+ K /Q+ K )E k (au) 1 1.5 2 2.5 3 3.5 0 0.5 1 1.5 Real Parts of 11 Ersatz Components: Re(P + K /Q+ K )E k ≡ Re{\|d+ k\|z/(z − z+ k,Q )} , k ∈ [1,K ] , K = 575(5)625 (b) NAA acNeu Lip 11 Sets of Ersatz Components Using the Reconstructed FID FPT(+) : Averaging & Extrapolation Chemical Shift (ppm) 10−3 × Re(P+ K /Q+ K )E k (au) 1 1.5 2 2.5 3 3.5 0 0.5 1 1.5 Real Parts of 6 Ersatz Components: Re(P + K /Q+ K )E k ≡ Re{\|d+ k\|z/(z − z+ k,Q )} , k ∈ [1,K ] , K = 575(10)625 (c) NAA acNeu Lip 6 Sets of Ersatz Components Using the Encoded FID FPT(+) : No Averaging & No Extrapolation Chemical Shift (ppm) 10−3 × Re(P+ K /Q+ K )E k (au) 1 1.5 2 2.5 3 3.5 0 0.5 1 1.5 Real Parts of 11 Ersatz Components: Re(P + K /Q+ K )E k ≡ Re{\|d+ k\|z/(z − z+ k,Q )} , k ∈ [1,K ] , K = 575(5)625 (d) NAA acNeu Lip 11 Sets of Ersatz Components Using the Encoded FID FPT(+) : No Averaging & No Extrapolation Chemical Shift (ppm) 10−3 × Re(P+ K /Q+ K )E k (au) Convergence Rate of Component Spectra for 2 Overlapping Sets of 6 and 11 Values of Model Orders K Ersatz Components (Phases of Amplitudes Nulled) With and Without Prior Averaging and Extrapolation Fig. 12 Ersatz component shape spectra using the FIDs that are either reconstructed (with spectra averaging and FID extrapolation) (a, b) or encoded (c, d) (without spectra averaging or FID extrapolation). 3.7 Component spectra Here, (a, c) are for 6 model orders K = 575(10)625 and (b,d) for 11 model orders K = 575(5)625 (Color online) 1 1.5 2 2.5 3 3.5 0 0.5 1 1.5 Real Parts of 6 Ersatz Components: Re(P + K /Q+ K )E k ≡ Re{\|d+ k\|z/(z − z+ k,Q )} , k ∈ [1,K ] , K = 575(10)625 (a) NAA acNeu Lip 6 Sets of Ersatz Components Using the Reconstructed FID FPT(+) : Averaging & Extrapolation Chemical Shift (ppm) 10−3 × Re(P+ K /Q+ K )E k (au) Convergence Rate of Component Spectra for 2 Overlapping Sets of 6 and 11 Values of Model Orders K Ersatz Components (Phases of Amplitudes Nulled) With and Without Prior Averaging and Extrapolation Convergence Rate of Component Spectra for 2 Overlapping Sets of 6 and 11 Values of Model Orders K Ersatz Components (Phases of Amplitudes Nulled) With and Without Prior Averaging and Extrapolation 1 1.5 2 2.5 3 3.5 0 0.5 1 1.5 Real Parts of 6 Ersatz Components: Re(P + K /Q+ K )E k ≡ Re{\|d+ k\|z/(z − z+ k,Q )} , k ∈ [1,K ] , K = 575(10)625 (a) NAA acNeu Lip 6 Sets of Ersatz Components Using the Reconstructed FID FPT(+) : Averaging & Extrapolation Chemical Shift (ppm) 10−3 × Re(P+ K /Q+ K )E k (au) 1 1.5 2 2.5 3 3.5 0 0.5 1 1.5 Real Parts of 11 Ersatz Components: Re(P + K /Q+ K )E k ≡ Re{\|d+ k\|z/(z − z+ k,Q )} , k ∈ [1,K ] , K = 575(5)625 (b) NAA acNeu Lip 11 Sets of Ersatz Components Using the Reconstructed FID FPT(+) : Averaging & Extrapolation Chemical Shift (ppm) 10−3 × Re(P+ K /Q+ K )E k (au) Convergence Rate of Component Spectra for 2 Overlapping Sets of 6 and 11 Values of Model Orders K Ersatz Components (Phases of Amplitudes Nulled) With and Without Prior Averaging and Extrapolation 1 1.5 2 2.5 3 3.5 0 0.5 1 1.5 Real Parts of 6 Ersatz Components: Re(P + K /Q+ K )E k ≡ Re{\|d+ k\|z/(z − z+ k,Q )} , k ∈ [1,K ] , K = 575(10)625 (a) NAA acNeu Lip 6 Sets of Ersatz Components Using the Reconstructed FID FPT(+) : Averaging & Extrapolation Chemical Shift (ppm) 10−3 × Re(P+ K /Q+ K )E k (au) 1 1.5 2 2.5 3 3.5 0 0.5 1 1.5 Real Parts of 11 Ersatz Components: Re(P + K /Q+ K )E k ≡ Re{\|d+ k\|z/(z − z+ k,Q )} , k ∈ [1,K ] , K = 575(5)625 (b) NAA acNeu Lip 11 Sets of Ersatz Components Using the Reconstructed FID FPT(+) : Averaging & Extrapolation Chemical Shift (ppm) 10−3 × Re(P+ K /Q+ K )E k (au) 1 1.5 2 2.5 3 3.5 0 0.5 1 1.5 Real Parts of 6 Ersatz Components: Re(P + K /Q+ K )E k ≡ Re{\|d+ k\|z/(z − z+ k,Q )} , k ∈ [1,K ] , K = 575(10)625 (c) NAA acNeu Lip 6 Sets of Ersatz Components Using the Encoded FID FPT(+) : No Averaging & No Extrapolation Chemical Shift (ppm) 10−3 × Re(P+ K /Q+ K )E k (au) 1 1.5 2 2.5 3 3.5 0 0.5 1 1.5 Real Parts of 11 Ersatz Components: Re(P + K /Q+ K )E k ≡ Re{\|d+ k\|z/(z − z+ k,Q )} , k ∈ [1,K ] , K = 575(5)625 (d) NAA acNeu Lip 11 Sets of Ersatz Components Using the Encoded FID FPT(+) : No Averaging & No Extrapolation Chemical Shift (ppm) 10−3 × Re(P+ K /Q+ K )E k (au) Ersatz Components (Phases of Amplitudes Nulled) With and Without Prior Averaging and Extrapolation Fig. 3.7 Component spectra 12 Ersatz component shape spectra using the FIDs that are either reconstructed (with spectra averaging and FID extrapolation) (a, b) or encoded (c, d) (without spectra averaging or FID extrapolation). Here, (a, c) are for 6 model orders K = 575(10)625 and (b,d) for 11 model orders K = 575(5)625 (Color online) 12 123 1151 J Math Chem (2017) 55:1110–1157 K = 575(10)625 for the six model orders, nearly all the absorptive Lorentzians are of blue color throughout. Only at about 3.35 and 3.4ppm, can miniscule green tips be seen above the blue peaks. Further, in panel (b) for the 11 model orders K = 575(5)625, the ﬁndings are almost indistinguishable from panel (a) except for a red tip to a peak at about 3.35 ppm. Overall, with spectra averaging and time signal extrapolation taken as a tandem, the Ersatz component spectra in both panels (a, b) for six and eleven model orders K, respectively, have fully stabilized and, thus, converged. However, without spectra averaging and extrapolation, in panels (c, d) for the six and eleven model orders, respectively, the situation is quite different. Here, all six colors are visible. Thus, the Ersatz components have not completely stabilized, when spectra averaging and time signal extrapolation are not performed. 4 Discussion and conclusions The results of the present paper show that for poles and zeros, as well as for magni- tudes and phases of complex amplitudes, spectra averaging and Padé-based time signal extrapolation are both needed to be performed together for fully accurate reconstruc- tion of stable, genuine resonances. This is critical, since poles and zeros are the key to the stability of the system. Conversely, with an underlying strong coupling, unstable zeros lead to unstable poles. It should be emphasized that at the end of data analysis, the clause “spectral param- eters and spectra have stabilized against changes in model order K,” should be taken to mean that such reconstructions are to be retained as physical. By implication, the lack of stabilization of the complementary reconstructions is interpreted as their rejection from the ﬁnal results. In a broad view, we can refer to “system theory,” whereby parametrization of a general complex system is vital for depicting the system’s performance with a rel- atively small set of the dominant features (the principle of parsimony). In quantum mechanics this and, in fact, the complete information is contained in the two equivalent concepts, the Schrödinger and the resolvent eigenvalue problems. They both gener- ate the frequency or energy spectrum as the Heaviside partial fraction decomposition which exactly sums up to the quotient of two polynomials, i.e. to the fast Padé trans- form, FPT. The parameters in the partial fractions are the fundamental frequencies and amplitudes that contain the complete information about the examined system. Thus, quantum mechanics parametrizes any system using the well-known complete- ness relation. The meaning of this relation is that everything which is informing about the system can be reconstructed from the eigenvalues and eigenfunctions as the solu- tion of the quantum-mechanical eigenproblems. The quantum-mechanical spectrum of a general system is the unique ratio of two polynomials. Consequently, the fast Padé transform, FPT, is indeed the method of choice for quantitative description of the system’s performance as well as for determining its structure. The key to robust performance of any system is its stability. This is, in turn, based upon the stability of their fundamental parameters. The averaging procedure with separation of the physical from unphysical poles and zeros is of vital importance in attaining the sought stability. This general strategy has “no borders” vis-à-vis cross disciplinary applications [59]. 4 Discussion and conclusions 12 3 3 1152 J Math Chem (2017) 55:1110–1157 In practical implementations from this work, the parametric analysis of the FPT is used to generate envelopes and component spectra. Although the spectral parameters are essential for quantitative assessment in MRS, the Usual and Ersatz component spectra, as well as the envelope spectra are also instructive for visual examination to aid clinical interpretation. The results of the present study further corroborate our previous conclusion that component spectra and total shape spectra generated from the Padé-reconstructed spectral parameters are trustworthy. Herein, we have shown that for in vivo MRS, Padé-based spectra averaging and extrapolation are needed to obtain converged results to the level of stochasticity. This is seen from the Usual and Ersatz component spectra for six versus eleven model orders with spectra averaging and extrapolation acting in concert. On the other hand, without spectra averaging and extrapolation, complete stability was not attained for six and eleven model orders. The present analyses and results have important implications for expediting the entire Padé methodology. This study further supports the earlier assertion [7,8] that with the FPT, short echo times, TEs, could also be conﬁdently employed for quantiﬁca- tion. Moreover, this would be recommended, due to the abundant spectral information which can be gleaned from short-lived resonances. The essential condition for using short TEs, is the unequivocal disentangling of overlapping resonances, a task for which the parametric FPT is fully capable, as seen herein and in our previous inves- tigations [7,8] on time signals encoded in vivo from the ovary at a TE of 30ms. As noted, dilemmas regarding the diagnostic importance of lipid, Lip, versus lactate, Lac, both resonating at about 1.3ppm, for distinguishing benign versus cancerous ovarian lesions, could best be addressed at short TEs, before Lip has decayed. Another chemical shift region of key diagnostic importance for ovarian cancer is that between 3.20 and 3.24ppm, where the components of total Cho lie in very close proximity. In our previous study [8], there was complete convergence of all the Padé- reconstructed complex frequencies and amplitudes for all physical resonances in that chemical shift region. In the present examination, the dense accumulation of non- physical poles and zeros in this chemical shift region were successfully delineated. 123 4 Discussion and conclusions The prime exam- ple to illustrate this occurrence is phsophocholine, PC, whose resonance although small, even relative to its neighbors (GPC and Cho), let alone vis-à-vis more dis- tant peaks (AcNeu, NAA) is, nevertheless, diagnostically of great importance, being one of the biomarkers of malignant transformation on the molecular level [54–56]. Only with the powerful parametric properties of the FPT, has this important cancer biomarker been identiﬁed and quantiﬁed in vivo, as seen in the present study and in Refs. [7,8,18,19,64]. Being a small and moving organ, encoding good quality MRS time signals from the ovary is very difﬁcult [7,50]. These technical challenges subsequently require advanced signal processing to effectively handle the high noise content engendered. Clearly, the fast Fourier transform, FFT, with any ﬁtting procedure is inadequate for this challenging task. The meager results from the studies using the FFT for in vivo MRS time signals from the ovary have certainly put a damper on efforts to explore the potential of MRS for early ovarian cancer detection. As a consequence, researchers within the MR community have not prioritized this problem area, despite the fact that the potential added value of improved MRS is perhaps nowhere more salient and urgent than for this problem area [64,65]. The Padé-generated results to date on the ovary [1–8] clearly indicate that this situation can, and should, change. In other words, there is now sufﬁcient evidence to conclude that Padé-optimized in vivo MRS (or FPT–MRS for short) indeed holds promise for early ovarian cancer detection and better identiﬁcation of benign ovarian lesions. A major advantage of MR-based methods is the lack of exposure to ionizing radiation. This is of critical importance for woman at increased ovarian cancer risk, for whom diagnostic radi- ation may be particularly deleterious [66]. For women at elevated ovarian cancer risk, due to heredity, ionizing radiation exposure, or other risk factors, FPT–MRS could also be suitable for surveillance. Aligned with this perspective is the view of women at high ovarian cancer risk [36,67], as well as of women from the general population [68], who have clearly expressed an interest and preference for screen- ing surveillance strategies vis-à-vis ovarian cancer. Most essentially, the survival for women afﬂicted with ovarian cancer would be markedly improved with early detection of this malignancy. To achieve this goal, effective diagnostic methods are vital. 4 Discussion and conclusions With averaging and extrapolation, the physical poles and zeros, as well as the physical magnitudes and phases were all entirely stable against changes in model order in this region between 3.20 and 3.24ppm (here, “physical” refers to the reconstructed positive imaginary frequencies). Together, these ﬁndings suggest that phosphocholine, PC, a recognized cancer biomarker, can be quantiﬁed with full reliability through Padé- optimized in vivo MRS. The chemical shift region between 2.0 and 2.1ppm is also of particular interest for ovarian cancer diagnostics. In our previous investigation [8], complete convergence of the spectral parameters of all the physical resonances was also achieved in that region, with quantiﬁcation by the FPT using the extrapolated time signals reconstructed by inverting the complex average envelope. Herein, we have also isolated the very dense accumulation of non-physical poles, zeros, magnitudes and phases between 2.0 and 2.1 ppm. Unequivocal identiﬁcation of such spurious information and its subsequent elimination clears the way for generating the ﬁnal linelist of exclusively genuine spectral parameters as the true constituents of the input time signal. We anticipate that existing dilemmas regarding the presence of NAA implied by the fast Fourier transform, FFT, for in vivo MRS of the ovary [51,60–63] could also be clariﬁed. 123 1153 J Math Chem (2017) 55:1110–1157 Namely, that the true signiﬁcance of NAA versus acNeu in identifying malignant as opposed to benign ovarian lesions can be ascertained. ( ) Namely, that the true signiﬁcance of NAA versus acNeu in identifying malignant as opposed to benign ovarian lesions can be ascertained. Overall, it was seen herein that the FPT(+) is able to reconstruct a large num- ber of resonances (of the order of 90) in the considered spectral range of interest, SRI, which is ν ∈[0, 75, 3.75]ppm. Admittedly, many of the resonant peaks are short, indicating that the corresponding pole strengths are weak. The peak height is directly proportional to the magnitude of the signal amplitude and inversely pro- portional to the full width at half maximum, FWHM. What counts here is not the relative smallness of a resonance, but rather its stability with respect to perturbations, such as alterations in noise levels, model order K, etc. Some of the found weak, but stable resonances can still have important diagnostic signiﬁcance. Conﬂict of interest The authors declare that they have no conﬂict of interest. Conﬂict of interest The authors declare that they have no conﬂict of interest. Conﬂict of interest The authors declare that they have no conﬂict of interest. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 Interna- tional License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. 4 Discussion and conclusions The contribution of in vivo FPT-MRS to this endeavor can be conﬁdently anticipated. 12 3 1154 J Math Chem (2017) 55:1110–1157 Acknowledgements This work was supported by The Marsha Rivkin Center for Ovarian Cancer Research, King Gustav the 5th Jubilee Fund, and FoUU through Stockholm County Council to which the authors are grateful. We would like to thank our colleagues, Professors Marinette van der Graaf, Leon Massuger, Ron Wevers, Eva Kolwijck, Udo Engelke, Arend Heerschap, Henk Blom, M’Hamed Hadfoune, and Wim A. 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W4247462534.txt	https://cloudfront.escholarship.org/dist/prd/content/qt9wc1t4k9/qt9wc1t4k9.pdf?t=pfzyg1	en		from the society: Update on the 6th Biennial Conference of the International Biogeography Society	Frontiers of biogeography	2,012	cc-by	427	UC Merced Frontiers of Biogeography Title from the society: Update on the 6th Biennial Conference of the International Biogeography Society Permalink https://escholarship.org/uc/item/9wc1t4k9 Journal Frontiers of Biogeography, 4(1) Author Gavin, Daniel Publication Date 2012 DOI 10.21425/F5FBG12531 Copyright Information Copyright 2012 by the author(s).This work is made available under the terms of a Creative Commons Attribution License, available at https://creativecommons.org/licenses/by/4.0/ eScholarship.org Powered by the California Digital Library University of California membership corner from the society Update on the 6th Biennial Conference of the International Biogeography Society Planning is well underway for the Sixth Biennial Conference of the International Biogeography Society, which will be held at the Kovens Convention Center located on Biscayne Bay campus of Florida International University in Miami Florida on January 9th to 13th, 2012. January climate in South Florida is mild, with temperatures ranging from 17 to 26 °C and only a small chance of rain. The main conference hotel is located directly on the beach. The preliminary program includes four plenary symposia: (1) Predicting species and biodiversity in a warmer world: are we doing a good job? (2) Beyond Bergmann: new perspectives on the biogeography of traits, (3) Island biogeography: new syntheses, and (4) Convergence of conservation paleontology and biogeography. Plenary speakers will include, among others, James Brown (University of New Mexico), Jonathan Losos (Harvard University), Elizabeth Hadley (Stanford University), Antoine Guisan (University of Lausanne), and Lauren Buckley (University of North Carolina). Over 70 contributed talks will address a variety of biogeographic themes, from marine biogeography to phylogeography. The spacious conference center will accommodate 300 posters. Graduate students will be able to engage senior scientists during specially organized lunch-time sessions. Pre-conference workshops (Jan 9th) in development include half-day sessions on the topics of (1) merging ecophysiological data into species distribution models, (2) popular science writing, (3) advice for early-career and non-native English speakers on preparing work for publication, (4) Bayesian modeling, and a full-day session on (5) biodiversity informatics. Field trips are being planned for the beginning (Jan 9th) and end (Jan 13th) of the conference. These tours, mostly for small groups of less than 20 people, may range from airboat rides, hikes in Everglades National Park, canoeing the historic Oleta River, birding tour of south Florida, sea kayaking, snorkeling, and tours of the Fairchild Tropical Botanic Garden. Abstract submission and registration will open mid-20121. Daniel Gavin Vice President for Conferences, International Biogeography Society. dgavin@uoregon.edu Edited by Matthew Heard 1. Further details are posted at http://www.biogeography.org/html/Meetings/2013/index.html. frontiers of biogeography 4.1, 2012 — © 2012 the authors; journal compilation © 2012 The International Biogeography Society 41

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